EFFECT OF ETHANOL EXPOSURE ON XENOPUS EMBRYO LIPID COMPOSITION

Clara Lindi,*, Gigliola Montorfano, Federica Rossi, Rosalba Gornati1 and Angela M. Rizzo

Institute of General Physiology and Biochemistry, Faculty of Pharmacy, University of Milan and
1 Department of Structural and Functional Biology, University of Insubria, Varese, Italy

Received 30 November 2000; in revised form 22 March 2001; accepted 24 April 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
— Exposure to ethanol is known to alter cell membranes both chemically and biophysically; these effects may be related to the development of tolerance and physical dependence. In the present study, the effects of various concentrations of ethanol (1–250 mM) on the lipid composition after the first 6 days of Xenopus embryo development were investigated, using an in vitro fertilization technique. Lipid analysis revealed: (1) a decrease of the cholesterol/phospholipid molar ratio mainly derived from a higher content of phospholipids; (2) an increase of phospholipid unsaturated fatty acids, especially C20:4 and C20:5, with ethanol concentration of 150–250 mM; (3) a decrease of lipid-bound sialic acid with ethanol concentrations of >=5 mM. These results underline that sialoglycoconjugates are a more sensitive target of alcohol in comparison with other lipid components. The cultured embryo method certainly represents a useful model for investigation of the direct effects of ethanol on lipid metabolism, excluding maternal interference which can lead to misinterpretation of data.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Ethanol contributes directly or indirectly to a variety of serious disorders in different body organs. Like other general anaesthetics, it is known to act primarily within the lipid bilayer of biological membranes, affecting many of its physico-chemical structural properties. Moreover, the deleterious effect of maternal ingestion of ethanol on embryos, fetuses and offspring of different animal species is well documented (Smith et al., 1973Go; Gallo and Weinberg, 1986Go; Gilani et al., 1986Go). In some of these studies, it has been shown that ethanol is most teratogenic during the organogenesis and the development of the nervous system (Pentney and Miller, 1992Go; West et al., 1994Go; Guerri, 1996Go).

Although ethanol may exert different effects at the molecular or biophysical level in most cell types, it is the developmental timing of the affected target cell that determines the biological consequences (Armant and Saunders, 1996Go). In spite of the usefulness of studying the effect of ethanol on biological membranes during embryogenesis, few studies in this area have been reported in the literature (Sanchez-Amate et al., 1991Go, 1992Go). It has not been determined whether alcohol acts directly on embryonic tissues to induce modifications, whether these result from altered maternal or placental functions involved in normal embryonic development, or whether different ethanol administration procedures in association with dietary regime affect the resultant anomalies.

To obtain further insight into the ethanol effects on embryo development, we have investigated the changes in the lipid composition of Xenopus embryos exposed to ethanol during the first 6 days of their development: this model system avoids confounding maternal ethanol influences and it can be readly manipulated without any intrusive technique of alcohol administration.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
In vitro fertilization and embryo culture
Xenopus, purchased from a local dealer (Rettili, Varese, Italy) were housed in aquaria (Tecnoplast, Varese, Italy), maintained under controlled conditions, and fed with Xenopus adult food (Carolina Biological Supply Company, Burlington, NC, USA) (Rizzo et al., 1999Go).

In vitro fertilization was performed according to the procedure of Bernardini et al. (1994). Five to six adult females per experiment were injected with 700–1000 IU of human chorionic gonadotropin; about 16 h later, females were made to lay eggs in Petri dishes. Eggs were immediately inseminated with sperm suspension, obtained by mincing the testes in 1–2 ml of cold DBT (De Boers–Tris, in mM: NaCl 119, KCl 2.5, CaCl2 1.8, Tris/HCl 15, pH 7.5). After 2 min, 10 ml of FETAX (Frog Embryo Teratogenesis Assay — Xenopus, containing in mg/l: NaCl 625, NaHCO3 96, KCl 30, CaCl2 15, CaSO4•2H2O 60, MgSO4 70, pH 7.8–8) solution were added to each Petri dish. Successful insemination was detected after a few minutes; only the eggs oriented with the dark side (animal pole) up were considered. All irregular segmented eggs were eliminated and eventually replaced; at the beginning of the treatments each Petri dish contained 15–20 embryos.

Each assay consisted of five to ten Petri dishes with embryos from the same female; to minimize ethanol evaporation the dishes were covered and then assigned at random to become controls or to be exposed to alcohol.

The established ethanol exposure protocols were similar to those reported in the literature for embryo and cell culture systems (Priscott, 1982Go; Dresser et al., 1992Go; Kotch et al., 1995Go; Kulyk and Hoffman, 1996Go): the tested concentrations ranged from 1 to 250 mM, because embryos did not survive at concentrations of >250 mM. The ethanol solutions were prepared each day. Controls, consisting of FETAX solution alone, were run concurrently. The treatment started 8 h post fertilization. Each day the solutions were renewed and the dead embryos counted and removed.

Embryos were kept in a thermostatic chamber at 23 ± 0.5°C. In order to assess the absence of ethanol evaporation from the embryo culture, samples from ethanol-containing Petri dishes were taken each day before solution replacement and subjected to an enzymatic assay (Boehringer–Mannheim Ethanol Test Combination, Italy). These analyses revealed that there was no loss of ethanol at any tested concentration. On day 6 of development, a determined number of embryos (usually 100) were collected with large-bore pipettes and stored at -20°C until used.

Lipid extraction
The frozen embryos were thawed, freed of the excess water with a pipette and extracted in chloroform (C)/methanol (M) according to the method of Suzuki (1965) with some slight modifications. Briefly, 100 embryos were first homogenized in 4 ml of C/M (1/2 by vol) followed by two extractions with 4 ml of C/M 2/1 and C/M 1/1. After the three extractions, the insoluble residue containing non-lipid components was removed by centrifugation (18 500 g for 15 min at 4°C) and used for determining protein by the bicinchoninic acid method of Smith et al. (1985). The three supernatants were pooled, dried under vacuum, and resuspended in 10 ml C/M (2/1 by vol); a partition step with water (W) followed by theoretical upper phase (C/M/W 3/48/47) resulted in an aqueous and an organic phase.

The aqueous phase was desalted with a C18 RP column (Bondelut, Analytichem International, Arbor City, USA) using the Williams and McCluer (1980) procedure and the total ganglioside content was determined as lipid-bound N-acetyl-neuraminic acid (NeuAc) by the resorcinol method (Svennerholm, 1957Go).

Aliquots of the organic phase were used for the determinations of phospholipid phosphorus (Bartlett, 1959Go) and total cholesterol (Pearson et al., 1953Go). The total phospholipids were then separated from the other lipid components of the extract by silicic acid chromatography according to Vance and Sweely (1967).

Individual phospholipid classes were separated by high-performance thin-layer chromatography (HPTLC) on silica gel 60 plates (Merck, Germany) developed with C/M/acetic acid/W (60/45/4/2 by vol). The phospholipid spots, visualized with iodine, were carefully scraped into Pyrex tubes and submitted to phosphorus determination (Dodge and Phillips, 1967Go).

The fatty acid composition of total phospholipids was determined by the conversion of phospholipid fatty acids to methyl esters using 0.5 M NaOH in methanol and a resulting analysis by gas–liquid chromatography (DANI 86.10, Monza, Italy; AT Silar, Alltech capillary column, 32 m x 0.32 mm i.d.; temperature programme 80°C for 2 min followed by a rise to 180°C at 8°C/min; helium flow rate was 0.7 ml/min).

Statistical analysis
Significant differences were determined by Student's t-test. All values are reported as means ± SD.


    RESULTS AND DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The overall mortality and malformation (axial and ocular abnormalities, generalized oedema, growth inhibition) of the Xenopus embryo controls during the first 6 days of development were 12.3% and 15% respectively. Ethanol did not cause an increase in mortality or malformation in the treated embryos below a concentration of 100 mM. Higher ethanol concentrations resulted in a dose–response reaching 70% mortality and 50% malformation at 250 mM.

Evaluation of the lipid composition of Xenopus embryos (Table 1Go) indicated that, when compared with control values, the total cholesterol, phospholipid and ganglioside NeuAc content showed different responses to the 6-day ethanol exposure. As reported in Table 1Go, the phospholipid content significantly increased steadily from 150 to 250 mM ethanol concentrations, whereas no changes in the cholesterol and protein contents were noted for all tested dosages. The increase in phospholipids accounts for the lower cholesterol/phospholipids molar ratio and for the higher lipid/protein ratio from the samples exposed to the same ethanol concentration range of 150–250 mM.


View this table:
[in this window]
[in a new window]
 
Table 1. Effect of 6-day ethanol (0–250 mM) exposure on protein, total cholesterol, phospholipid and ganglioside N-acetyl-neuraminic acid (NeuAc) content of Xenopus laevis embryos
 
Similarly studies performed in chicks have indicated that, in agreement with our results, chronic ethanol administration does not affect the embryo total cholesterol content in liver or plasma (Marco et al., 1986aGo; Sanchez-Amate et al., 1991Go). These results are different from those reported by other authors in mammalian species, in which an increase in total cholesterol levels in liver was found after ethanol treatment (Lakshman et al., 1988Go; Baraona and Lieber, 1998Go).

It has been reported that chronic ethanol consumption also increases the total phospholipid levels in membranes of different experimental animals (Koivusaari et al., 1981Go), though this finding is controversial because other authors did not find such an effect (Cunningham et al., 1982Go; Marco et al., 1986bGo).

Analysis of the individual phospholipid content (Table 2Go) indicated that the 6-day ethanol exposure affected only the two major phospholipid classes, which account for ~80% of the total species: in fact the observed increase in the phospholipid content corresponded to an increase in only phosphatidylcholine (PC) and phosphatidylethanolamine (PE).


View this table:
[in this window]
[in a new window]
 
Table 2. Effect of 6-day ethanol (0–250 mM) exposure on phospholipid distribution of Xenopus laevis embryos (µg phospholipid/embryo)
 
Unlike the cholesterol and phospholipid contents, that of ganglioside NeuAc (Table 1Go) was significantly decreased already at a 5 mM ethanol concentration. It has been shown, in general, that chronic exposure to ethanol reduces the levels of sialic acid in several tissues of the developing organism through impairment of ganglioside and glycoprotein metabolism, affecting the sialylation or desialylation systems (Renau-Piqueras et al., 1989Go; Prasad, 1992Go; Ghosh et al., 1993Go; Mihas and Tavassoli, 1993Go; Omodeo-Salè and Palestini, 1994Go; Rosenberg and Noble, 1994Go). Our results possibly indicate that the biosynthetic pathway of gangliosides is a more sensitive target in comparison with those of the other lipids; in fact the ethanol-induced changes on gangliosides are evident at a very low (5 mM) concentration. Sialic acid residues are important for the conformation of glycolipids, their structural stability and their functions. As acidic glycolipids are involved in neurodifferentiation (Rosner, 1998Go), their changes during embryogenesis may represent a mechanism for development of ethanol-dependent behavioural dysfunction, such as the fetal-alcohol syndrome.

Table 3Go shows the embryo phospholipid fatty acid composition: for a simplified reading of the results, we excluded from the reported data some ethanol concentrations (1, 2, 5 and 10 mM), where no changes were found. Embryo phospholipid fatty acid composition was affected significantly from a concentration of 150–250 mM: a decrease of palmitic acid (C16:0) and an increase of arachidonic (C20:4) and eicosapentaenoic (C20:5) acids were detected, resulting in an increase of polyunsaturated/saturated fatty acid ratio.


View this table:
[in this window]
[in a new window]
 
Table 3. Effect of 6-day ethanol (0–250 mM) exposure on phospholipid fatty acid composition of Xenopus laevis embryos
 
Similar results have been reported in studies on membranes from cultured cells (Keegan et al., 1983Go), chick (Marco et al., 1986aGo) and rat (Rovinski and Hosein, 1983Go) tissues exposed to ethanol during development. Since ethanol causes disruption of biological membranes (Seeman, 1972Go; Hunt, 1975Go; Goldstein and Chin, 1981Go) it is postulated that changes in the degree of unsaturation observed here might result in generalized alteration of the intrinsic ‘fluidity’ of the membranes of these embryos (Oldfield and Chapman, 1971Go; Quinn and Chapman, 1980Go).

During embryogenesis, several processes must operate harmoniously to complete the developing plan and such cooperation is easily perturbable by many molecules with toxicological potentials. Xenopus is an advantageous system for studying possible modifications of lipid pattern during development induced by these molecules. For example, embryos can be easily cultured in lipid-free media and their development can be followed with a dissecting microscope. The amphibian embryo, during its early development, is a closed system that operates the rearrangement of stored materials of maternal origin; in nature, feeding starts when the embryos are approximately 4 or 5 days old. In our study, however, embryos have not been fed; accordingly, we can also exclude interferences of dietary factors that make the interpretation of the effects of ethanol on membranes difficult. In fact, the differences in the results concerning the effects of ethanol on biological membranes obtained by some authors could be due in part to the different composition of external diets administered simultaneously with ethanol (Abel and Reddy, 1997Go). In addition, differences in the chemical composition of membranes could be responsible for differences in the response to ethanol (Marco et al., 1986bGo; Sanchez-Amate et al., 1992Go).

It is also possible to exclude maternal metabolism in studies with embryo culture, so that variables such as ethanol-induced changes in placental function are eliminated, and the direct effects of ethanol on developing embryos can therefore be easily studied. Moreover in X. laevis, alcohol dehydrogenase activity is not observed until stage 47 (day 5 of development) when larval feeding has commenced (Nieuwkoop and Faber, 1957Go): because there was no ethanol oxidation during the treatment period, the effects observed can therefore be linked to ethanol per se.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The authors are thankful to Professor Giovanni Bernardini for his suggestions and to Prof. Bruno Berra for his critical reading of the manuscript. This work was supported by a research grant from MURST (60%).


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed at: Institute of General Physiology and Biochemistry, University of Milan, Via Trentacoste 2, 20134 Milan, Italy. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Abel, E. L. and Reddy, P. P. (1997) Prenatal high saturated fat diet modifies behavioral effects of prenatal alcohol exposure in rats. Alcohol 14, 25–29.[ISI][Medline]

Armant, D. R. and Saunders, D. E. (1996) Exposure of embryonic cells to alcohol: contrasting effects during preimplantation and postimplantation development. Seminars in Perinatology 20, 127–139.[ISI][Medline]

Baraona, E. and Lieber, C. S. (1998) Alcohol and lipids. Recent Developments in Alcoholism 14, 97–134.[Medline]

Bartlett, G. R. (1959) Phosphorus assay in column chromatography. Journal of Biological Chemistry 234, 466–468.[Free Full Text]

Bernardini, G., Vismara, C., Boracchi, P. and Camatini, M. (1994) Lethality, teratogenicity and growth inhibition of heptanol in Xenopus assayed by a modified frog embryo teratogenesis assay-Xenopus (FETAX) procedure. Science of the Total Environment 151, 1–8.[ISI][Medline]

Cunningham, C. C., Filus, S., Bottenus, R. and Spach, P. I. (1982) Effect of ethanol consumption on the phospholipid composition of rat liver microsomes and mitochondria. Biochimica et Biophysica Acta 712, 225–233.[ISI][Medline]

Dodge, J. T. and Phillips, G. B. (1967) Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. Journal of Lipid Research 8, 667–675.[Abstract/Free Full Text]

Dresser, T. H., Rivera, E. R., Hoffmann, F. J. and Finch, R. A. (1992) Teratogenic assessment of four solvents using the frog embryo teratogenesis assay–Xenopus (FETAX). Journal of Applied Toxicology 12, 49–56.[ISI][Medline]

Gallo, P. V. and Weinberg, J. (1986) Organ growth and cellular development in ethanol-exposed rats. Alcohol 3, 261–267.[ISI][Medline]

Ghosh, P., Okoh, C., Liu, Q. J. and Lakshman, M. R. (1993) Effects of chronic ethanol on enzymes regulating sialylation and desialylation of transferrin in rats. Alcoholism: Clinical and Experimental Research 17, 576–579.[ISI][Medline]

Gilani, S. H., Sachdev, R. and Persaud, T. V. N. (1986) Chick embryonic development following exposure to ethanol and pyrazole. Anatomischer Anzeiger 162, 79–82.[ISI][Medline]

Goldstein, D. B. and Chin, J. H. (1981) Interaction of ethanol with biological membranes. Federation Proceedings 40, 2073–2076.[ISI][Medline]

Guerri, C. (1996) Teratogenic effects of alcohol: current status of animal research and in vitro models. Archives of Toxicology 18, 71–80.

Hunt, W. A. (1975) The effects of aliphatic alcohols on the biophysical and biochemical correlates of membrane function. Advances in Experimental Medicine and Biology 56, 195–210.[Medline]

Keegan, R., Wilce, P. A., Ruczkal-Pietrzak, E. and Shanley, B. C. (1983) Effect of ethanol on cholesterol and phospholipid composition of Hela cells. Biochemical and Biophysical Research Communications 114, 985–990.[ISI][Medline]

Koivusaari, U., Norling, A., Lang, M. and Spach, P. I. (1981) Structural and biotransformational membrane changes in the liver and intestine during chronic ethanol administration. Toxicology 20, 173–183.[ISI][Medline]

Kotch, L. E., Chen, S. Y. and Sulik, K. K. (1995) Ethanol-induced teratogenesis: free radical damage as a possible mechanism. Teratology 52, 128–136.[ISI][Medline]

Kulyk, W. M. and Hoffman, L. M. (1996) Ethanol exposure stimulates cartilage differentiation by embryonic limb mesenchyme cells. Experimental Cell Research 223, 290–300.[ISI][Medline]

Lakshman, M. R., Chirtel, S. J. and Chambers, L. L. (1988) Role of omega-3 fatty acids and chronic ethanol in the regulation of plasma and liver lipids and plasma apoproteins A1 and E in rats. Journal of Nutrition 118, 1299–1303.[ISI][Medline]

Marco, C., Ceacero, F., Gonzalez-Pacanowska, D., Garcia-Peregrin, E. and Segovia, J. L. (1986a) Alterations induced by chronic ethanol treatment on lipid composition of microsomes, mitochondria and myelin from neonatal chick liver and brain. Biochemical International 12, 51–60.

Marco, C., Ceacero, F., Garcia-Peregrin, E. and Segovia, J. L. (1986b) Effect of ethanol on lipid composition of neonatal chick liver. Nutrition Research 6, 1386–1389

Mihas, A. A. and Tavassoli, M. (1993) Ethanol enhances desialylation of transferrin by liver endothelial cells in the rat. The American Journal of Medical Sciences 305, 12–17.

Nieuwkoop, P. D. and Faber, J. (1957) Normal Table of Xenopus laevis (Daudin). North-Holland, Amsterdam.

Oldfield, E. and Chapman, D. (1971) Effects of cholesterol and cholesterol derivatives on hydrocarbon chain mobility in lipids. Biochemical and Biophysical Research Communications 43, 610–616.[ISI][Medline]

Omodeo-Salè, F. and Palestini, P. (1994) Chronic ethanol effects on glycoconjugates and glycosyltransferases of rat brain. Alcohol 11, 301–306.[ISI][Medline]

Pearson, S., Stern, S. and Gavak, T. M. (1953) A rapid method for the determination of total cholesterol in serum. Analytical Chemistry 25, 813–814.[ISI]

Pentney, R. J. and Miller, M. W. (1992) Effect of ethanol on neuronal morphogenesis. In Development of the Central Nervous System: Effects of Alcohol and Opiates, Miller, M. W. ed., pp. 71–108. Wiley–Liss, New York.

Prasad, V. V. (1992) Effect of prenatal and postnatal exposure to ethanol on rat central nervous system gangliosides and glycosidases. Lipids 27, 344–348.[ISI][Medline]

Priscott, P. K. (1982) The effect of ethanol on rat embryos developing in vitro. Biochemical Pharmacology 31, 3641–3643.[ISI][Medline]

Quinn, P. J. and Chapman, D. (1980) The dynamics of membrane structure. CRC Critical Reviews in Biochemistry 8, 1–117.[ISI][Medline]

Renau-Piqueras, J., Sancho-Tello, M., Baguena Cervellera, R. and Guerri, G. (1989) Prenatal exposure to ethanol alters the synthesis and glycosylation of proteins in fetal hepatocytes. Alcoholism: Clinical and Experimental Research 13, 817–823.[ISI][Medline]

Rizzo, A. M., Gornati, R., Rossi, F., Bernardini, G. and Berra, B. (1999) Effect of maternal diet on the distribution of phospholipids and their fatty acid composition in Xenopus laevis embryos. Journal of Nutritional Biochemistry 10, 44–48.[ISI]

Rosenberg, A. and Noble, E. P. (1994) Ethanol attenuation of ganglioside sialylation and neuritogenesis. Alcohol 11, 565–569.[ISI][Medline]

Rosner, H. (1998) Significance of gangliosides in neuronal differentiation of neuroblastoma cells and neurite growth in tissue culture. Annals of the New York Academy of Sciences 845, 200–214.[Abstract/Free Full Text]

Rovinski, B. and Hosein, E. A. (1983) Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion. Biochimica et Biophysica Acta 735, 407–417.[ISI][Medline]

Sanchez-Amate, M. C., Segovia, J. L. and Marco, C. (1991) Influence of ethanol on liver and plasma lipid levels during chick embryo development. Biochemistry International 24, 299–306.[ISI][Medline]

Sanchez-Amate, M. C., Marco, C. and Segovia, J. L. (1992) Physical properties, lipid composition and enzyme activities of hepatic subcellular membranes from chick embryo after ethanol treatment. Life Sciences 51, 1639–1646.[ISI][Medline]

Seeman, P. (1972) The membrane actions of anesthetics and tranquilizers. Pharmacological Reviews 24, 583–655.[ISI][Medline]

Smith, K. L., Jones, D. W., Ulleland, C. N. and Stressguth, A. P. (1973) Pattern of malformation in offspring of chronic alcoholic mothers. Lancet i, 1268–1271.

Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985) Measurement of protein using bincinchonininc acid. Analytical Biochemistry 150, 76–85.[ISI][Medline]

Suzuki, K. (1965) Pattern of mammalian brain gangliosides. Journal of Neurochemistry 12, 629–638.[ISI][Medline]

Svennerholm, L. (1957) Quantitative estimation of sialic acid: a colorimetric resorcinol–hydrochloric acid method. Biochimica et Biophysica Acta 24, 604–611.[ISI]

Vance, D. E. and Sweely, C. C. (1967) Quantitative determination of the neutral glycosil-ceramides in human blood. Journal of Lipid Research 8, 621–630.[Abstract/Free Full Text]

West, L. R., Chen, W. A. and Pantazis, N. J. (1994) Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage. Metabolic Brain Disease 9, 291–322.[ISI][Medline]

Williams, M. A. and McCluer, R. H. (1980) The use of Sep-Pack C18 cartridges during the isolation of gangliosides. Journal of Neurochemistry 35, 266–269.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Lindi, C.
Articles by Rizzo, A. M.
PubMed
PubMed Citation
Articles by Lindi, C.
Articles by Rizzo, A. M.