Effect of neonatal undernutrition on rat brain gangliosides

Effects of age and neonatal undernutrition were studied on total and fractions of gangliosides (GT1, GD1b, GD1a and GM1) in rat brain. GT1, GD1b + GD1a and GM1 are being presented here as polysialo-, disialo-, and monosialo-gangliosides. Undernutrition was induced by feeding mothers a low protein diet during lactation. The concentration of gangliosides increased to its maximum level by the age of 3 weeks and then decreased to its adult value by the age of 8 weeks. Polysialo gangliosides (GT1) comprised the maximum amount of gangliosides at birth (40%) and decreased to an adult value by 3 weeks of age. Disialo ganglioside GD1b increased to its maximum by 2 weeks and then leveled off. Disialo ganglioside GD1a and GM1 showed a decrease by 2 weeks and then reached to its adult value by 4 weeks. Neonatal undernutrition results in a decreased body and brain weight at all ages studied (7, 14 and 21 days) as well as total ganglioside concentration by 36%, 15% and 55% respectively at 7, 14 and 21 days. Polysialo gangliosides (GT1) constitute the major fraction of gangliosides at 21 days in undernourished rats as compared to controls, whereas mono- and di-sialo gangliosides remained decreased at all three ages compared to controls. This indicates the possibility of an underlying metabolic defect in undernourished animals.     

Profiling gangliosides from milk products and other biological membranes using LC/MS

Gangliosides are an important class of glycosphingolipids involved in numerous biological processes such as neuronal development, host–pathogen interactions and gastrointestinal health. Due to the highly heterogeneous nature and relatively low abundance of gangliosides, characterization of gangliosides in biological membranes is challenging. Existing methods for ganglioside analysis are quite time consuming and require expensive high resolution mass spectrometers. A rapid method combining reversed phase chromatography and mass spectrometry was developed using a triple-quadrupole mass spectrometer operating in multiple reaction monitoring mode. The ganglioside species were separated with a Poroshell 120 EC-C18 column and analysed under the negative ion mode. This method allows a sensitive, specific, and quantitative assay for profiling gangliosides. The method is developed for analysis of gangliosides in the milk fat globule membrane of whole milk and applied to other biological membranes. Application includes the cellular membrane of prostate cancer cells. In summary, the method allows various biological membranes to be screened for over 600 gangliosides from 12 classes (GM1, GM2, GM3, GM4, GD1, GD2, GD3, GD4, GT1, GT2, GT3, and GT4) in less than three hours. In summary, expressed as % of relative amounts: 1.5% GM3, 80.2% GD3, 14.4% GT3, 1.5% GM1, 2.4% GD1 were observed in whole milk; 2.5% GD1, 88.2% GD3, 2.5% GM1, 2.2% GM3, 0.2% GT2, 4.2% GT3 were observed in buttermilk and 10.6% GD1, 55.6% GD3, 1.6% GM1, 12.2% GM3, 19.2% GT3, 0.9% GT4 were observed in colostrum.     

human and bovine milk gangliosides differ in their fatty acid composition

Gangliosides are considered bioactive components in human infant nutrition, and their fatty acid composition alters their biological effects. We used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS and GLC to analyze the fatty acid composition of the predominant gangliosides, the monosialoganglioside GM(3) [sialic acid (Sia) alpha2-3 galactose (Gal) beta1-4 glucose (Glc) beta1-1 ceramide] and the disialoganglioside GD(3) (Sia alpha2-8 Sia alpha2-3 Gal beta1-4 Glc beta1-1 ceramide), in pooled human and bovine milk, the latter being a source for gangliosides in infant formula. Compared with whole milk lipids, both human and bovine milk gangliosides were selectively enriched with certain fatty acids, and the fatty acid composition of milk gangliosides in the 2 species was significantly different. The amount of long-chain fatty acids (> or =20 C atoms) was higher in bovine milk gangliosides (GM(3): 73.71 +/- 3.39%; GD(3): 79.19 +/- 2.79%) than in human milk gangliosides (GM(3): 51.25 +/- 0.65%; GD(3): 34.04 +/- 1.80%). Tricosanoic acid (23:0) dominated in bovine milk gangliosides (GM(3): 24.05 +/- 1.37%; GD(3): 26.66 +/- 1.24%), whereas it only played a minor role in human milk gangliosides (GM(3): 2.88 +/- 0.10%; GD(3): 1.84 +/- 0.29%). We hypothesized that the differences in the fatty acid composition of milk gangliosides result in physiological distinctions between breast-fed and formula-fed infants and therefore are of importance for human infant nutrition.     

Ganglioside long-chain base composition of rat brain subcellular fractions after chronic ethanol administration

Rats of two different ages (2 and 7 months) were treated with an ethanol-containing liquid diet for 24 days and changes of the ceramide composition of gangliosides were studied in the brain synaptosomal, microsomal and myelin fractions. Greater differences were observed in the younger age, where ethanol treatment caused a significant increase of C20: 1 LCB in GM1 ganglioside of synaptosomes and microsomes and in GDla of myelin.     

Ethanol disordering of GM1-enriched Short-Sleep synaptosomal plasma membranes

The Long-Sleep (LS) and Short-Sleep (SS) mouse synaptosomal plasma membranes differ in ethanol sensitivity at superficial membrane regions, which corresponds with the behavioral response of the mice to ethanol hypnosis. The only significant difference between these synaptosomal plasma membranes is the synaptosomal monosialoganglioside (G_M1) content, LS > SS. Here, G_M1 was examined as a parameter for increasing membrane sensitivity to ethanol effects in the ethanol-resistant SS membranes. Synaptosomal plasma membranes from SS mice were allowed to incorporate exogenous G_M1. Membrane order was then studied at the surface, intermediate, and interior regions of the membranes by delayed Fourier transform proton NMR in the presence and absence of perdeuterated ethanol. Differences in membrane order were observed in all three membrane regions with increasing perdeuterated ethanol concentrations depending on the synaptosomal G_M1 content.     

应用高效薄层色谱法检测母乳中神经节苷脂

目的：检测泌乳期母乳中神经节苷脂成分,并评价其生物学意义。方法：应用高效薄层色谱法对68份母乳标本进行了检测。结果：母乳中总神经节苷脂结合唾液酸含量为7．8－10．4μg/ml。在母乳中检测出4种神经节苷脂,分别为GM3、GD3、GX1及GX2。GX1及GX2为新发现的神经节苷脂,对GA1抗体呈阴性反应,推测其可能为C－通路神经节能苷脂。GM3与GD3为主要神经节苷脂（占总量50％－65％）。GM3在分娩后第8天时明显增高,GD3则明显降低,随分娩后天数增加GM3与GD3变化有显性。结论：母乳中存在GM3、GD3、GX1及GX24种神经节苷脂;GM3与GD3的含是随分娩后天数增加变化有显性。     

Enhancement of the immune response to poorly immunogenic gangliosides after incorporation into very small size proteoliposomes (VSSP).

Certain gangliosides are tumor-associated antigens that constitute potential targets for cancer immunotherapy. A major drawback in the design of ganglioside-based cancer vaccines, however, is the poor immunogenicity of these glycolipids. Here we report the immunological and physicochemical properties of very small size proteoliposomes (VSSP) obtained by using anionic detergents to incorporate gangliosides into the outer membrane protein complex (OMPC) of N. meningitidis. VSSP of three different gangliosides, GM3, NGcGM3 and GD3, were tested. These gangliosides differ in level of expression in normal tissues and in immunogenicity in different animal species. We show that the immunization with VSSP in an oil adjuvant consistently induced both IgM and IgG anti-ganglioside antibodies. In the mouse, the anti-ganglioside IgG fraction was not restricted to the typical T-independent isotype IgG3. Unexpectedly, significant levels of the T-dependent IgG1, IgG2a and particularly IgG2b were also found. VSSP-mediated enhancement of the immunogenicity was not restricted to the relatively immunogenic ganglioside GD3, satisfactory immune responses against highly tolerated GM3 and NGcGM3 were also obtained. Similar results were achieved in chickens and monkeys. No reactogenicity was observed even when self-gangliosides were used for immunization. VSSP overcame natural tolerance to gangliosides in an adjuvant dependent fashion.     

Brain neurotensin receptors in mice bred for differences in sensitivity to ethanol

The hypothesis that some of ethanol's acute effects are mediated via neurotensinergic systems was investigated by characterizing neurotensin (NT) receptors in mice (LS and SS) selectively bred for differences in sensitivity to ethanol. [3H]Neurotensin binding in brain membranes from both mouse lines was specific, saturable, reversible, and linear with protein concentrations. Subcellular localization studies showed specific NT binding to be concentrated in the microsomal/synaptosomal fractions. Scatchard analyses of [3H]NT binding indicated similar KD values for membranes from various brain regions of LS and SS mice. However, Bmax values in frontal cortex, cerebellum, and striatum were greater in SS than in LS mice. In competitive binding studies IC50 values were lower for NT8-13 than for NT1-13, and IC50 values for NT1-8, NT1-11, D-Trp11-NT, and D-Tyr11-NT were greater than 1000 nM. Association and dissociation rate constants for [3H]NT and resulting KD values (0.8 nM) were similar for LS and SS brain membranes. Ethanol, in vitro, had no effect on NT binding characteristics, but as expected various cations markedly increased KD values.     

4,8-Sphingadienine and 4-hydroxy-8-sphingenine activate ceramide production in the skin

ABSTRACT: BACKGROUND: Ingestion of glucosylceramide improves transepidermal water loss (TEWL) from the skin, but the underlying mechanism by which a small amount of dietary glucosylceramide can vastly improve skin conditions remains unclear. In a previous report, glucosylceramides were shown to be digested to sphingoids, which were shown to be absorbed through the intestinal epithelium. Based on these observations, we hypothesized that sphingoids are the key molecules facilitating endogenous ceramide production. In this study, we assessed the effect of 4,8-sphingadienine (d18:2) and 4-hydroxy-8-sphingenine (t18:1), derived from konjac glucosylceramide, on stimulating ceramide production. METHODS: Konjac glucosylceramide acidolysis was performed using hydrochloric acid; the resulting d18:2 and t18:1 were fractionated by column chromatography. Real-time quantitative RT-PCR was performed to assess the effect of d18:2 and t18:1 on gene expression in normal human epidermal keratinocytes, while their effect on the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)gamma, was measured using a receptor-cofactor assay system. The effect of d18:2 and t18:1 on stimulating ceramide production was evaluated using HPTLC analysis in a 3-dimensional human skin model. RESULTS: We noted the upregulation of genes related to de novo ceramide synthesis as well as of those encoding the elongases of very long-chain fatty acids by d18:2 and t18:1, but not by glucosylceramide and 4-sphingenine. Both these sphingoids also facilitated the expression of PPARbeta/delta and PPARgamma; moreover, they also demonstrated ligand activity for PPARgamma. These results indicated that d18:2 and t18:1 promote the differentiation of keratinocytes. Analysis of the lipids within the 3-dimensional human skin model indicated that treatment with d18:2 and t18:1 not only upregulated gene expression but also increased ceramide production. CONCLUSIONS: The sphingoids d18:2 and t18:1 activated genes related to de novo ceramide synthesis and increased ceramide production, whereas glucosylceramide and 4-sphingenine could not. These results suggest that the effect of dietary glucosylceramides on the skin is mediated by d18:2 and t18:1.     

Characterization and chronological changes of preterm human milk gangliosides

Objective

Gangliosides are present in high concentrations in the nervous tissue, and some are observed in small amounts in many extraneural tissues and body fluids. Human milk may play important roles in energy supplementation, prophylaxis of infection, and brain development. For preterm infants, human milk gangliosides are also very important substances during the early lactation stage. However, there are no data on human milk gangliosides from mothers at preterm delivery. We investigated the characterization of gangliosides and chronologic changes in human preterm milk earlier than 30 wk of gestation from 1 to 60 d after birth.

Methods

Forty-one samples were analyzed by high-performance thin-layer chromatography and a microtechnique using 1 mL of milk from each lactation and compared with 61 full-term human milk samples.

Results

Total lipid-bound sialic acid of human milk gangliosides after preterm delivery showed a peak concentration at 2 to 3 d postpartum and then remained at a high concentration until approximately 10 d. GD3 was the major ganglioside in the colostrum until approximately 7 to 10 d postpartum. GM3 was scarcely detected until 7 d postpartum and then increased gradually. There was no difference in the GD3 concentration per 1 mL of human milk between preterm and full-term human milk until approximately 5 to 8 d postpartum. After that time, the GD3 concentration decreased sharply. In contrast, the total concentrations of GM3 per 1 mL of human milk from mothers after preterm delivery were lower than those from mothers after full-term delivery throughout the entire period examined.

Conclusion

This finding is essential to elucidate the composition of human milk gangliosides after preterm delivery, which may contribute to the analysis of the physiologic composition and formulation appropriate preterm infant nutrition.     

Ganglioside composition of differentiated Caco-2 cells resembles human colostrum and neonatal rat intestine

Gangliosides are glycosphingolipids found in cell membranes and human milk with important roles in cell proliferation, differentiation, growth, adhesion, migration, signalling and apoptosis. Similar changes in ganglioside composition occur during embryonic development, lactation and cancer cell differentiation. It is not known, however, whether ganglioside compositional changes that occur in differentiating colon cancer cells reflect changes that occur during intestinal development. The Caco-2 cell line is commonly used to study physiological and pathophysiological processes in the small intestine and colon. Therefore, to examine this question, undifferentiated and differentiated Caco-2 cells were grown and total lipid was extracted from cell supernatant fractions using the Folch method. The upper aqueous phase containing gangliosides was collected and purified. Total gangliosides were measured as ganglioside-bound N-acetyl neuraminic acid, while individual ganglioside content was quantified via a colorimetric assay for sialic acid and scanning densitometry. The total ganglioside content of differentiated Caco-2 cells was 2.5 times higher compared with undifferentiated cells. Differentiated Caco-2 cells had significantly more (N-acetylneuraminyl) 2-galactosylglucosyl ceramide (GD3) and polar gangliosides, and a lower N-acetylneuraminylgalactosylglucosylceramide (GM3):GD3 ratio than undifferentiated cells. The present study demonstrates that the total ganglioside content and individual ganglioside composition of differentiated Caco-2 cells are similar to those of human colostrum and neonatal rat intestine. Differentiated Caco-2 cells may therefore be an alternative model for studying physiological and pathological processes in the small intestine and colon, and may help to elucidate possible functions for specific gangliosides in development and differentiation. Further research using more sensitive techniques of ganglioside analysis is needed to confirm these findings.     

Dietary n-3 and n-6 fatty acids alter avian metabolism: molecular-species composition of breast-muscle phospholipids

The effects of diets high in n-3 polyunsaturated fatty acids (PUFA; provided by fish oil), n-6 PUFA (sunflower oil) or in more-saturated fatty acids (tallow) on the distribution of subclasses of choline phospholipids (PC) and ethanolamine phospholipids (PE) from the breast muscle of broiler chickens were examined. Supplementation with the different fatty acids had no effect on the distribution of phospholipid subclasses. Feeding sunflower oil or tallow gave a molecular-species profile similar in both fatty acid subtype and proportion. In the diacyl PC phospholipids, 16 : 0-18 : 1n-9 and 16 : 0-18 : 2n-6 accounted for approximately 60 % of the total molecular species, whereas for the alkylenyl PC the predominant species were 16 : 0-18 : 1n-9 and 16 : 0-20 : 4n-6. Of the diacyl PE the dominant species was 18 : 0-20 : 4n-6 which accounted for 50 % of the molecular species, and of the alkylenyl PE the dominant species were 16 : 0-18 : 1n-9, 16 : 0-20 : 4n-6 and 18 : 0-20 : 4n-6. Supplementation with fish oil significantly increased levels of both eicosapentaenoic acid (20 : 5n-3) and docosahexaenoic acid (22 : 6n-3) in PC and PE when compared with either sunflower oil or tallow supplementation. The increase in the n-3 PUFA incorporation was associated with a corresponding decrease in the proportion of arachidonic acid (20 : 4n-6) in both PC and PE. Different dietary fats induce different patterns of fatty acid incorporation and substitution in the sn-2 position of the diacyl and alkylenyl PC and PE of avian breast muscle, and this finding is indicative of selective acyl remodelling in these two phospholipids.     

Differential expression of the Wnt/β-catenin pathway in the genital tubercle (GT) of fetal male rat following maternal exposure to di-n-butyl phthalate (DBP)

Di-n-butyl phthalate (DBP) is one of the most abundantly produced endocrine disruptors that leaches out from polyvinyl chloride plastics and can cause hypospadias in male rats during maternal exposure. The objective of this study was to first explore the roles of Wnt/β-catenin pathway in the fetal rat genital tubercle (GT) following in-utero exposure to DBP. Timed-pregnant rats were given DBP by gastric intubation at a dose of 750?mg/kg body weight (bw)/day from gestation day (GD) 14 to GD18 to establish a rat model of hypospadias. On GD19, genital tubercle down-regulation of β-catenin, Phospho-GSK-3β, and up-regulation of GSK-3β (glycogen synthase kinase-3β), NFκB in fetal male rats was observed by western blot analysis. β-catenin was located in the urethral plate epithelium (UPE). Immunochemistry showed that the relative expression of β-catenin decreased in the DBP-treated fetal rat GT compared to the normal control. These findings, for the first time, indicate that DBP may affect the development of GT by down-regulating the Wnt/β-catenin pathway in fetal male rats.     

One year of oral calcium supplementation maintains cortical bone density in young adult female distance runners

We conducted a double-blind, placebo-controlled, randomized trial to determine whether 1 year of supplemental calcium intake would augment hip [greater trochanter, GT, femoral neck (FN), total hip (TH)], spine (LS), and femoral mid-shaft (Fmr) BMD in female distance runners. Twenty-three women (age: 23.7 +/- 4.7 yrs, height: 165.6 +/- 6.3 cm, weight: 55.7 +/- 6.1 kg) were randomly assigned to receive either 1000 mg/d of supplemental calcium (N = 13) or placebo tablets (N = 10) for 1 year. BMD was determined by DXA (Hologic 1000-W) and tablet compliance by self-report logs. Compliance averaged 79% and 71% for supplement and placebo groups, respectively. Calcium supplementation did not affect hip or spine BMD, but did prevent loss at the femoral mid-shaft (GT: -0.5% vs. 0.2%, FN: 0.9% vs. 1.1%, TH: -0.3% vs. 0.2%, LS: 0.3% vs. 1.2%, Fmr: 0.1% vs. -1.8%, for calcium vs. placebo, respectively). We conclude that the addition of 800 mg/d of supplemental calcium to the diet of young adult female distance runners with habitual calcium intakes of approximately 1000 mg/d, prevents cortical but not trabecular bone loss.     

Chronic ethanol effects on glycoconjugates and glycosyltransferases of rat brain

We studied the effects of a four week administration of low doses of ethanol on glycoconjugates of the synaptosomal and microsomal fraction prepared from the brain of rats aged 2 and 7 months. Synaptosomes were the more sensitive to ethanol treatment. Total lipid bound sialic acid and neutral glycolipid and glycoprotein content were significantly reduced only in the synaptosomal fraction, with greater differences in the younger age, while glycoprotein sialic acid was not affected. None of the above differences were statistically significant in the microsomal fraction. Ganglioside pattern was altered only in the 2 month rats, showing a reduction of GM1 and GD1a in the synaptosomal fraction and of GD1a in the microsomal fraction. UDP-Gal: asialo-mucin galactosyltransferease, UDP-Gal: GlcCer galactosyltransferase, and UDP-Gal: GM2 galactosyltransferase activities were decreased and could account for the observed modifications in glycoconjugate content and distribution.     

The effect of the anti-progestin RU 486 on early pregnancy in the long-tailed macaque (Macaca fascicularis)

The efficacy of various doses of RU 486 in terminating pregnancy before and after the luteal-placental shift (LPS) in the long-tailed macaque (Macaca fascicularis) was assessed through sonographic examination and measurements of steroid hormones and their metabolites. Intramuscular injection of 1.0, 2.5, 12.5, or 25.0 mg/kg was administered either from gestational day (GD) 15-18 (Group 1; N = 11) or GD 23-26 (Group 2; N = 9). The timing of treatment was determined by the detection of the preovulatory estrogen peak via daily urinary estrone conjugate (E1C) measurements. In Group 1, a 90.9% pregnancy loss was observed (10/11); seven animals aborted during GD 15-20, two animals indicated early embryonic death with retained gestational sacs, one animal aborted on GD 56, and one pregnancy was maintained. In Group 2, an 88.9% pregnancy loss was observed (8/9); eight animals aborted between GD 26-29, and one pregnancy was unaffected. Hormone profiles appeared to fall secondarily to the loss of trophoblast function. These results indicate: (a) RU 486 was more effective after the LPS; and (b) the primary effect of RU 486 appeared to be at the level of the products of conception.     

Medium-chain fatty acids: evidence for incorporation into chylomicron triglycerides in humans

The purpose of this study was to evaluate the fatty acid composition of chylomicron triglycerides isolated from subjects fed liquid-formula diets containing 40% of total energy as medium- (C8:0 and C10:0) or long-chain (C16-C18) triglycerides (MCT, LCT) for 6 d. Medium-chain fatty acids (MCFA) comprised 8% of total chylomicron triglyceride fatty acids after the first MCT meal. After 6 d of continued MCT feeding, chylomicron triglyceride MCFA content increased to 13%. When subjects were fed the LCT (soybean oil) diet, C16:0, C18:1, and C18:2 comprised nearly 90% of the chylomicron triglyceride fatty acids. The mass of triglyceride transported in chylomicrons isolated from subjects fed the MCT diet was approximately 20% of that found when subjects consumed the LCT diet. We conclude that although total triglyceride production during MCT ingestion is low, the chylomicron triglycerides that are synthesized contain significant amounts of MCFA.     

Ethanol-induced inhibition of GABA release from LS and SS mouse brain slices

The effects of ethanol on K+-stimulated and spontaneous release of gamma-aminobutyric acid (GABA) were studied in cortical and cerebellar brain slices obtained from long-sleep (LS) and short-sleep (SS) mice. A superfusion technique was used. Tissue slices were perfused with ethanol (0-515 mM) for 20 min. Ethanol inhibited K+-stimulated 3H-GABA release at lower concentrations in LS cortical slices than in SS slices. Little or no inhibition of K+-stimulated 3H-GABA release was seen in both LS and SS cerebellar slices. The spontaneous release of 3H-GABA was inhibited to equal degrees in LS and SS cerebellar slices but was unaffected in cortical slices, at the concentrations used. The possibility that ethanol inhibits 3H-GABA release by stimulating prostaglandin production was partially tested by assessing the effects of prostaglandin F2 alpha (PGF2 alpha) on 3H-GABA release in cortical and cerebellar slices. No effect of PGF2 alpha on 3H-GABA release was seen in either brain region. These results support the notion that ethanol may elicit some of its actions by inhibiting neurotransmitter release. This effect appears to be influenced by genotype and brain region.     

The role of dietary gangliosides on immunity and the prevention of infection

Gangliosides are acid glycosphingolipids widely distributed in most vertebrate tissues and fluids. They are present in mammalian milk, where they are almost exclusively associated with the membrane fraction of the fat globule. In human milk, the content and individual distribution of gangliosides changes during lactation, GD3 being the most abundant ganglioside in colostrum, while in mature milk, GM3 is the major individual species. Gangliosides function as "unintended" target receptors for bacterial adhesion in specific tissues. After oral administration, they can be putative decoys that interfere with pathogenic binding in the intestine, this being the main mechanism by which these compounds can prevent infection. Ganglioside-supplemented infant formula has been reported to modify the intestinal ecology of preterm newborns, increasing the Bifidobacteria content and lowering that of Escherichia coli. In addition, the influence of dietary gangliosides on several parameters related to the development of intestinal immune system, such as cytokine and intestinal IgA production, has also been described in animal models. Recently, the influence of GM3 and GD3 on dendritic cell maturation and effector functionalities has also been reported, suggesting a role for these milk gangliosides, especially GD3, in modulating the process of oral tolerance during first stages of life. In summary, dietary gangliosides may have an important role in the modification of intestinal microflora and the promotion of intestinal immunity development in the neonate, and consequently in the prevention of infections during early infancy.     

Developmental profile of hepatic alcohol and aldehyde dehydrogenase activities in long-sleep and short-sleep mice

Ethanol is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (ADH). The product, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (AHD). Mouse liver contains five major constitutive AHD isoenzymes: mitochondrial high Km (AHD-1), mitochondrial low Km (AHD-5), cytosolic high Km (AHD-7), cytosolic low Km (AHD-2) and microsomal high Km (AHD-3). The Long-Sleep (LS) and Short-Sleep (SS) mice differ in their sleep time response to ethanol as early as 10 days of age, and this difference increases with increasing age. Age- and genotype-related differences in metabolism could account for the pattern of responses seen in these mice. We measured the activity of hepatic ADH and the five AHD isoenzymes in LS and SS mice from 3 days of age to adulthood to determine if there were differences in the developmental profiles of these enzyme activities. We found no sex differences in the developmental profile of either ADH or AHD, and the LS and SS mice have nearly identical ADH and AHD activities with the possible exception of the high Km mitochondrial enzyme activity between days 3 and 6, and the low Km mitochondrial enzyme between days 28 and 32. Thus, it appears that differences in ethanol or acetaldehyde metabolism do not contribute significantly to the differential sensitivity to ethanol between young LS and SS mice or to the differential sensitivity between young and adult mice.