Neutral amino acids monitoring in phenylketonuric plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Neutral and non-polar amino acids such as phenylalanine (Phe), valine (Val), tyrosine (Tyr), threonine (Thre) and GABA are hard to resolve by capillary zone electrophoresis (CZE). Their separation is possible by adding a surfactant to the mobile phase. This method is called micellar electrokinetic chromatography (MEKC). We used MEKC with laser-induced fluorescence detection (LIFD) to separate and quantitate these amino acids in plasma microdialysates of patients with phenylketonuria (PKU). This disease is an inborn enzymatic defect with decreased conversion of Phe to Tyr that causes severe neurological damage and mental deterioration, which is diagnosed by measuring plasma Phe and Phe/Tyr ratio. The amino acids tested had linear concentration-signal relation. PKU patients had significantly higher Phe, lower Tyr, 21 times higher Phe/Tyr ratio and decreased values of Val and Thre than controls. These results show that microdialysis of biological fluids coupled with MEKC-LIFD is a convenient technique to measure neutral amino acids in clinical disorders such as PKU.     

Electrophoretic polymorphism of glutathione peroxidase

A method for the detection of glutathione peroxidase after electrophoresis on starch gel has been devised. Blood samples from 780 persons of Caucasian or Negro origin were studied. An electrophoretically fast enzyme, designated as the Thomas variant, was found in 6·4 % of 392 Negro donors and 0·8 % of 388 Caucasian donors. This variant was inherited in an autosomal dominant fashion. A slightly rapid electrophoretic mobility was noted in some cord blood samples and in a father and daughter with a refractory anaemia. Because this variant, designated ‘Musi’ differed so little from the normal variant, it was difficult to further characterize it, but it did not appear that this variant was genetically determined. Glutathione peroxidase from most other tissues had the same electrophoretic mobility as the enzyme from erythrocytes, but the enzyme from cultured human fibroblasts moves more rapidly. Various laboratory animals were found to have electrophoretically distinguishable glutathione peroxidase, but efforts to hybridize hamster enzyme to human enzyme were unsuccessful.     

汉族人群血管紧张素原基因的新单核苷酸多态性位点

应用改进的PCR－SSCP技术、多种聚丙烯酰胺凝胶电泳和PCR产物直接测序等方法，分析人的血管紧张素原（AGT）基因启动子区5远侧端的单核苷酸多态性（SNP）。在非变性胶上，发现不同样品的PCR产物（双链DNA分子）电泳迁移率不同；而在变性胶上，对应的单链DNA分子电泳迁移率相同；PCR产物直接测序的结果表明，在同一PCR扩增片段中同时存在两个SNPs位点（G－1074T，A－1179G）；通过476个不同样品的实验结果，进一步证实两个SNPs位点处于完全的连锁不平衡关系。GenBank同源性比较结果显示：A－1179G为中国汉族人群所特有。     

Simplified cell microelectrophoretic method applied to the macrophage electrophoretic mobility test for cancer diagnosis.

Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.     

Simplified Cell Microelectrophoretic Method Applied to the Macrophage Electrophoretic Mobility Test for Cancer Diagnosis

Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.     

Achondrogenesis type IB (Fraccaro): Study of collagen in the tissue and in chondrocytes cultured in agarose

A lethal chondrodysplasia characterized by extreme micromelia was diagnosed by ultrasound examination in two sibs whose nonconsanguineous parents were healthy. Radiographic and histopathologic data indicated that the two foetuses (18 and 21 weeks old) had achondrogenesis type IB (Fraccaro). Quantitation of total collagen extractable from dried cartilage samples demonstrated a 50% decrease when compared to an age-related control. This decrease was essentially related to type II collagen. Nevertheless, the α chains and the CB peptides of type II collagen had a normal electrophoretic mobility. A significant amount of collagen type I was also detected. The electrophoretic pattern of collagens type IX and XI did not differ significantly from control sample. The extracellular matrix elaborated by patient chondrocytes cultured in agarose for 10–12 days, contained less collagen type II than normal cells. Labelling with ¹⁴C-proline of cultured cells showed the presence of pro-collagen and type II collagen chains with a normal electrophoretic mobility, but an α2(I) chain was detectable in the patient material, indicating the presence of collagen type I which supported the tissue findings. The significance of the type II collagen reduction in the patient's cartilage is unclear but it is unlikely to be the primary defect in achondrogenesis type I. © 1994 Wiley-Liss, Inc.     

Change in electrophoretic mobility of PC12 cells after culturing on PVA membranes modified with different diamines

The cell-biomaterial interaction is of extreme importance in regulating the numerous functions necessary for cell adhesion, growth, and differentiation. In the current study, electrophoresis was used to investigate the interactions between cells and biomaterials by measuring the change in electrophoretic mobility of pheochromocytoma (PC12) cells after they were cultured on the poly (vinyl alcohol) membranes modified with different diamines. Variations in cellular activity and electrophoretic mobility of cultured cells were compared. It was found that the intracellular metabolism and the cell surface charge properties were altered after cells contacting biomaterials and the variation of the latter occurred earlier than that of the former. Although the precise mechanism by which the variation of electrophoretic mobility of cultured PC12 cells was unknown, the biomaterials could influence the cell mobility within a short incubation time. It was hypothesized that changes in extracellular matrix components of cell surface may be in part responsible.     

Determination of the extracellular basal levels of glutamate and GABA at dentate gyrus of streptozotocin-induced diabetic rats

Previous studies have indicated an association between diabetes mellitus and impairments in synaptic plasticity in the hippocampus. However, it is not clear if the impairments of synapses are pre- or post-synaptic or both. The aim of this study was to evaluate the extracellular basal levels of glutamate and GABA at dentate gyrus of anesthetized streptozotocin-induced diabetic rats, after 12 weeks of diabetes induction. Extracellular levels of glutamate and GABA were investigated by using the microdialysis technique coupled to high performance liquid chromatography (HPLC) with fluorescent detection. Experimental groups were the control group and the diabetes group. The results showed that glutamate levels were significantly decreased in diabetes group compared to the control group, while GABA levels showed no changes. The findings support the possibility that alterations in transmission may account, in part, for synaptic plasticity deficits induced in diabetes.     

Change of electrophoretic mobility of purified alternative pathway factor D

The electrophoretic mobility of factor D (D) of the alternative pathway of human complement activation was examined by the method of lysoelectrophoresis. Purified D was found to have beta-mobility, while D in fresh serum showed alpha-mobility. Addition of D-depleted serum to D induced a change of electrophoretic mobility from beta to alpha. Addition of guinea pig serum to D did not produce this change. The change of electrophoretic mobility of D was not due to complex formation between D and other known alternative factors, P, C3NeF, B and C3. The Factor(s), which mediated the change in the electrophoretic mobility of D, had pseudoglobulin properties and was distributed around the third peak on Sephadex G-200 gel filtration of human serum with a mol. wt. of 50,000-80,000. This phenomenon might be restricted to a semi-solid state reaction, because complexes containing D were not observed upon analyses by sucrose density gradient ultracentrifugation and isoelectrofocusing.     

Factors affecting stability of isozymes of creatine phosphokinase.

Human brain-type (BB) creatine phosphokinase (CPK) and skeletal muscle (MM) CPK were purified from autopsy tissues. The stability of each isozyme with and without the reducing agent mercaptoethanol in human plasma, saline solution, and saline solution with bovine serum albumin was studied. Human BB CPK, at 37 C, without added mercaptoethanol, lost 50% of its activity in 15 minutes during incubation in plasma. It was much more stable at both room temperature and 4 C. BB CPK was also unstable in saline solution plus bovine serum albumin in the absence of mercaptoethanol but was much more stable in the presence of mercaptoethanol. Plasma factors with molecular weights less than 1,000 were potent inhibitors of CPK activity. Electrophoresis following incubation of BB CPK in plasma without mercaptoethanol revealed a new band with CPK activity with the electrophoretic mobility close to that of cardiac muscle (MB) CPK, but no band with the electrophoretic mobility of MM CPK was noted. A new band whose electrophoretic mobility was intermediate between that of MM CPK and that of MB CPK appeared after 21 hours of incubation of MM CPK in human plasma. Modifications of methods for handling blood samples in order to increase the possibility of preserving CPK isozymes in human plasma are proposed. Immunologic methods for determining CPK isozymes may provide more definitive answers as to the nature of the isozyme species present in human sera in various diseases.     

Dependence of erythrocyte electrophoretic mobility of human and rat blood on cell volume in health and pathology

To study correlations between electrophoretic mobility of erythrocytes and their volume, we compared blood samples from intact rats, animals exposed to extreme impacts, healthy humans and cancer patients. In rats, the above erythrocytes indices were closely related polynomially--deviations of the cell volume in both directions from physiological values are accompanied with a fall of mean cell mobility in electric field. In healthy subjects electrophoretic mobility of erythrocytes and their volume vary independently, in cancer these parameters are related curvilinearly.     

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

AIMS--To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS--DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS--Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS--The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.     

Stability of the lyophilized F(ab')2 fragments of horse tetanus antibodies isolated by affinity chromatography.

F(ab')2 fragments of horse tetanus antibodies were obtained from horse hyperimmune sera after peptic digestion. The digest was passed through a column of tetanus toxoid coupled with Sepharose 4B, F(ab')2 fragments were eluted with a solution of 5 mM HCl in 150 mM NaCl and the eluates were concentrated by ultrafiltration and lyophilized. Glycine and human serum albumin were used as stabilizing agents. Polyacrylamide gel electrophoretic mobility and molecular weight of the fragments remained unchanged after lyophilization. Freeze-dried preparations stored two months at 56 degrees C showed only a slight decrease in antitetanus activity. The ORD measurements and spectrophotometric determinations of unfolding over pH range 2.1-5.0 show that the F(ab')2 fragment structure is highly stable in acid medium.     

The macrophage electrophoretic mobility (MEM) test for malignant disease. Further clinical investigations and studies on macrophage slowing factors

This paper describes further confirmation of the successful application of the macrophage electrophoretic mobility (MEM) test to the detection of malignant disease. Results from fifty-one patients and eighteen controls are presented. The apparatus and technique used for the measurement of macrophage electrophoretic mobility differed from those previously used in other investigations. Further information is presented on the nature and behaviour of macrophage slowing factor(s), and experiments concerning the possible use of cells other than guinea-pig macrophages as indicators of slowing factor production are also described.     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C(18) cartridge using a mixture of methanol-water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0+/-4.2% and 123.3+/-4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5-20 mg l(-1) and detection limits were between 1.1 and 2.4 mg l(-1).     

Effect of ischaemia and reperfusion on the extra- and intracellular distribution of glutamate,glutamine, aspartate and GABA in the rat hippocampus,with a note on the effect of the sodium channel blocker BW1003C87

The redistribution of neurotransmitter amino acids resulting from 20 min of ischaemia was studied in the rat hippocampus by quantitative, electron microscopic immunocytochemistry and by in vivo microdialysis. Changes in the distribution of glutamate, glutamine, aspartate and GABA in various cell compartments of CA1 were analysed immediately after ischaemia or after 60 min of reperfusion, by incubating ultrathin sections with antisera raised against protein glutaraldehyde conjugates of the respective amino acids and subsequently with a secondary antibody coupled to colloidal gold particles. Transverse microdialysis probes coupled with HPLC and implanted in the same animals were used to determine the extracellular concentration of amino acids in the left hippocampus and to apply a drug (BW 1003C87) believed to modify the extracellular release of amino acids induced by ischaemia. Forebrain ischaemia was induced by temporary occlusion of the common carotid arteries in rats with permanently occluded vertebral arteries. The extracellular concentrations of glutamate, aspartate and GABA increased markedly during ischaemia, but returned rapidly to normal during reperfusion. BW 1003C87 (250 M, in the dialysis fluid) did not modify the increase in extracellular concentration of amino acids during ischaemia. Glutamate-like immunoreactivity was reduced in pyramidal cell somata both immediately after ischaemia and after 60 min of reperfusion. This reduction appeared to be somewhat less pronounced for cells in the left hemisphere (perfused with BW 1003C87) than in the contralateral hemisphere. Ischaemia caused no consistent changes in terminals. The ratio between the intracellular levels of glutamate and glutamine was assessed by double-labelling immunocytochemistry, using two different gold particle sizes. The glutamate-glutamine ratio in glial cells was greatly increased after ischaemia, but recovered to a normal level within 1 h of reperfusion. Aspartate-like immunoreactivity was substantially reduced in pyramidal cell somata both immediately and 60 min after ischaemia, while profiles that were immunopositive for GABA in control brains showed increased GABA immunolabelling. These results suggest that postsynaptic neuronal elements as well as glial cells contribute to the extracellular overflow of excitatory amino acids during an ischaemic event: post-synaptic elements by leaking or releasing glutamate and aspartate, and glial cells by losing their ability to convert glutamate to glutamine effectively. The temporal association between the changes in the glial contents of glutamate and glutamine, and the extracellular amino acid fluctuations recorded by microdialysis in the same animals, underline the strategic role of glia in regulating the extracellular level of glutamate.     

Ionic mechanisms of GABA-induced long-term potentiation in the rat superior colliculus

GABA-induced excitation and long-term potentiation (LTPG) have been demonstrated recently in the superficial layers of the superior colliculus (SC). In other regions of the nervous system, GABA elicits excitatory responses via ionotropic GABA receptors under certain conditions. This excitation is proposed to be due to either a high neuronal chloride concentration favouring a depolarising chloride efflux, or to a bicarbonate efflux coupled to a chloride influx. The aim of this study was to characterise the mechanisms underlying excitation and prolonged increase in synaptic transmission induced by GABA in the SC. Extracellular field potentials were recorded from 1-month-old rat SC slices, and LTPG of these responses was evoked by application of 3 mM GABA. GABA-induced excitation and LTPG were significantly reduced by lowering the extracellular calcium concentration, but not by a decreased potassium concentration. Replacing the extracellular bicarbonate-buffered perfusion medium with a HEPES-buffered solution had no effect on LTPG but blocking the bicarbonate-generating enzyme carbonic anhydrase both intra- and extracellularly with ethoxyzolamide (50 microM) prevented LTPG. The chloride transport inhibitor bumetanide (50 microM) reduced but did not block LTPG. We therefore suggest that the contribution of the chloride equilibrium to LTPG is only of minor importance. The intracellular bicarbonate pool and related efflux provides the basis for the excitatory action of GABA, leading to a subsequent depolarisation and calcium influx through voltage-dependent calcium channels, thus causing long-lasting changes in synaptic transmission.     

Purification and partial characterization of a putative precursor to staphylococcal enterotoxin B.

A putative precursor to staphylococcal enterotoxin B (SEB) has been identified as a component of purified membranes from Staphylococcus aureus S6. Agarose gel immunodiffusion analysis of the solubilized membranes demonstrated an immunoreactive protein that formed complete lines of identity with purified extracellular SEB. This putative precursor (pSEB) also had a different electrophoretic mobility from that of extracellular SEB when analyzed by immunoelectrophoresis. When membrane proteins from S6 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose sheets and probed with I-125 labeled, affinity-purified anti-SEB, the pSEB band was identified. The pSEB was approximately 3,500 daltons larger than extracellular SEB. This component was purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional peptide maps of the putative SEB precursor revealed that most of the tryptic peptides were identical to those of mature extracellular SEB. When purified membranes of other SEB+ (DU4916 and 10-275) and SEB- (RN450, RN451, S6R, and FR1100) S. aureus strains were analyzed by the nitrocellulose blot procedure, only the SEB+ strains contained this putative SEB precursor on their membranes.     

Optimization and application of electrophoretic mobility analysis of human red blood cells to study their in vitro stability, interaction with polycations and proteolytic enzymes

Electrophoretic light scattering method has been considered to determine both the mean and polydispersity of electrophoretic mobility of normal human red blood cells. The final goal of our study was to optimize an in vitro test allowing us to investigate the interaction of xenobiotics, in particular polyelectrolytes, with blood cells. The feasibility of our method has been evaluated based on the reproducibility of our technique to analyze the native electrophoretic mobility of human RBCs, as well as their evolution in the presence of polycationic compounds, i.e. a natural polyamine, spermine, and a synthetic polycation, a polyethylenimine (PEI). Additionally, the follow-up of the human RBCs electrophoretic mobility has been controlled after their incubation with neuraminidase.     

The mucolipidoses: Identification by abnormal electrophoretic patterns of lysosomal hydrolases

The human mucolipidoses (ML) are characterized by abnormal activities and abnormal electrophoretic patterns of fibroblast lysosomal hydrolases. These altered mobility patterns can be used to confirm the clinical diagnosis of the four mucolipidoses. The mobility patterns of one nonlysosomal and seven lysosomal enzymes were tested in fibroblasts from two ML I (sialidosis type 2, infantile), fifteen ML II (I-cell disease), eight ML III (pseudohurler poly-dystrophy), and one ML IV patients. A single sialidosis type 2, juvenile, line was also examined. Characteristic mobility patterns were found which identify each of the four mucolipidoses. Both the ML I and sialidosis type 2 juvenile lines displayed anodal mobility patterns, but distinct differences between the two disorders were observed. Lysosomal hydrolases from ML II lines demonstrated reduced activities or had altered mobilities. Differing electrophoretic patterns demonstrated the presence of at least two groups within the clinical phenotype diagnosed as ML II, indicating heterogeneity. The ML III lines showed normal electrophoretic patterns for most lysosomal hydrolases. The ML IV line expressed normal mobilities for every enzyme studied, with a single exception. The electrophoretic patterns of only β-hexosaminidase, acid phosphatase-2, α-galactosidase, and esterase A₄ were sufficient to identify and distinguish the different mucolipidosis types. Electrophoretic variation was also seen in liver but not kidney extracts from three ML II patients. β-Hexosaminidase and α-mannosidase B secreted into the medium by ML II and ML III fibroblasts had mobility patterns different from normal and from their intracellular patterns. These data suggest that the mucolipidoses are genetically distinct with heterogeneity within them.