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Breakdown of choline-containing phospholipids in rat brain during severe weight loss
Neuroscience Letters 326 (2002) 21–24 www.elsevier.com/locate/neulet
Breakdown of choline-containing phospholipids in rat brain during severe weight loss Christoph Go¨pel a, Martin H. Schmidt a, Marianne Campanini b, Jochen Klein c,* b
a Central Institute of Mental Health, Department of Child and Adolescent Psychiatry, Mannheim, Germany Department of Pharmacology, Johannes Gutenberg University of Mainz, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany c Department of Pharmaceutical Sciences, Texas Tech School of Pharmacy, 1300 Coulter Drive, Amarillo, TX 79106, USA
Received 12 December 2001; received in revised form 17 March 2002; accepted 22 March 2002
Abstract Recent investigations in human anorectic patients indicated changes of brain choline metabolism. We used starved rats to investigate possible changes of brain choline metabolites during severe weight loss. Reductions of body weight by 15, 30 and 45% resulted in significant decreases of cerebral phosphatidylcholine and sphingomyelin levels. Concomitantly, the brain tissue content of glycerophosphocholine was increased while phosphocholine and free choline were unchanged. We conclude that severe weight loss is accompanied by phospholipase activation and breakdown of choline-containing phospholipids in the brain. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Choline; Glycerophosphocholine; Phosphatidylcholine; Phosphocholine; Sphingomyelin
Undernutrition during the first weeks of life is associated with delayed maturation of the brain and reductions of brain phospholipids in rats and humans [8,15]. It is not known, however, whether similar changes can be caused by undernutrition in the mature brain. In adolescents, severe weight loss is a characteristic of the eating disorder, anorexia nervosa. Anorectic patients are characterized by an abnormally low body weight and a high mortality due to somatic pathology [13]. Anorectic patients also develop brain atrophy, enlargement of the external cerebrospinal fluid spaces, and cognitive dysfunctions [5]. The cellular and molecular correlates of these changes are unknown. We recently observed consistent increases of the choline-containing compounds signal (‘choline peak’) by proton magnetic resonance (PMR) spectroscopy in anorectic patients [3,12]. Our findings were at variance with a study by Roser et al. [11] who failed to confirm an increase of the choline peak. Unfortunately, it is not entirely clear what the ‘choline peak’ denotes [6]. As the brain levels of free choline and acetylcholine are very small (,30 nmol/g), and the large pools of choline-containing phospholipids (.20 mmol/g) are invisible by nuclear magnetic resonance, the choline peak probably reflects tissue levels of phosphocholine * Corresponding author. Tel.: 11-806-356-4000, ext. 252; fax: 11-806-356-4034. E-mail address: [email protected] (J. Klein).
(tissue level 200–300 nmol/g) and glycerophosphocholine (GPCh; 500–600 nmol/g) [6,7]. To clarify possible changes of brain choline metabolites during undernutrition, we turned to the animal model of starved rats and applied neurochemical analysis to quantify choline-containing metabolites. For this purpose, female Wistar rats (Charles River, Sulzbach, Germany) at 90–110 days of age were kept on a body weight-reducing diet by gradually feeding lower amounts of standard diet (Altromin C1320) for 3 months. According to the manufacturer, this diet contains 56.6% carbohydrates, 17.3% crude protein, 5.4% crude fat, 3.1% crude fibre, 5.5% ash, 10% moisture, vitamins and mineral supplements. The concentration of choline in this diet is 60–70 mg of total choline per 100 g (analyzed in our laboratory), and the methionine and folic acid contents are 0.3 g and 0.2 mg per 100 g, respectively (data from manufacturer). Control rats (N ¼ 12) were kept on standard diet fed ad libitum for 90 days. Animals in groups A and B (N ¼ 12 each) received reduced amounts of food to reduce their body weight by an average of 15 and 30%, respectively, within 3 months. Rats in group C (N ¼ 6) were kept on a reduced diet to lose 45% of their body weight within 3 months, and this group of rats was also kept underweight for an additional 2 weeks before sacrifice. All animals were housed individually with free access to water. Control rats always had access to food while groups A–C were fed once daily in the afternoon.
0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 2) 00 30 4- X
C. Go¨pel et al. / Neuroscience Letters 326 (2002) 21–24
22
Table 1 Body weight of animal groups before and at the end of the experiment Group
Controls A B C
Body weight (g) before experiment
Body weight (g) at end of experiment
Mean ^ SD
Median
Mean ^ SD
Median
269.4 ^ 17.9 277.3 ^ 16.5 247.6 ^ 8.6 288.3 ^ 12.0
269 281 248.5 285.5
384.9 ^ 49.9 233.8 ^ 6.6 167.7 ^ 12.1 159.9 ^ 8.0
378 233.5 169.5 158.5
The weight loss of the animals as a group is summarized in Table 1. We noted that rats in groups B and C, but not in group A, displayed increasingly aggressive behaviour during starvation. No other abnormalities in appearance, behaviour or physical health were observed in any of the groups. At the end of the experiment, rats were sacrificed, and the cerebellar hemispheres were quickly removed and weighed. Brain hemisphere weights were 0.52 ^ 0.08 g (controls), 0.59 ^ 0.11 g (group A), 0.59 ^ 0.09 g (group
B) and 0.51 ^ 0.10 g (group C) and were not significantly different between treatment groups. Analysis of cholinecontaining compounds was done as previously described [2,7]. We first analyzed the brain contents of choline-containing phospholipids, namely phosphatidylcholine (PC), the major membrane phospholipid representing the largest store of choline in the brain, and sphingomyelin (SM), which is enriched in myelin sheaths. We observed reductions of PC and SM contents in the brains of all test groups although losses of SM were significant only in the heavily starved group C (Fig. 1). Thus, losses of cerebral PC and SM were already induced by a 3 month diet reducing the body weight by 15% (Fig. 1, group A) whereas highly significant reductions were seen in group C suffering a severe (45%) weight loss. It should be noted that these changes cannot be due to dietary choline deficiency because we have shown in a previous study that choline deficiency alone did not affect choline-containing phospholipids in the brain over a period of several months [2]. Rather, the loss of PC and SM may indicate a general loss of phospholipid membrane as a consequence of a general hypometabolic state. The loss of
Fig. 1. Cerebral tissue contents of phosphatidylcholine (A) and sphingomyelin (B) in control rats (‘Ctr’) and in rats having lost 15% (group A, N ¼ 12), 30% (group B, N ¼ 12) and 45% (group C, N ¼ 6) of their body weight. Data are given as (mmol/g tissue) and are means ^ SEM of six to 12 experiments. Statistical significance was tested by analysis of variance followed by t-test (two-sided): *P , 0:05; **P , 0:01 vs. controls.
C. Go¨pel et al. / Neuroscience Letters 326 (2002) 21–24
Fig. 2. Cerebral tissue contents of glycerophosphocholine (GPCh) in control rats (‘Ctr’) and in rats having lost 15% (group A, N ¼ 12), 30% (group B, N ¼ 12) and 45% (group C, N ¼ 6) of their body weight. Data are given as individual data points of six to 12 determinations. Means are indicated by horizontal lines. Statistical significance was tested by analysis of variance followed by t-test (two-sided): *P , 0:05 vs. controls.
SM is compatible with a reduction of myelination [8,10] although further studies are required to clarify this issue. In addition to phospholipids, we investigated the tissue levels of the PC breakdown products, GPCh, phosphocholine, and free choline. The levels of free choline were 56.2 ^ 9.1 nmol/g (N ¼ 12) in controls and were unchanged in starved groups (free choline values were high because of the well-known post-mortem increase of free choline). Similarly, tissue levels of phosphocholine (199.8 ^ 9.8 nmol/g, N ¼ 12) in controls were not significantly affected by loss of body weight. However, starved rats displayed increases of GPCh levels as illustrated in Fig. 2. Severe weight loss induced a marked albeit variable increase of GPCh levels which was significant (P , 0:05) in groups B (178% vs. controls) and C (187% vs. controls). Thus, PC breakdown is reflected in an increase of its breakdown product, GPCh, whereas the levels of free choline and phosphocholine, which essentially reflect anabolic choline metabolism [6], remained unchanged. By analogy, increases of GPCh in human anorexia would be in agreement with our previous PMR observations of an increased ‘choline peak’ in this condition. Moreover, increased GPCh would also explain the enhanced signal for phosphodiesters in anorectic patients recently observed by Kato et al. [4]. The rather wide variability of GPCh levels in our experimental study remains unexplained at present but may be due to variations of GPCh formation or hydrolytic breakdown in individual animals. Individual variations in GPCh metabolism may
23
also explain some of the discrepancies reported in human PMR studies. Importantly, the increases in GPCh observed in the present study also give hints as to the mechanism of PC breakdown. As GPCh is only formed by phospholipase Amediated hydrolysis of PC, PC breakdown during fasting is obviously initiated by activation of phospholipase A (most likely phospholipase A2) [6]. Our present results have implications for metabolic as well as clinical issues. In metabolic terms, fatty acids released from phospholipids may serve as an energy supply in periods of extended starvation. Alternatively, it has been proposed that ‘autocannibalism’ of choline-containing phospholipids may be initiated to guarantee a sufficient supply of choline for acetylcholine synthesis in cholinergic neurons. In both cases, oral therapy with cytidinediphosphate-choline, a precursor of PC, may be therapeutically useful [1]. With respect to clinical issues, our findings offer an explanation for the decrease of cognitive abilities observed in severely starved anorectics [9] which may be caused by loss of phospholipids causing a general disturbance of neuronal membranes or a more specific reduction of central cholinergic function. While valid animal models in the field of eating disorders are scarce due to the complex psychological, biological and psychosocial aspects of the disease [14], the present model of long-term starvation may be useful to investigate cerebral consequences of severe weight loss including the development of cognitive dysfunction and the possible reversibility of these conditions. [1] Babb, S.M., Appelmans, K.E., Renshaw, P.F., Wurtman, R.J. and Cohen, B.M., Differential effect of CDP-choline on brain cytosolic choline levels in younger and older subjects as measured by proton magnetic resonance spectroscopy, Psychopharmacology, 127 (1996) 88–94. [2] Gonzalez, R., Bohl, J., Lo¨ ffelholz, K. and Klein, J., Influence of dietary choline deficiency on choline metabolites in the rat brain, Neurosci. Res. Commun., 18 (1996) 29–37. [3] Hentschel, F., Mo¨ ckel, R., Schlemmer, H.P., Markus, A., Go¨ pel, C., Guckel, F., Ko¨ pke, J., Georgi, M. and Schmidt, M.H., 1H-MR spectroscopy in anorexia nervosa: the characteristic differences between patients and healthy subjects, Rofo Fortschr. Geb. Rontgenstr. Neuen Bildgebenden Verfahren, 170 (1999) 284–289. [4] Kato, T., Shioiri, T., Murashita, J. and Inubushi, T., Phosphorus-31 magnetic resonance spectroscopic observations in 4 cases with anorexia nervosa, Prog. Neuro-Psychopharmacol. Biol. Psychiatry, 21 (1997) 719–724. [5] Katzman, D.K., Christensen, B., Young, A. and Zipursky, R.B., Starving the brain: structural abnormalities and cognitive impairment in adolescents with anorexia nervosa, Semin. Clin. Neuropsychiatry, 6 (2001) 146–152. [6] Klein, J., Membrane breakdown in acute and chronic neurodegeneration: focus on choline-containing phospholipids, J. Neural Transm., 107 (2000) 1027–1063. [7] Klein, J., Gonzalez, R., Ko¨ ppen, A. and Lo¨ ffelholz, K., Free choline and choline metabolites in rat brain and body fluids: sensitive determination and implication for choline supply to the brain, Neurochem. Int., 22 (1993) 293–300.
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C. Go¨pel et al. / Neuroscience Letters 326 (2002) 21–24
[8] Krigman, M. and Hogan, E., Undernutrition in the developing rat: effect upon myelination, Brain Res., 107 (1976) 239–255. [9] Lauer, C.J., Gorzewski, B., Gerlinghoff, M., Backmund, H. and Zihl, J., Neuropsychological assessments before and after treatments in patients with anorexia nervosa and bulimia nervosa, J. Psychiatr. Res., 33 (1999) 129–138. [10] Reddy, P.V. and Sastry, P.S., Effect of undernutrition on the metabolism of phospholipids and gangliosides in developing rat brain, Br. J. Nutr., 40 (1978) 403–411. [11] Roser, W., Bubl, R., Buergin, D., Seelig, J., Radue, E.W. and Rost, B., Metabolic changes in the brain of patients with anorexia and bulimia nervosa as detected by proton resonance spectroscopy, Int. J. Eat. Disord., 26 (1999) 119– 136.
[12] Schlemmer, H.P., Mo¨ ckel, R., Marcus, A., Hentschel, F., Go¨ pel, C., Becker, G., Ko¨ pke, J., Guckel, F., Schmidt, M.H. and Georgi, M., Proton magnetic resonance spectroscopy in acute, juvenile anorexia nervosa, Psychiatry Res., 82 (1998) 171–179. [13] Sharp, C.W. and Freeman, C.P.L., The medical complications of anorexia nervosa, Br. J. Psychiatry, 162 (1993) 452–462. [14] Smith, G.P., Animal models of human eating disorders, Ann. N. Y. Acad. Sci., (1989) 63–72. [15] Yusuf, H.K., Dickerson, J.W. and Waterlow, J.C., Changes in content and composition of brain phospholipids in malnourished children, Am. J. Clin. Nutr., 32 (1979) 2227– 2232.
Breakdown of choline-containing phospholipids in rat brain during severe weight loss Christoph Go¨pel a, Martin H. Schmidt a, Marianne Campanini b, Jochen Klein c,* b
a Central Institute of Mental Health, Department of Child and Adolescent Psychiatry, Mannheim, Germany Department of Pharmacology, Johannes Gutenberg University of Mainz, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany c Department of Pharmaceutical Sciences, Texas Tech School of Pharmacy, 1300 Coulter Drive, Amarillo, TX 79106, USA
Received 12 December 2001; received in revised form 17 March 2002; accepted 22 March 2002
Abstract Recent investigations in human anorectic patients indicated changes of brain choline metabolism. We used starved rats to investigate possible changes of brain choline metabolites during severe weight loss. Reductions of body weight by 15, 30 and 45% resulted in significant decreases of cerebral phosphatidylcholine and sphingomyelin levels. Concomitantly, the brain tissue content of glycerophosphocholine was increased while phosphocholine and free choline were unchanged. We conclude that severe weight loss is accompanied by phospholipase activation and breakdown of choline-containing phospholipids in the brain. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Choline; Glycerophosphocholine; Phosphatidylcholine; Phosphocholine; Sphingomyelin
Undernutrition during the first weeks of life is associated with delayed maturation of the brain and reductions of brain phospholipids in rats and humans [8,15]. It is not known, however, whether similar changes can be caused by undernutrition in the mature brain. In adolescents, severe weight loss is a characteristic of the eating disorder, anorexia nervosa. Anorectic patients are characterized by an abnormally low body weight and a high mortality due to somatic pathology [13]. Anorectic patients also develop brain atrophy, enlargement of the external cerebrospinal fluid spaces, and cognitive dysfunctions [5]. The cellular and molecular correlates of these changes are unknown. We recently observed consistent increases of the choline-containing compounds signal (‘choline peak’) by proton magnetic resonance (PMR) spectroscopy in anorectic patients [3,12]. Our findings were at variance with a study by Roser et al. [11] who failed to confirm an increase of the choline peak. Unfortunately, it is not entirely clear what the ‘choline peak’ denotes [6]. As the brain levels of free choline and acetylcholine are very small (,30 nmol/g), and the large pools of choline-containing phospholipids (.20 mmol/g) are invisible by nuclear magnetic resonance, the choline peak probably reflects tissue levels of phosphocholine * Corresponding author. Tel.: 11-806-356-4000, ext. 252; fax: 11-806-356-4034. E-mail address: [email protected] (J. Klein).
(tissue level 200–300 nmol/g) and glycerophosphocholine (GPCh; 500–600 nmol/g) [6,7]. To clarify possible changes of brain choline metabolites during undernutrition, we turned to the animal model of starved rats and applied neurochemical analysis to quantify choline-containing metabolites. For this purpose, female Wistar rats (Charles River, Sulzbach, Germany) at 90–110 days of age were kept on a body weight-reducing diet by gradually feeding lower amounts of standard diet (Altromin C1320) for 3 months. According to the manufacturer, this diet contains 56.6% carbohydrates, 17.3% crude protein, 5.4% crude fat, 3.1% crude fibre, 5.5% ash, 10% moisture, vitamins and mineral supplements. The concentration of choline in this diet is 60–70 mg of total choline per 100 g (analyzed in our laboratory), and the methionine and folic acid contents are 0.3 g and 0.2 mg per 100 g, respectively (data from manufacturer). Control rats (N ¼ 12) were kept on standard diet fed ad libitum for 90 days. Animals in groups A and B (N ¼ 12 each) received reduced amounts of food to reduce their body weight by an average of 15 and 30%, respectively, within 3 months. Rats in group C (N ¼ 6) were kept on a reduced diet to lose 45% of their body weight within 3 months, and this group of rats was also kept underweight for an additional 2 weeks before sacrifice. All animals were housed individually with free access to water. Control rats always had access to food while groups A–C were fed once daily in the afternoon.
0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 2) 00 30 4- X
C. Go¨pel et al. / Neuroscience Letters 326 (2002) 21–24
22
Table 1 Body weight of animal groups before and at the end of the experiment Group
Controls A B C
Body weight (g) before experiment
Body weight (g) at end of experiment
Mean ^ SD
Median
Mean ^ SD
Median
269.4 ^ 17.9 277.3 ^ 16.5 247.6 ^ 8.6 288.3 ^ 12.0
269 281 248.5 285.5
384.9 ^ 49.9 233.8 ^ 6.6 167.7 ^ 12.1 159.9 ^ 8.0
378 233.5 169.5 158.5
The weight loss of the animals as a group is summarized in Table 1. We noted that rats in groups B and C, but not in group A, displayed increasingly aggressive behaviour during starvation. No other abnormalities in appearance, behaviour or physical health were observed in any of the groups. At the end of the experiment, rats were sacrificed, and the cerebellar hemispheres were quickly removed and weighed. Brain hemisphere weights were 0.52 ^ 0.08 g (controls), 0.59 ^ 0.11 g (group A), 0.59 ^ 0.09 g (group
B) and 0.51 ^ 0.10 g (group C) and were not significantly different between treatment groups. Analysis of cholinecontaining compounds was done as previously described [2,7]. We first analyzed the brain contents of choline-containing phospholipids, namely phosphatidylcholine (PC), the major membrane phospholipid representing the largest store of choline in the brain, and sphingomyelin (SM), which is enriched in myelin sheaths. We observed reductions of PC and SM contents in the brains of all test groups although losses of SM were significant only in the heavily starved group C (Fig. 1). Thus, losses of cerebral PC and SM were already induced by a 3 month diet reducing the body weight by 15% (Fig. 1, group A) whereas highly significant reductions were seen in group C suffering a severe (45%) weight loss. It should be noted that these changes cannot be due to dietary choline deficiency because we have shown in a previous study that choline deficiency alone did not affect choline-containing phospholipids in the brain over a period of several months [2]. Rather, the loss of PC and SM may indicate a general loss of phospholipid membrane as a consequence of a general hypometabolic state. The loss of
Fig. 1. Cerebral tissue contents of phosphatidylcholine (A) and sphingomyelin (B) in control rats (‘Ctr’) and in rats having lost 15% (group A, N ¼ 12), 30% (group B, N ¼ 12) and 45% (group C, N ¼ 6) of their body weight. Data are given as (mmol/g tissue) and are means ^ SEM of six to 12 experiments. Statistical significance was tested by analysis of variance followed by t-test (two-sided): *P , 0:05; **P , 0:01 vs. controls.
C. Go¨pel et al. / Neuroscience Letters 326 (2002) 21–24
Fig. 2. Cerebral tissue contents of glycerophosphocholine (GPCh) in control rats (‘Ctr’) and in rats having lost 15% (group A, N ¼ 12), 30% (group B, N ¼ 12) and 45% (group C, N ¼ 6) of their body weight. Data are given as individual data points of six to 12 determinations. Means are indicated by horizontal lines. Statistical significance was tested by analysis of variance followed by t-test (two-sided): *P , 0:05 vs. controls.
SM is compatible with a reduction of myelination [8,10] although further studies are required to clarify this issue. In addition to phospholipids, we investigated the tissue levels of the PC breakdown products, GPCh, phosphocholine, and free choline. The levels of free choline were 56.2 ^ 9.1 nmol/g (N ¼ 12) in controls and were unchanged in starved groups (free choline values were high because of the well-known post-mortem increase of free choline). Similarly, tissue levels of phosphocholine (199.8 ^ 9.8 nmol/g, N ¼ 12) in controls were not significantly affected by loss of body weight. However, starved rats displayed increases of GPCh levels as illustrated in Fig. 2. Severe weight loss induced a marked albeit variable increase of GPCh levels which was significant (P , 0:05) in groups B (178% vs. controls) and C (187% vs. controls). Thus, PC breakdown is reflected in an increase of its breakdown product, GPCh, whereas the levels of free choline and phosphocholine, which essentially reflect anabolic choline metabolism [6], remained unchanged. By analogy, increases of GPCh in human anorexia would be in agreement with our previous PMR observations of an increased ‘choline peak’ in this condition. Moreover, increased GPCh would also explain the enhanced signal for phosphodiesters in anorectic patients recently observed by Kato et al. [4]. The rather wide variability of GPCh levels in our experimental study remains unexplained at present but may be due to variations of GPCh formation or hydrolytic breakdown in individual animals. Individual variations in GPCh metabolism may
23
also explain some of the discrepancies reported in human PMR studies. Importantly, the increases in GPCh observed in the present study also give hints as to the mechanism of PC breakdown. As GPCh is only formed by phospholipase Amediated hydrolysis of PC, PC breakdown during fasting is obviously initiated by activation of phospholipase A (most likely phospholipase A2) [6]. Our present results have implications for metabolic as well as clinical issues. In metabolic terms, fatty acids released from phospholipids may serve as an energy supply in periods of extended starvation. Alternatively, it has been proposed that ‘autocannibalism’ of choline-containing phospholipids may be initiated to guarantee a sufficient supply of choline for acetylcholine synthesis in cholinergic neurons. In both cases, oral therapy with cytidinediphosphate-choline, a precursor of PC, may be therapeutically useful [1]. With respect to clinical issues, our findings offer an explanation for the decrease of cognitive abilities observed in severely starved anorectics [9] which may be caused by loss of phospholipids causing a general disturbance of neuronal membranes or a more specific reduction of central cholinergic function. While valid animal models in the field of eating disorders are scarce due to the complex psychological, biological and psychosocial aspects of the disease [14], the present model of long-term starvation may be useful to investigate cerebral consequences of severe weight loss including the development of cognitive dysfunction and the possible reversibility of these conditions. [1] Babb, S.M., Appelmans, K.E., Renshaw, P.F., Wurtman, R.J. and Cohen, B.M., Differential effect of CDP-choline on brain cytosolic choline levels in younger and older subjects as measured by proton magnetic resonance spectroscopy, Psychopharmacology, 127 (1996) 88–94. [2] Gonzalez, R., Bohl, J., Lo¨ ffelholz, K. and Klein, J., Influence of dietary choline deficiency on choline metabolites in the rat brain, Neurosci. Res. Commun., 18 (1996) 29–37. [3] Hentschel, F., Mo¨ ckel, R., Schlemmer, H.P., Markus, A., Go¨ pel, C., Guckel, F., Ko¨ pke, J., Georgi, M. and Schmidt, M.H., 1H-MR spectroscopy in anorexia nervosa: the characteristic differences between patients and healthy subjects, Rofo Fortschr. Geb. Rontgenstr. Neuen Bildgebenden Verfahren, 170 (1999) 284–289. [4] Kato, T., Shioiri, T., Murashita, J. and Inubushi, T., Phosphorus-31 magnetic resonance spectroscopic observations in 4 cases with anorexia nervosa, Prog. Neuro-Psychopharmacol. Biol. Psychiatry, 21 (1997) 719–724. [5] Katzman, D.K., Christensen, B., Young, A. and Zipursky, R.B., Starving the brain: structural abnormalities and cognitive impairment in adolescents with anorexia nervosa, Semin. Clin. Neuropsychiatry, 6 (2001) 146–152. [6] Klein, J., Membrane breakdown in acute and chronic neurodegeneration: focus on choline-containing phospholipids, J. Neural Transm., 107 (2000) 1027–1063. [7] Klein, J., Gonzalez, R., Ko¨ ppen, A. and Lo¨ ffelholz, K., Free choline and choline metabolites in rat brain and body fluids: sensitive determination and implication for choline supply to the brain, Neurochem. Int., 22 (1993) 293–300.
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C. Go¨pel et al. / Neuroscience Letters 326 (2002) 21–24
[8] Krigman, M. and Hogan, E., Undernutrition in the developing rat: effect upon myelination, Brain Res., 107 (1976) 239–255. [9] Lauer, C.J., Gorzewski, B., Gerlinghoff, M., Backmund, H. and Zihl, J., Neuropsychological assessments before and after treatments in patients with anorexia nervosa and bulimia nervosa, J. Psychiatr. Res., 33 (1999) 129–138. [10] Reddy, P.V. and Sastry, P.S., Effect of undernutrition on the metabolism of phospholipids and gangliosides in developing rat brain, Br. J. Nutr., 40 (1978) 403–411. [11] Roser, W., Bubl, R., Buergin, D., Seelig, J., Radue, E.W. and Rost, B., Metabolic changes in the brain of patients with anorexia and bulimia nervosa as detected by proton resonance spectroscopy, Int. J. Eat. Disord., 26 (1999) 119– 136.
[12] Schlemmer, H.P., Mo¨ ckel, R., Marcus, A., Hentschel, F., Go¨ pel, C., Becker, G., Ko¨ pke, J., Guckel, F., Schmidt, M.H. and Georgi, M., Proton magnetic resonance spectroscopy in acute, juvenile anorexia nervosa, Psychiatry Res., 82 (1998) 171–179. [13] Sharp, C.W. and Freeman, C.P.L., The medical complications of anorexia nervosa, Br. J. Psychiatry, 162 (1993) 452–462. [14] Smith, G.P., Animal models of human eating disorders, Ann. N. Y. Acad. Sci., (1989) 63–72. [15] Yusuf, H.K., Dickerson, J.W. and Waterlow, J.C., Changes in content and composition of brain phospholipids in malnourished children, Am. J. Clin. Nutr., 32 (1979) 2227– 2232.