Routine analysis of ascorbic acid in citrus juice using capillary electrophoresis.

A procedure to monitor citrus juice samples was established to quantitate vitamin C by capillary electrophoresis using a previously developed method. Dilution and filtration were the only preparation requirements and separation was achieved with an uncoated capillary using a 35mM sodium borate buffer (pH 9.3) containing 5% (v/v) acetonitrile at 21 kV and 23 degrees C. Detection was performed by high speed scanning between 200 and 360 nm. From the multiwave length scan, the electropherogram at 270 nm was extracted and used to quantitate ascorbic acid. The ascorbic acid concentration was calculated with an internal standard method, with ferulic acid as internal standard. The level of ascorbic acid during analysis was stabilized with ethylenediaminetetraacetic acid and dithiothreitol was used to reduce dehydroascorbic acid to ascorbic acid to estimate the total vitamin C level. Results were similar to those obtained by liquid chromatography and the method is now used to determine routinely the level of ascorbic acid in citrus juices.

Analytical monitoring of citrus juices by using capillary electrophoresis.

A capillary electrophoretic method was developed to analyze simultaneously most citrus juice components in a single procedure. After filtration, sample components are separated with an uncoated capillary tubing and a 35 mM sodium borate buffer (pH 9.3) containing 5% (v/v) acetonitrile. Analyses were run at 21 kV and 23 degrees C. Compounds monitored regularly were the biogenic amine synephrine, some flavonoids (didymin, hesperidin, narirutin, neohesperidin, and naringin), the polyphenol phlorin, 3 UV-absorbing amino acids (tryptophan, phenylalanine, and tyrosine), ascorbic acid, an unidentified peak generated by heat and storage, and the preservatives sorbate and benzoate that can be added to citrus products. Separation can be achieved in 20 min, and each compound can be subsequently quantitated. Didymin, narirutin, and phlorin peaks were used with an artificial neural network to assess the volume of added pulp wash, a by-product of juice preparation. This method allows rapid monitoring of citrus juices, giving information on quality, freshness, and possible adulteration of the product. Similar procedures could be used to monitor other fruit juices and quantitate diverse juice blends.

Use of capillary electrophoresis for monitoring citrus juice composition.

New trends in adulteration monitoring, favor the development of methods analyzing simultaneously as many compounds as possible. Capillary electrophoresis has been applied to the examination of a broad spectrum of citrus juice molecules that absorb in the UV and in the visible light. Depending on the conditions up to thirty compounds could be separated. The identified molecules included phenolic amines, amino acids, flavonoids, polyphenols and vitamin C. Samples can be analyzed without specific preparation and the best separations were obtained with diluted solutions due to a stacking effect. This method has been applied to the comparison of pure orange juice and pulpwash, a major adulterant of orange juice. Several significant quantitative differences were seen and it is hoped that this procedure will provide a more precise way of estimating pulpwash in orange juice.

Alteration of cytoskeletal organization during axonal elongation and its role in regulating the growth process.

The composition and organization of the axonal cytoskeleton vary from neuron to neuron and the ability of a nerve to regenerate may, at least in part, depend on the pre-existing nature of the cytoskeleton. In axons, where the cytoskeletal elements are loosely organized enough material may be mobilized after an injury to allow elongation to proceed. However, if microtubules and neurofilaments interact closely with each other, it may not be possible for enough material to be released from or to migrate through a complex network. As a consequence material is not provided to the growing tip and elongation cannot proceed beyond the initial sprouting.

Axonal transport in the garfish optic nerve: comparison with the olfactory system.

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.

Slow transport in a nerve with embryonic characteristics, the olfactory nerve.

The kinetics for slowly transported polypeptides have been examined in intact garfish olfactory nerves. The shape of the slow peak is essentially determined by alpha-and beta-tubulin which are by far the major polypeptides of the entire wave. The proximal area of the peak is similar to the slow component a (Sca) subcomponent defined in other nerves and contains discretely moving neurofilament proteins. The distal peak area, however, is more reminiscent of Scb. The two subcomponents were found to overlap considerably. Traces of polypeptides comigrating with tubulin and actin move far ahead of the slow wave at rates similar to the rate of slow transport measured in growing fibers and to the maximal velocity of axonal elongation. One of the most striking properties of slow transport in this nerve is the difference in the spreading of the various transported polypeptides along the axon, following their release from the perikarya. Labeled tubulin and actin can cover more than 20 cm of nerve; while neurofilament proteins can be found only on a 6 cm segment. Comparisons between slow transport in garfish olfactory axons and other vertebrate nerves indicate that despite major differences, the basic characteristics of slow transport are conserved. The features specific to the olfactory nerve may reflect its specialized properties. The constant turnover of olfactory neurons implies that these cells have an excellent growth potential but a short life span and, therefore, never reach full maturity. It can, therefore, be expected that their molecular composition is reminiscent of that embryonic neurons with a high level of plasticity but a slow stability.

Survival and subsequent regeneration of olfactory neurons after a distal axonal lesion.

If an axonal lesion is made close enough to the cell body, injured olfactory neurons degenerate and are replaced by new nerve cells arising from undifferentiated mucosal basal cells. Therefore, under these conditions neural regeneration occurs through a process similar to neuronal development. The use of the long garfish olfactory nerve has revealed that neuronal death is not an inevitable consequence of an axonal injury and that the extent of cell death depends on the distance between the site of injury and the perikaryon. A lesion located up to 40 mm from the cell body induces the death of all mature neurons. Between 60 and 100 mm an increasing proportion of neurons survive the injury (from 60 to more than 90%) and are able to regenerate their distal segment. During regeneration, two populations of growing axons have been characterized. The fastest growing fibres (5.8 mm per day) correspond to the small population of neurons which were already growing at the time of the lesion and are able to survive an injury at any distance along the nerve. The majority of the regenerating fibres grow at 0.8 mm per day and corresponds to the damaged mature neurons. Elongation velocities were not affected by the distance from injury to cell body or by the mode of neural repair (development or regeneration). The initial delay between the injury and the beginning of elongation increases linearly with distance at a rate of 1 day cm-1 and is independent of the elongation velocity of the growing neurons. This indicates that the mechanism responsible for the beginning of axonal growth is initiated at or near the cell body and involves the entire axonal stump and not only the area surrounding the crush site. The ability of nerve cells to survive an injury may depend on the length of the axonal stump remaining attached to the cell body and on the level of protein synthesis at the time of the crush. From preliminary results it can be hypothesized that the length of the initial delay is determined by a reorganization of the cytoskeletal elements in the proximal axonal stump.

Study of a floating fraction obtained during preparation of myelin from degenerating goldfish optic tract.

A floating fraction that layers on top of 0.25 sucrose has been obtained during the preparation of myelin from intact and 9 day degenerating goldfish optic tracts. The proportion of total tract protein isolated in floating fraction rises from 6.6% to 11.0% during degeneration. This increase is paralleled by a morphologically observed splitting of myelin lamellae. Floating fraction contains all of the major myelin proteins but shows a 40% increase in the proportion of basic protein and a 2-3 fold decrease in the proportion of IP proteins (intermediate molecular weight glycoproteins) and a 36 Kd (X) protein. The lipid to protein ratio is slightly higher in floating fraction than myelin. Lipid composition is characterized by 1/2-1/3 the myelin levels of galactolipids and twofold increased levels of triglycerides and cholesterol esters. Electron microscopy of floating fraction shows a mixture of myelin fragments with few lamellae and single membrane fragments. Taken together the results indicate that floating fraction in the degenerating goldfish optic tract is at least partially derived from the breakdown of myelin.

Influence of temperature on slow flow in populations of regenerating axons with different elongation velocities.

The slow intra-axonal transport mechanisms may be subdivided into 4 elementary processes: (a) protein synthesis; (b) release of the slow material from the cell body; (c) transport of the molecules along the axon; and (d) deposition of the material from the moving phase into the immobile axonal constituents. These 4 components of the slow flow mechanisms are differently affected by temperature and react differently to axonal regeneration. They have been studied at temperatures ranging from 10 to 35 degrees C, in 3 different populations of regenerating C-fibers of the garfish olfactory nerve. These 3 classes of axons have very different elongation properties as determined by elongation rates ranging from a minimum of 0.3 mm/day at 14 degrees C to a maximum of 12.4 mm/day at 35 degrees C. From this study several properties of the slow intra-axonal transport mechanism can be underlined: (a) in regenerating nerves the rate of slow flow is multiplied by a factor of 3.3 as compared to the rate characteristic of an intact nerve. A similar increase was measured in every regenerating phase regardless of the specific elongation velocity of the growing axons. This accelerated rate of slow flow has been measured previously in degenerating axons detached from their perikarya: therefore the rate increase appears to be triggered by the injury rather than the needs of the neuron; (b) protein synthesis represents one of the main steps controlling axonal elongation. Neurons with the most active level of protein synthesis grow with the highest elongation velocity; (c) the somal releasing times decrease exponentially with temperature but are identical to the values measured in intact neurons; (d) finally the height of the slow flow peak decreases exponentially as a function of the distance, as seen previously in intact nerves, but a significantly larger amount of radioactivity reaches the end of the nerve. It can be concluded that slow flow is intimately linked with the regeneration process. The rate of slow flow and more specifically of Scb represents the upper limit that can be reached by the elongation velocities of regenerating fibers when all the growth conditions are maximal. When the regenerating conditions are not at their peak, the axonal elongation proceeds at velocities which can be very significantly slower than the rate of slow flow and might be determined by the level of protein synthesis in the perikarya. Axonal elongation, however, can never proceed at a rate faster than slow flow.

Regeneration of three populations of olfactory axons as a function of temperature.

Following injury near the mucosa, 3 populations of regenerating axons have been shown to invade successively the olfactory nerve. At 21 degrees C, each of the first 2 phases (I and II) represents 3-5% of the original axonal population. Phase III corresponds to the massive arrival of new axons and contains 50-70% of an intact nerve axonal population. The influence of temperature on the elongation rates of these 3 phases of growing fibers has been followed from 10 to 35 degrees C. No regeneration occurs at 10 degrees C. Between 14 and 31 degrees C the elongation velocity of the most rapidly growing fibers (phase I) increases linearly from 1.6 to 10.3 mm/day. These values are identical to the rates of slow intra-axonal flow measured in all 3 phases of regenerating axons. In the same range of temperature, the elongation velocity of the other two populations, increases exponentially from 0.5 to 9 mm/day for phase II and from 0.3 to 6.3 mm/day for phase III. Above 33 degrees C, rates of phases II and III fibers do not reach the values predicted from the exponential functions. The 3 phases fused as a single axonal population whose elongation velocity was shown at 34 and 35 degrees C to increase linearly according to the equation defined for phase I. Since a similar rate of slow flow was measured in all 3 populations, it can be concluded that slow flow defines the upper limit of the elongation velocity of a regenerating fiber. However, other factors acting on the regenerating neuron (mainly protein synthesis), might prevent it from reaching the maximal possible elongation velocity.

Proximodistal degeneration of C-fibers detached from their perikarya.

Degeneration was followed in the garfish olfactory nerve after removal of the mucosa containing the cell bodies. Degeneration, as measured by a decrease in the weight of consecutive 3-mm nerve segments, spreads at constant velocity from the site of injury toward the synaptic area. The proximodistal degeneration is temperature dependent and progresses from 0.3 mm/d at 10 degrees C to 13.0 mm/d at 35 degrees C. Between 14 and 35 degrees C, the velocity increases linearly with temperature. At all the temperatures investigated, these proximodistal degeneration velocities are identical to the rates of slow intraaxonal flow measured in axons detached from their cell bodies, or to the rates measured in regenerating fibers, and, except at 10 degrees C, are 3.3 times faster than the rate of slow flow in intact nerves. These results were confirmed by light and electron microscopy. We hypothesize that the collapse and subsequent degeneration of the axons is the result of a proximodistal depletion of cytoskeletal elements no longer provided by the cell body to the axon by slow intraaxonal flow. A significant number of axons disappeared rapidly from the nerve before the arrival of the slow degenerative wave. From studies by other groups, this rapid degeneration may be the result of a lack of rapidly transported, mainly membranous components.

Slow flow in regenerating C-fibers.

An identical rate of slow flow was measured in 3 different populations of regenerating C-fibers which have very different growth abilities as expressed by a 6-fold difference in their elongation velocities. At 21 degrees C, this rate of slow flow is 3.5 times faster than the rate measured in intact nerves and is identical to the elongation velocity of the most rapidly regenerating fibers. Slow flow may constitute the upper limit of the elongation velocity of a regenerating axon but other factors determined by the perikaryon (such as protein synthesis) might prevent the neuron from reaching the maximal growth rate.

Influence of a detergent on the catfish olfactory mucosa.

The olfactory mucosa of the catfish (Ictulurus punctatus) has been briefly exposed to various concentrations of the non-ionic detergent Triton X-100. At high concentrations (1-4%) the upper layer of cells constituting the sensory and non-sensory areas of the lamellae is extensively damaged and new receptor cells do not appear in significant number before 2 months after treatment. Respiratory cells regenerate first followed by sustentacular and olfactory receptors. The regenerative process is very similar to that described previously after prolonged contact between the mucosa and ZnSO4. Low detergent concentrations 0.03-0.1% affect only the sensory area. Olfactory and sustentacular microvilli and cilia, are immediately severed by the chemical. Regeneration occurs within the next 4 days. The cellular membranes appear also to be affected. From anatomical, electrophysiological and biochemical studies both in vivo and in vitro, it can be hypothesized that a receptors involved in the transduction process are solubilized by the detergent but reappear at a level corresponding to 50-60% of their original activity within 2 h. Proteins, having an amino acid binding effectiveness correlated to the amino acid electrophysiological activities measured in vivo, can be isolated from the solubilized material. Further studies will be necessary to confirm that some of these molecules are involved in the olfactory transduction mechanism.

Slow flow in axons detached from their perikarya.

Slow flow was followed in unmyelinated olfactory axons, severed from their cell bodies, at 14 degrees C, 21 degrees C, and 31 degrees C. Slow flow does not stop after axotomy but rather accelerates to a value 3.3 times faster than the rates measured in an intact nerve. These velocities are equivalent to the rates of slow flow characteristic of regenerating fibers. The injury appears to have an influence on the contralateral intact nerve, where slow flow velocity increases to severed nerve values for several days before reverting to intact nerve rates. It can be hypothesized that the increase in the rate of slow flow is triggered by a factor repressed in intact nerve but released into the blood stream following injury.

Degeneration and regeneration of olfactory cells induced by ZnSO4 and other chemicals.

The necrotic effect of various salt solutions was tested on the catfish olfactory mucosa. Only zinc cations were able to induce an extensive degeneration of the olfactory cells. Two different modes of irrigation of the mucosa with zinc sulfate were investigated. (1) The olfactory cavity is flushed with the chemical for not more than a few seconds. At concentrations above 30 mM, the resulting damage is very reproducible, largely concentration independent and almost completely specific for the olfactory receptor cells. The non-sensory respiratory cells are unaffected, the sustentacular cells surrounding the receptor cells are affected mainly by a loss of microvilli. The olfactory receptor cells, on the contrary, start to degenerate within a few hours and by day 4 only 20% of the original receptor population remains. Division of the mucosal basal cells increases during days 3 and 4 on and day 6 olfactory receptor cells reach the bare surface of the lamella. After day 7, the receptor population reaches a level of more than 80% of its original value. Because of the absence of sustentacular processes covering the olfactory cell's knobs on day 6, it has been possible to confirm that each of the two types of olfactory receptor cells previously characterized are concentrated on each half of the mucosa (2) The salt is maintained in contact with the tissue for several days. After this treatment most of the lamellae are irreversibly destroyed, some regeneration occurs in limited areas of the mucosa. In these small areas, indifferent respiratory cells reappear first between 20 and 35 days. It is only when the structure of the olfactory tissue is completely reorganized that the new receptor cells reappear between days 45 and 55. Regeneration is not completed before 60-65 days.

Rate of movement and composition of rapidly transported proteins in regenerating olfactory nerve.

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.

Study of regeneration in the garfish olfactory nerve.

Previous studies of the olfactory nerve, mainly in higher vertebrates, have indicated that axonal injury causes total degeneration of the mature neurons, followed by replacement of new neuronal cells arising from undifferentiated mucosal cells. A similar regeneration process was confirmed in the garfish olfactory system. Regeneration of the nerve, crushed 1.5 cm from the cell bodies, is found to produce three distinct populations of regenerating fibers. The first traverses the crush site 1 wk postoperative and progresses along the nerve at a rate of 5.8 +/- 0.3 mm/d for the leading fibers of the group. The second group of fibers traverses the crush site after 2 wk postcrush and advances at a rate of 2.1 +/- 0.1 mm/d for the leading fibers. The rate of growth of this group of fibers remains constant for 60 d but subsequently falls to 1.6 +/- 0.2 for the leading population of fibers. The leading fibers in the third group of regenerating axons traverse the crush site after 4 wk and advance at a constant rate of 0.8 +/- 0.2 mm/d. The multiple populations of regenerating fibers with differing rates of growth are discussed in the context of precursor cell maturity at the time of nerve injury and possible conditioning effects of the lesion upon these cells. Electron microscopy indicates that the number of axons decreases extensively after crush. The first two phases of regenerating axons represent a total of between 6 and 10% of the original axonal population and are typically characterized by small fascicles of axons surrounded by Schwann cells and large amounts of collagenous material. The third phase of fibers represents between 50 and 70% of the original axonal population.

Subcellular and polypeptide distributions of slowly transported proteins in the garfish olfactory nerve.

In the garfish olfactory nerve proteins labeled with [3H]leucine are transported by slow axonal flow as a well-defined crest of radioactivity. At 21 degrees C slow flow moves along the axon with a velocity of 0.92 +/- 0.02 mm/day. It has been possible to analyze 4 subcellular fractions (soluble, mitochondrial and 2 membranous) as well as their polypeptide composition, in areas of the nerve containing (1) the slow moving crest, (2) the material remaining in the nerve behind the crest, and (3) the labeling present in front of the slow crest. Analyses were done 70 and 110 days after isotope deposition. The crest of slow moving radioactivity is characterized by a close parallelism between labeling and protein concentration in the subcellular fractions as well as among the polypeptides constituting these fractions. The radioactivity is mainly associated with mol. wt. of 14,000, 30-45,000, 58,000 and 68,000. This last peak corresponds to a protein not labeled by fast transport, present only in the light membranous fraction. The composition of the moving crest remains essentially constant during the 40-day period investigated. Most of the slow-moving molecules remain in the axon behind the moving crest. This deposited material appears to be redistributed and/or to be turning over more rapidly than the molecules still moving in the crest. A large amount of radioactivity was recovered in front of the moving crest. This might be produced by molecules deposited by fast transport and by material released from the cell body at rates intermediate between the fast and slow phases of transport. The subcellular and polypeptide compositions of this area of the nerve remain constant and are intermediate between the compositions of fast and slow flow. The slowly transported labeled polypeptides in the mitochondrial fraction are of low molecular weight, and were found to be similar in the various areas of the nerve and at the two time points studied, and were even similar to the polypeptide distribution determined for fast transport.