[Method of quantitative analysis by HPLC and confirmation by LC-MS of sugar alcohols in foods].

A reliable quantitative determination method of sugar alcohols, D-mannitol, xylitol and D-sorbitol, in food samples by HPLC, and a simple confirmation method by LC-MS were developed. Quantitative HPLC analysis was performed using a separation column packed with polystyrene cation exchange resin of sulfonic acid type, and with pure distilled water as the mobile phase. This column, operated at 0.85 mL/min flow rate of mobile phase and 50℃ column oven temperature, completely separated the three sugar alcohols. Further, these three sugar alcohols were well separated from erythritol and other sugars (sucrose, D-glucose, D-xylose and D-fructose). Recoveries of the three sugar alcohols spiked into food samples, such as orange juice, yogurt, chewing gum and milk, exceeded 91% and the values of coefficient of variance were below 3.1%. A triple extraction process with 80% ethanol was needed for biscuit to achieve recoveries exceeding 82%. LC-MS was carried out on a NH2 column with acetonitrile-water (9 : 1) as the mobile phase, and this afforded partial but acceptable separation of the three sugar alcohols with in 10 minutes. Ion peaks derived from [M-H](-) and [M+Cl](-) were clearly detected for all three sugar alcohols in the negative electrospray inization mode at 30 V cone voltage. The positive electrospray ionization mode produced the ions [M+Na](+) and [M+Na+CH3CN](+). These characteristic ions served to confirm the presence of the sugar alcohols in food samples.

[Structural analyses of unknown red dyes detected in dried strawberry].

We examined two unknown red dyes (designated as red dyes "A" and "B") from a dried strawberry package with a label that indicated the presence of food red No. 40 (R40). Red dye "A" was identified as trisodium 3-hydroxy-4-[(2'-methoxy-5'-methyl-4'-sulfonatophenyl)azo]-2,7-naphthalenedisulfonate (CSA-R) by HPLC, UV-VIS spectra and MS spectra. This compound is one of the four reported subsidiary colors of R40. Detailed analyses of red dye "B" by MS and NMR demonstrated that its structure was disodium 3-hydroxy-4-[(2'-methoxy-5'-methyl-4'-sulfonatophenyl)azo]-2-naphthalenesulfonate. Red dye "B" is a structural isomer of R40, that has not been reported previously. Our results suggest that the two minor red dyes were subsidiary colors contained in R40, which had been added to the dried strawberries.

Preparation of (13)C-labeled ceramide by acetic acid bacteria and its incorporation in mice.

We prepared 2-hydroxypalmitoyl-sphinganine (dihydroceramide) labeled with a stable isotope by culturing acetic acid bacteria with (13)C-labeled acetic acid. The GC/MS spectrum of the trimethylsilyl derivative of (13)C-labeled dihydroceramide gave molecular ions with an increased mass of 12-17 Da over that of nonlabeled dihydroceramide. The fragment ions derived from both sphinganine base and 2-hydroxypalmitate were confirmed to be labeled with the stable isotope in the spectrum. Therefore, (13)C-labeled dihydroceramide can be an extremely useful tool for analyzing sphingolipid metabolism. The purified [(13)C]dihydroceramide was administered orally to mice for 12 days, and the total sphingoid base fractions in various tissues were analyzed by GC/MS. The spectrum patterns specific to (13)C-labeled sphingoids were detected in the tissues tested. Sphinganine pools in skin epidermis, liver, skeletal muscle, and synapse membrane in brain were replaced by [(13)C]sphinganine at about 4.5, 4.0, 1.0, and 0.3%, respectively. Moreover, about 1.0% of the sphingosine pool in the liver was replaced by [(13)C]sphingosine, implying that exogenous dihydroceramide can be converted to sphingosine. These results clearly indicate that ingested dihydroceramide can be incorporated into various tissues, including brain, and metabolized to other sphingolipids.

Acetyl-L-carnitine improves aged brain function.

The effects of acetyl-L-carnitine (ALCAR), an acetyl derivative of L-carnitine, on memory and learning capacity and on brain synaptic functions of aged rats were examined. Male Fischer 344 rats were given ALCAR (100 mg/kg bodyweight) per os for 3 months and were subjected to the Hebb-Williams tasks and AKON-1 task to assess their learning capacity. Cholinergic activities were determined with synaptosomes isolated from brain cortices of the rats. Choline parameters, the high-affinity choline uptake, acetylcholine (ACh) synthesis and depolarization-evoked ACh release were all enhanced in the ALCAR group. An increment of depolarization-induced calcium ion influx into synaptosomes was also evident in rats given ALCAR. Electrophysiological studies using hippocampus slices indicated that the excitatory postsynaptic potential slope and population spike size were both increased in ALCAR-treated rats. These results indicate that ALCAR increases synaptic neurotransmission in the brain and consequently improves learning capacity in aging rats.

Effect of continuous ingestion of acetic Acid bacteria on memory retention and the synaptic function in aged rats.

We administered Acetobacter malorum NCI1683 (S24), containing a high concentration of dihydroceramide (7.2 mg/g of dry cell weight), consecutively to aged rats (male Crlj:Wistar rats, 22 months old). The ingestion of Acetobacter malorum for 89 d significantly extended the memory retention in passive avoidance tests, increased the release of acetylcholine with depolarization of brain synaptosomes and decreased the causative agents of neurodegenerative diseases in the cerebral cortices.

Acetic acid bacterial lipids improve cognitive function in dementia model rats.

Acetic acid bacteria, fermentative microorganisms of traditional foods, have unique alkali-stable lipids (ASL), such as dihydroceramide which is a precursor of sphingolipids. Sphingolipids are important components of the brain tissue. We examined the effect of oral administration of ASL in a rat model of dementia (7-week-old, male) with a basal forebrain lesion. In a water maze test, the dementia model rats demonstrated poor spatial orientation. The administration of ASL (165 or 1650 mg/kg of body weight per day, for 14 days) produced a significant improvement in learning ability in the dementia model rats. In vitro experiments showed ASL had the ability to promote neurite outgrowth in pheochromocytoma (PC12) cells. Among the ASL components, dihydroceramide has the most potent effect on the differentiation of PC12 cells. It is highly possible that oral administration of dihydroceramide-containing ASL reverses the decline in cognitive function in dementia.

Vitamin C is not essential for carnitine biosynthesis in vivo: verification in vitamin C-depleted senescence marker protein-30/gluconolactonase knockout mice.

Carnitine is an essential cofactor in the transport of long-chain fatty acids into the mitochondrial matrix and plays an important role in energy production via beta-oxidation. Vitamin C (VC) has long been considered a requirement for the activities of two enzymes in the carnitine biosynthetic pathway, i.e., 6-N-trimethyllysine dioxygenase and gamma-butyrobetaine dioxygenase. Our present study using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo, led to the conclusion that this notion is not true. After weaning at 40 d of age, SMP30/GNL KO mice were fed a diet lacking VC and carnitine, then given water containing 1.5 g/l VC (VC(+) mice) or no VC (VC(-) mice) for 75 d. Subsequently, total VC and carnitine levels were measured in the cerebrum, cerebellum, liver, kidney, soleus muscle, extensor digitorum longus muscle, heart, plasma and serum. The total VC levels in all tissues and plasma from VC(-) SMP30/GNL KO mice were negligible, i.e., <2% of the levels in SMP30/GNL KO VC(+) mice; however, the total carnitine levels of both groups were similar in all tissues and serum. In addition, carnitine was produced by incubated liver homogenates from the VC-depleted SMP30/GNL KO mice irrespective of the presence or absence of 1 mM VC. Collectively, these results indicate that VC is not essential for carnitine biosynthesis in vivo.

Mass spectrometric studies on brain metabolism, using stable isotopes.

In fields related to biomedicine, mass spectrometry has been applied to metabolism research and chemical structural analysis. The introduction of stable isotopes has advanced research related to in vivo metabolism. Stable-isotope labeling combined with mass spectrometry appears to be a superior method for the metabolism studies, because it compensates for the shortcomings of conventional techniques that use radioisotopes. Biomolecules labeled with stable isotopes have provided solid evidence of their metabolic pathways. Labeled large molecules, however, cannot homogeneously mix in vivo with the corresponding endogenous pools. To overcome that problem, small tracers labeled with stable isotopes have been applied to in vivo studies because they can diffuse and attain a homogeneous distribution throughout the inter- and intracellular spaces. In particular, D(2)O-labeling methods have been used for studies of the metabolism in different organs, including the brain, which is isolated from other extraneural organs by the blood-brain barrier (BBB). Cellular components, such as lipids, carbohydrates, proteins, and DNA, can be endogenously and concurrently labeled with deuterium, and their metabolic fluxes examined by mass spectrometry. Application of the D(2)O-labeling method to the measurements of lipid metabolism and membrane turnover in the brain is described, and the potential advantages of this method are discussed in this review. This methodology also appears to have the potential to be applied to dynamic and functional metabolomics.

Age-related changes in the levels of voltage-dependent calcium channels and other synaptic proteins in rat brain cortices.

Neurotransmitter release from synapses is one of the most important interneuronal signaling in the nervous system. We previously reported that aging decreases depolarization-induced acetylcholine release in rat brain synaptosomes. To investigate the mechanisms underlying the age-related decrements of neurotransmission, we determined the levels of the alpha1 subunit proteins of voltage-dependent calcium channels (VDCCs) and three synaptic proteins that relate to exocytotic processes using synaptosomes prepared from cerebral cortices of young (6-month-old) and aged (27-month-old) rats. Immunoblotting analyses revealed that the protein levels of alpha1A (P/Q-type) and alpha1B (N-type) subunits in aged rats were 38% and 43% lower than the levels of young rats, respectively, but the levels of the alpha1C (L-type) subunit were not different between young and aged. On the contrary, the levels of synaptotagmin-1, synaptophysin and syntaxin were not significantly different between the two age groups in the synaptosomal preparations. These results suggest that synaptic density does not change much in the cerebral cortex in normal aging, and that the reduction of P/Q-type and N-type VDCCs, both of which participate in neurotransmitter release, is one of the causes for the decrease of neurotransmission at aged synapses.

Synaptic function of cholinergic-specific Chol-1alpha ganglioside.

The function of a cholinergic-specific ganglioside, Chol-1alpha, was investigated. The release of acetylcholine from synaptosomes was inhibited by anti-Chol-1alpha monoclonal antibody but not by monoclonal antibodies against other brain gangliosides tested. Chol-1alpha ganglioside stimulated the high-affinity choline uptake by synaptosomes and consequently enhanced acetylcholine synthesis, resulting in an increased release of acetylcholine from synaptosomes. The memory and learning abilities of rats given anti-Chol-1alpha antibody were remarkably suppressed. These in vitro and in vivo studies suggest that Chol-1alpha ganglioside plays a pivotal role in cholinergic synaptic transmission and participates in cognitive function.

alpha-Sialylcholesterol enhances the depolarization-induced release of acetylcholine and glutamate in rat hippocampus: in vivo microdialysis study.

The effects of alpha-sialylcholesterol (alpha-SC), a synthetic ganglioside analogue, on synaptic neurotransmission were studied using in vivo microdialysis technique. Application of alpha-SC through a microdialysis probe enhanced high potassium-evoked release of acetylcholine and glutamate in the hippocampal CA3 region of Wistar rats. The experiments using synaptosomes and FM1-43, a fluorescent styryl dye used for studies of neurotransmitter release mechanisms, showed that alpha-SC increased depolarization-induced loss of dye but it did not evoke the dye loss at resting condition. These results indicate that alpha-SC promotes a depolarization-induced exocytotic neurotransmitter release in the brain under in vivo conditions. Application of alpha-SC increased the level of glutamate but not that of acetylcholine, suggesting that alpha-SC affects spontaneous glutamate release and/or transport system at the brain region.

Acetyl-L-carnitine supplementation restores decreased tissue carnitine levels and impaired lipid metabolism in aged rats.

The effects of long-term carnitine supplementation on age-related changes in tissue carnitine levels and in lipid metabolism were investigated. The total carnitine levels in heart, skeletal muscle, cerebral cortex, and hippocampus were approximately 20% less in aged rats (22 months old) than in young rats (6 months old). On the contrary, plasma carnitine levels were not affected by aging. Supplementation of acetyl-l-carnitine (ALCAR; 100 mg/kg body weight/day for 3 months) significantly increased tissue carnitine levels in aged rats but had little effect on tissue carnitine levels in young rats. Plasma lipoprotein analyses revealed that triacylglycerol levels in VLDL and cholesterol levels in LDL and in HDL were all significantly higher in aged rats than in young rats. ALCAR treatment decreased all lipoprotein fractions and consequently the levels of triacylglycerol and cholesterol. The reduction in plasma cholesterol contents in ALCAR-treated aged rats was attributable mainly to a decrease of cholesteryl esters rather than to a decrease of free cholesterol. Another remarkable effect of ALCAR was that it decreased the cholesterol content and cholesterol-phospholipid ratio in the brain tissues of aged rats. These results indicate that chronic ALCAR supplementation reverses the age-associated changes in lipid metabolism.

Turnover of myelin lipids in aging brain.

Turnover rates of myelin membrane components in mouse brains were determined by a method using stable isotope-labeling and mass spectrometry. The half-replacement times based on incorporation rates of newly synthesized molecules for young adult mice were 359 days for cholesterol, 20 days for phosphatidylcholine, 25 days for phosphatidylethanolamine, 94 days for cerebroside and 102 days for ganglioside GM1. The turnover rates of half-lives of myelin components were calculated from the decay curves of initially labeled molecules, and they were about the same as the half-replacement times. Individual components were thus revealed to be metabolized at different rates, and their turnover rates were differently affected by aging. As was observed with phospholipids, myelin pools appeared to be compartmentalized into rapidly and slowly exchanging pools. The turnover rates of cerebroside and GM1 decreased between the young and adult periods and slightly increased in senescence. The latter phenomenon may indicate an enhanced myelin turnover in senescence. The present study reveals the dynamic aspects of myelin membrane turnover during the life span of mouse.

Assessment of choline uptake for the synthesis and release of acetylcholine in brain slices by a dynamic autoradiographic technique using [11C]choline.

The uptake of choline for the synthesis and release of acetylcholine was investigated in brain slices by dynamic positron autoradiography using [11C]choline. Brain slices (330 microm) were incubated with [11C]choline in oxygenated Krebs-Ringer medium at 34 degrees C and serial two-dimensional time-resolved images of the uptake and release of radioactivity were recorded on Storage Phosphor screens. [11C]choline uptake increased with the period of incubation and was 1.9 times higher in the striatum than cerebral cortex. The uptake in the striatum was significantly diminished by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline uptake. Pretreatment of brain slices with 50 mM K(+) for 20 min enhanced the uptake in striatum. The uptake of [11C]choline in brain slices was saturable using nonlabeled choline. Two uptake systems, a high-affinity and a low-affinity system, were confirmed to exist by kinetic analysis using Lineweaver-Burk plots. The 11C radioactivity that had accumulated in the striatum disappeared on treatment with veratridine, a depolarization agent, in the presence of HC-3. This pattern of disappearance was consistent with that of the appearance of unlabeled and labeled acetylcholine in the medium. These results indicate that this method is useful for obtaining information regarding the uptake of choline for the synthesis and release of acetylcholine in live brain tissues.

Turnover of synaptic membranes: age-related changes and modulation by dietary restriction.

We examined age-related changes in the turnover rates of synaptic membrane components that might underlie the decrease in synaptic functions in senescence. Synaptic membrane constituents were labeled in vivo with deuterium and the disappearance of the deuterated molecules from synaptic membranes was measured by mass spectrometry. The turnover rates of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and synaptophysin were all shown to slow down with aging. Dietary restriction, which is known to retard various aging processes, was found to decrease the turnover rates of membrane lipid species. Consequently, the fatty acid composition in phospholipids remained unchanged in the synaptic plasma membranes of food restricted mice. In contrast, the turnover rate of synaptophysin was accelerated under dietary restriction. This may mean that increased turnover enhances the removal of damaged proteins from membranes.

Light microscopic study of the alligator (Alligator mississippiensis) spleen with special reference to vascular architecture.

The spleen of the American alligator (Alligator mississippiensis) was studied histologically. The alligator spleen is a bean-shaped organ covered by a thick capsule. The concave side of the spleen faces the pancreas. Many venous vessels are present in the capsule. The stem segment of a large intestinal artery, the lieno-intestinal artery, enters the organ from its upper pole, runs within the organ at the axial center (axial artery) and leaves it from the lower pole. Many peripheral branches originate from the axial artery towards the capsule, but the artery has no associated collateral veins. The capsule/trabecula and white and red pulp may be distinguished. The capsular veins appear to be continuous with venous vessels that sheathe the axial artery and its peripheral branches. Surrounding the axial artery are trabeculae containing leiomyocytes and nerves. The white pulp consists of lymphoid tissue and occurs around terminal arterioles and sheathed capillaries. The materials examined do not show germinal centers. The large red pulp is composed of venous vessels and splenic cords rich in reticular fibers. Two venous routes, hilar and capsular, are present. The structural characteristics of the alligator spleen are similar to spleens of other reptiles; however, its vascular architecture is primitive, suggesting that the alligator spleen may be a portal spleen. J Morphol 233:43-52, 1997. © 1997 Wiley-Liss, Inc.

Spleen of the snake (Elaphe climacophora) and intrasplenic vascular architecture.

The spleen of the blue-green snake (Elaphe climacophora) lies at the head of the pancreas and is separated from it by a fibrous capsule. A hilus is not clearly distinguished. Arteries and veins enter or leave the spleen through the capsule, but no collateral relationship is observed between these vessels. Histologically, the spleen consists of the capsule-septal tissue, lymphoid tissue (the white pulp), and a fibrous zone (the perilymphoid fibrous zone: PLFZ) around the lymphoid tissue that includes many small veins. The PLFZ probably represents the involuted red pulp. The border between the white pulp and PLFZ is unclear in routine histological sections due to diffuse infiltration of lymphocytes into the latter region, but the border could be distinguished clearly in silver-impregnated specimens. Arteries enter the spleen, run within the septa, and divide into terminal arteries in the lymphoid tissue that form end branches. There are no ellipsoids around the terminal arteries. The end branches communicate with veins of the PLFZ through transitional vessels within the lymphoid tissue (closed circulation). The veins of the PLFZ anastomose with drainage veins in the capsule. The snake spleen retains the basic histological characteristics of a spleen and is morphologically distinguishable from a lymph node. It may represent an extreme example of a spleen modified by the remodelling of the intrasplenic vasculatures during evolution. © 1995 Wiley-Liss, Inc.