Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.

[Alterations in neuroepithelial intermediate filaments during neurogenesis in the chick cervical spinal cord].

Intermediate filament proteins including nestin, vimentin and neurofilament were immunohistochemically studied during neurogenesis in the chick cervical spinal cord from stages 8 to 28. At stage 8, neuroepithelial cells of the neural groove contained a large amount of nestin in their cytoplasm and a little vimentin in the basal cytoplasmic areas, and no neurofilaments could be recognized at all. At stage 10, there was a marked decrease in nestin expression in the neural groove, and there was an increase in vimentin in neuroepithelial cells. At stage 15, when the neural tube was formed, small oval neuroblasts appeared in the peripheral area of the neuroepithelium. By employing double-immunostaining, three different neuroblasts could be identified; vimentin-positive and neurofilament-negative cells, neurofilament- and vimentin-double-positive cells, and neurofilament-positive and vimentin-negative cells. During the neuroblast stage, intracellular intermediate filaments were relayed from vimentin to neurofilaments. At stage 20, large polygonal cells containing a large number of neurofilaments could be recognized in the enlarged basal plate of the neural tube. At stage 28, neuronal processes developed in large polygonal cells and, although the staining intensity of the neurofilaments was slightly decreased in the soma, the neuronal processes contained a large number of neurofilaments. During neurogenesis in the chick cervical spinal cord, the intermediate filaments, nestin and vimentin, are present in neuroepithelial cells. During the neuroblast stage, vimentin and neurofilaments are observed together for a short time. Finally, in polygonal neurons, only neurofilaments are observed.

[Keratin filaments in epithelial cells of the excretory ducts of rabbit submandibular glands--an immunohistochemical and ultraimmunohistochemical study].

To clarify the roles of various keratin proteins, the distributions of eight keratin intermediate filament proteins (keratins 7, 8, 10, 13, 14, 18, 19 and 20) in the epithelial cells of the excretory ducts of rabbit submandibular glands were studied immunohistochemically and ultraimmunohistochemically. The epithelia of excretory ducts were composed of columnar cells and basal cells. In the columnar cells, intermediate filaments formed a network at the apical area, that is, an apex network connected with desmosomes. Keratins 7, 18 and 20 were detected in the upper layer of the network and keratins 8, 18 and 20 in the lower layer. The intermediate filaments containing keratin 7 were also connected with hemidesmosomes on the basal side. Keratins 7, 18 and 20 were found throughout the entire cytoplasm of the columnar cells. Keratins 8 and 14 were expressed near the nucleus, forming a ring-like structure around the Golgi apparatus in the columnar cells. In the basal cells, by contrast, the intermediate filaments were concentrated around the nucleus, forming a juxtanuclear network which contained keratin 10. Keratin 13 was detected between the juxtanuclear network and the cell membrane, and was connected with both desmosomes and hemidesmosomes. Kratin 7 filaments were contained throughout the entire cytoplasm of the basal cells. These results suggested that different functional subsets of keratin filaments could be distinguished in the epithelial cells of the excretory ducts of the submandibular glands. In the columnar cells, keratins 7, 8, 18 and 20 play a role in cell-cell contact or cell-matrix contact, and both keratins 8 and 14 seem to be involved in the structure of the Golgi apparatus. In the basal cells, keratin 10 may serve to position and anchor the nucleus within the cell, and keratin 13 plays a role in cell-cell and cell-matrix contacts.

'In vivo perfusion Turnbull's reaction' for Fe(II) histochemistry in non-anoxic/non-ischemic and anoxic/ischemic cat brains.

We developed a simple but ingenious histochemical method, 'in vivo perfusion-Turnbull's reaction', for the visualization of non-heme Fe(II) of the brain; in situ release of Fe(2+) ions was coupled with formation of insoluble reaction product (Turnbull's blue) by in vivo perfusion of acid ferricyanide through the abdominal (non-anoxic/non-ischemic brain) or ascending (anoxic/ischemic brain) aorta in the deeply anesthetized adult cats. Frozen sections of the brain were treated according to the method of Nyguen-Legros et al. [12] to intensify Turnbull's reaction. The method revealed that cytoplasmic Fe(III) was reduced to Fe(II) in oligodendroglias in anoxic/ischemic (for 20 min) brains, and that Fe(II) was concentrated in the neuronal and glial cell nuclei regardless of the presence or absence of blood supply impairment.

Intrastriatal targets of projection fibers from the central lateral nucleus of the rat thalamus.

We examined light and electron microscopically intrastriatal targets of projection fibers from the central lateral thalamic nucleus (CL), which is a major relay of cerebello-striatal projections. The study was done in the rat by combining the anterograde tract-tracing with immunohistochemistry for parvalbumin (PV); an anterograde tracer (biotin dextran amine: BDA) was injected into the CL. In the striatum, 91% of BDA-labeled axon terminals made asymmetrical synapses on PV immunonegative dendritic spines (assumed to be those of striatal projection neurons); only 0.5% of BDA-labeled axon terminals made synapses on PV immunopositive dendritic shafts. The remaining BDA-labeled axon terminals were in synaptic contact with PV immunonegative dendritic shafts. The results suggest that the cerebello-striatal projections through the CL predominantly access to striatal projection neurons, with only minor access to PV immunopositive (assumed to be GABAergic) interneurons in the striatum.

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria.

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.

Vimentin intermediate filament protein as differentiation marker of optic vesicle epithelium in the chick embryo.

For the study of the differentiation process of optic vesicle epithelium into neural retina, pigment epithelium and pars caeca retinae, vimentin intermediate filament protein in retinal epithelial cells was detected immunohistochemically in chick embryo at stages 11-21. In the late stage of optic vesicle development (stage 14), optic vesicle epithelium was classified into the following 3 different portions on the basis of vimentin staining intensity: latero-central epithelium under the lens placode, medio-central epithelium facing the latero-central epithelium, and peripheral epithelium connecting the latero-central and medio-central epithelia. Latero-central epithelium, the future neural retina, exhibited strongest staining of vimentin of the 3 portions. In contrast, medio-central epithelium, the future pigment epithelium, showed weakest staining. Moderate staining was observed in peripheral epithelium, the future pars caeca retinae. These differences in levels of vimentin expression were observed during optic cup formation. The present results clearly demonstrate that differentiation of retinal epithelium into neural retina, pigment epithelium and pars caeca retinae occurs in the late stage of the optic vesicle, and that retinal differentiation is reflected by the amount of vimentin in epithelial cells.

[Vimentin and neuroepithelial cell differentiation in the spinal cord of chick embryos: an immunohistochemical study].

Using vimentin immunohistochemical staining, the differentiation processes of neuroepithelium in the neural tube were examined in chick embryos from stages 8 through 28. At an early stage of the neural groove, stage 8, no morphological differences could be found among the neuroepithelial cells, but vimentin staining allowed us to identify four different regions in the groove wall. The epithelial cells in the ventral wall exhibited moderate staining of vimentin, and vimentin was detected in the basal and middle cytoplasmic areas. A weak staining limited to the basal cytoplasm was observed in the dorsal wall. In contrast, epithelial cells in the median hinge region and in the lateral edge of the neural groove had little vimentin. On the basis of this vimentin staining, four similar regions could also be observed in the neuroepithelium of the neural tube at stage 12; the ventral wall, dorsal wall, floor plate and roof plate. Prior to the morphological changes in the neuroepithelial cells, vimentin expression showed dramatic changes, and our immunohistochemical data suggest that cell differentiation into motor areas and sensory areas starts at an early stage of the neural groove.

Effects of colchicine on the enucleation of erythroid cells and macrophages in the liver of mouse embryos: ultrastructural and three-dimensional studies.

Enucleation is the last event in the development of a definitive erythroid line, and extruded nuclei are phagocytosed by macrophages. Both colchicine and cytochalasin have been known to exert a great influence on the enucleation process, but the relationship between enucleation and these agents has not yet been clearly revealed in vivo. Our aim was to clarify the significance of the enucleation in liver erythropoiesis and macrophage phagocytosis by colchicine and cytochalasin administration to embryonic mice. Pregnant mice were intraperitoneally injected with colchicine or cytochalasin at 13 days of gestation. Embryonic livers were removed at intervals of 3, 6 and 12 h after injection for processing for light and electron microscopy, and, to obtain three-dimensional morphology of erythroids at enucleation, computer-aided reconstructions were performed by light microscopy. Colchicine injections had cytolytic effects on hepatocytes and macrophages, and numerous erythroblasts were observed in the process of enucleation after colchicine injection. However, the extruding nuclei were irregularly shaped, and some erythroblasts at mitosis showed extreme peripheralization of their chromosomal masses and cell membrane constriction. Enucleation behavior could also be observed in immature erythroblasts. Liver macrophages engulfed extruded nuclei and erythroblasts in mitosis. Cytochalasin injections, on the other hand, had no significant effect on embryonic livers. The progress of erythroblast mitosis was clearly stopped by colchicine injection, and numerous erythroblasts at mitosis were extruding their nuclear compartment. Following colchicine injection, erythroid enucleation also took place in immature erythroblasts, and mitotic erythroids were phagocytosed. In enucleation, more attention should be paid to hematopoietic environmental factors than to hemopoietic cell factors.

Origin and fate of the central macrophages of erythroblastic islands in the fetal and neonatal mouse liver.

Erythroblastic islands were examined by ultrastructure and ultrastructural histochemistry in fetal and early neonatal livers of the mouse. The liver primordium of day 11 embryos contained not only immature hemopoietic cells in the hepatic cords but also macrophages in expanded sinusoids. At 12 and 13 days of gestation, macrophages bearing large cytoplasmic inclusions increased in number, and some of them moved from the sinusoids into the hepatic cords. A ring of erythroblasts surrounded the macrophages, and erythroblastic islands could be identified at 14 days of gestation. Fetal livers contained two kinds of macrophages: sinusoidal macrophages and central macrophages of the erythroblastic islands. These macrophages exhibited a similar binding pattern to Griffonia simplicifolia isoagglutinin-IB4 (GS-IB4) and soybean agglutinin (SBA). Fetal hepatocytes, however, did not appear to engage in active phagocytosis, and the binding patterns of GS-IB4 and SBA differed significantly between hepatocytes and the two kinds of macrophages. In the early postnatal mouse, a marked decrease in the number of erythroblastic islands occurred. Erythroblasts left the central macrophages, and the macrophages subsequently underwent degeneration. The erythroblastic islands finally disappeared at the end of liver hemopoiesis, and the degenerated central macrophages were removed by scavenger macrophages in the perisinusoidal space. Our data demonstrate that scavenger macrophages play an essential role in the development of hepatic hemopoiesis, with special reference to the formation and dissolution of erythroblastic islands.

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates.

To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells.

Death process of primitive erythrocytes and phagocytosis by liver macrophages of the mouse embryo.

Primitive erythrocytes from the yolk sac are nucleated cells which have a short life span of several days in the embryonic circulation of the mouse. The death process of primitive erythrocytes was ultrastructurally investigated in embryonic mice. At 12 days of gestation, primitive erythrocytes accounted for 96.1% of the circulating erythrocytes. The percentages in 13-, 14-, 15- and 18-day-embryos were 43.8%, 15.4%, 7.7% and 0.0%, respectively. Between 13 and 15 days, anucleate primitive erythrocytes made up from 1.5% to 5.9% of circulating erythrocytes. During the gestational period, the nuclei of primitive erythrocytes markedly decreased in volume, and the chromatin underwent condensation and marginated to crescents along the nuclear envelope. Following nuclear fragmentation and dissolution of condensed chromatin, nuclear residues were finally formed in aging primitive erythrocytes. Formation of annulate lamellae was also observed, associated with the nuclear shrinkage. The nuclei had an apoptotic-like appearance, but TUNEL-positive reactions could not be identified in any nuclei of the primitive erythrocytes. Dying erythrocytes with nuclear residues were phagocytosed in toto by hepatic macrophages and disposed of from embryonic circulation without enucleation.

[Scanning and transmission microscopic observations on circulating primitive erythroblasts of yolk sac origin in the mouse embryo].

The earliest hemopoietic tissues which appear during the ontogeny of mammals are the blood islands of the yolk sac, and the blood cells produced therein begin to circulate between the embryo and visceral yolk sac at the establishment of the circulatory system. Primitive erythroblasts derived from the yolk sac have a short life span of only several days, and they form a majority of the embryonic blood cells prior to the start of liver hemopoiesis. To clarify cell fragmentation of primitive erythroblasts at the ultrastructural level, using 18 embryos of ICR-mice at 10 and 11 days of gestation, we observed circulating erythroblasts by scanning and transmission electron microscopy. The circulating erythroblasts generally had an irregularly ovoid contour, and they showed a great deal of micropinocytosis on their cell surface. The nuclei of the erythroblasts were round and possessed one or two nucleoli which were in contact with the nuclear membrane. Their nuclear chromatin was dispersed, and the cytoplasm was rich in polyribosomes and mitochondria. The majority of circulating erythroblasts were at the stage of either basophilic or polychromatophilic erythroblasts. Cytoplasmic projections could occasionally be seen extending from the erythroblast surface, and some of the projections appeared to be liberated into the vascular lumen as cell fragments. On the basis of their size and shape, the cytoplasmic projections could be classified into three types; finger-like projections, vesicular projections and microvesicular projections. The finger-like projections were approximately 1 micron in diameter and 3 microns in length. The vesicular projections, connected with the cell by a narrow stalk, were teardrop in shape, and approximately 0.8 microns in diameter and 1.5 microns in length. The microvesicular projections were approximately 0.2 microns in diameter and 0.2-0.5 microns in length. The finger-like projections had micropinocytotic invaginations on their surface, but no invaginations could be seen on the vesicular and microvesicular projections. Not only the finger-like but also the vesicular projections contained cytoplasmic matrix with a few polyribosomes. The microvesicular projections, on the other hand, occasionally contained myelinic-like figures. These projections were seen on the surface of erythroblasts at various maturation stages. The cytoplasmic fragments released from the erythroblasts were engulfed and eliminated from the embryonic peripheral blood by intravascular macrophages. The fragmentation of cytoplasmic projections was considered to be related to the development of microfilaments involved in the cytoskeleton of the erythroid elements.

The ultrastructure of intravascular and coelomic scavenger macrophages of the mouse embryo.

The aim of this study was to establish whether or not mononuclear cells which appear in both the vitelline vessels and embryonic coelom in mice prior to liver hemopoiesis are specialized scavengers. Before the initiation of liver hemopoiesis, the majority of the embryonic blood cells were primitive erythroblasts derived from yolk sac hemopoietic foci. In addition, the peripheral blood contained a few free phagocytes as early as 10 days of gestation. The phagocytes devoured various cell elements such as degenerating erythroblasts and cell fragments. Ultrastructurally, they had long filamentous cytoplasmic projections on their cell surface, clear subsurface vacuoles or vesicles, lipid droplets, a few lysosomal granules, large heterogeneous phagolysosomes and residual bodies. Mononuclear phagocytes with ultrastructural features similar to those of the intravascular phagocytes also could be observed in the intraembryonic peritoneal cavities at 10 days of gestation; they sometimes engulfed possible mesothelial cells undergoing degeneration. Based on fine structural criteria, these intravascular and coelomic phagocytes were considered to be specialized scavenger macrophages with the function of clearing the blood and tissue fluid of whatever has been ingested. In so doing, they serve as the most primitive discriminating filter set in embryonic circulation.

Membrane-bound glycoconjugates of fetal mouse erythropoietic cells with special reference to phagocytosis by hepatic macrophages.

Using lectin and colloidal iron (CI) stainings in combination with neuraminidase digestion, glycoconjugates on the surface of erythropoietic cells of the yolk sac and liver in fetal mice were examined. Fetal hepatic macrophages were capable of distinguishing between phagocytozed and non-phagocytozed erythroid elements as described in our previous study. Marked differences between these two elements could be ultrahistochemically detected on their cell surface. The phagocytozed elements, such as nuclei expelled from erythroblasts and degenerating primitive erythroblasts, faintly bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a weak peanut agglutinin (PNA) binding. In contrast, erythroblasts at various maturation stages, erythrocytes and normal primitive erythroblasts heavily bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a moderate PNA binding. No differences in binding of either concanavalin agglutinin, Ricinus communis agglutinin-I or PNA were noted between phagocytozed and non-phagocytozed erythroid elements. Desialylation appears to be one of the most important signs for the recognition mechanism of fetal macrophage phagocytosis. During maturation of hepatic erythroblasts, sialic acid changes its affinity for Limax flavus agglutinin from strong to weak, and soybean agglutinin binding sites disappear at the basophilic erythroblast stage. Glycoconjugates on polychromatophilic erythroblasts acquire similar compositions to those of erythrocytes.

Accumulation and massive cell death of polymorphonuclear neutrophils in the developing bone marrow of the mouse: a histological study.

Accumulation and cell death of neutrophils were studied by light and electron microscopy in neonatal mouse bone marrow. At the beginning of bone marrow hematopoiesis, the marrow cavity contained a large number of polymorphonuclear leukocytes. Polymorphs comprised approximately 75% of the total nucleated cells in the hematopoietic compartment of the newborn marrow, the majority being neutrophils. Mature neutrophils were sometimes crossing the endothelium of the marrow blood sinus. Neutrophils in neonatal marrow show features typical of apoptosis, e.g. formation of nuclear pockets and blebs, margination of compact nuclear chromatin to form sharply circumscribed masses, condensation of cytoplasm, and convolution of cell outlines. Dying neutrophils were devoured and digested by phagocytes. The occurrence of large-scale neutrophil death and removal of neutrophils by phagocytes in neonatal bone marrow are discussed in relation to programmed cell death in development of the fetal hematopoietic system.

Cell differentiation of alveolar epithelium in the developing rat lung: ultrahistochemical studies of glycoconjugates on the epithelial cell surface.

Glycoconjugates on the surface of pulmonary epithelial cells were ultrahistochemically examined in the fetal, neonatal and adult rat lung. Lectin and colloidal iron staining procedures were performed in combination with digestion using carbohydrate-degrading enzymes or methylation. The glycoconjugate composition of columnar cells at 16 days gestation was similar to that of cuboidal cells at 19 days gestation. Glycoconjugate differentiation on the cell surface occurred at 20 days gestation, and especially the loss of soybean agglutinin (SBA) binding sites could be detected on type II cells. The contents of Ricinus communis agglutinin-I (RCA-I) and Concanavalin A (Con A) binding sites on type II cells also began to decrease. On the contrary, the content of sulfated saccharides decreased on the surface of type I cells during development. Glycoconjugate differentiation on both type I and II cells was completed with the disappearance of hyaluronic acid and peanut agglutinin (PNA) binding sites; type I and II cells acquired a similar histochemical composition to that on adult type I and II cells at 5 days after birth. Both type I and II cells share a common early precursor cell, that is, the cuboidal epithelial cell at the canalicular stage.

Cell death and phagocytosis of haematopoietic elements at the onset of haematopoiesis in the mouse spleen: an ultrastructural study.

The spleen in fetal and early postnatal mice contains a variety of proliferating haematopoietic cells as well as 2 kinds of phagocytes, scavenger macrophages and mast cells, laden with large heterogeneous inclusions. Their phagocytotic activity is directed towards extruded erythrocyte nuclei, erythrocytes and dying haematopoietic cells. The splenic cords after 18 d of gestation become filled with proliferating haematopoietic cells, and the cords contain a small number of free haematopoietic cells undergoing degeneration. The early signs of cell death can be observed in the nuclear structures: hyperchromasia of the nuclear membrane or nuclear dissolution. Erythroblast nuclei are amongst the most frequent elements engulfed. Phagocytes also take up and digest degenerating blood cells, i.e. erythrocytes, erythroblasts and neutrophil granulocytes. Since the digestion processes are ultrastructurally different for the various haematopoietic elements, the origins of heterolysosomes enclosed by phagocytes can be identified by electron microscopy. Mast cells, originally classified as secretory cells, phagocytose erythroid line cells in the spleen. Cell death in several haematopoietic cell lines is discussed in relation to programmed cell death in the developing spleen.

Scavenger macrophages and central macrophages of erythroblastic islands in liver hemopoiesis of the fetal and early postnatal mouse: a semithin light- and electron-microscopic study.

The livers of 12-day mouse embryos contain many hemopoietic cells in the hepatic cords and macrophages which are laden with large heterophagosomes in the sinusoids. Macrophages, as scavengers, engulfed circulating primitive erythroblasts from the yolk sac, as well as nuclei expelled from erythroblasts. At the 14th day of gestation, scavenger macrophages in mitosis were seen, extending long and thin cytoplasmic projections from their cell surface. At the 13-14 day of gestation, macrophages migrated from sinusoids into hepatic cords, and erythroblasts gathered around them, thus forming cell clusters designated as erythroblastic islets. Central macrophages formed a cell-socket-like structure on their cell surface for surrounding erythroblasts, reflecting the close association between macrophages and erythroblasts. In the erythroblastic islands, contained in late fetal and neonatal livers, erythroblasts have dissociated from the central macrophages having a starfish-shaped cell profile. The macrophage-erythroblastic association in the islands became less marked rapidly after birth.

Glassy carbon pre-column for direct determination of acetylcholine and choline in biological samples using liquid chromatography with electrochemical detection.

The determination of acetylcholine and choline has been quite successfully accomplished using liquid chromatography with electrochemical detection following the original reports of Potter et al. [J. Neurochem., 41 (1984) 188]. A post-column reactor containing acetylcholinesterase and choline oxidase allows conversion of the desired species into hydrogen peroxide, an electrochemically active substance. However, the direct injection of tissue homogenates and other biological samples into such a system exhibits quite large solvent fronts and unidentified peaks. Using a pre-column packed with glassy carbon particles, we were able to dramatically decrease the size of the solvent front for such injections and tentatively identify the unknown peaks to be caused, at least in part, by common catecholamines. The glassy carbon pre-column, in addition to increasing the selectivity of the results, allowed the required chromatographic time per sample to be decreased from 20 to 10 min.