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Although studies on snow algae and macroinvertebrates have been frequently conducted on snow patches, only few surveys have been focused on microinvertebrates which reach high biomass and play various trophic roles in other cold habitats. The aims of this study were (1) to search for microinvertebrates in seasonal surface snow patches located on the slope of Mt. Gassan, in northern Japan, and (2) to identify factors determining their distribution associated with snow algal blooms of various colorations (orange, green, and golden-brown) collected from the same sampling site over two seasons (2018, 2019). Microscopic observation revealed presence of two major groups of microinvertebrates: Tardigrada and Rotifera. They were concentrated in green snow colored by blooms of Chloromonas sp. in comparison to orange or golden-brown snow and only a few were found in white snow. Mean body length of tardigrades increased throughout the melt season, their intestine content was green and they laid eggs on colored snow. These results suggest that tardigrades preferentially grew and reproduced on green snow patches. Population densities of tardigrades, rotifers and concentration of chlorophyll a were significantly correlated. Our study indicates that green snow patches in temperate mountainous forests constitute important and unique low-temperature ecosystems for microinvertebrates. Snow covered by algae is an unrecognized novel habitats for tardigrades and rotifers.

Snow algae are photosynthetic microbes that inhabit the melting snow surface in alpine and polar regions. We analyzed the pigment and species composition of colored snow collected on Mt. Tateyama in Japan during the melting seasons of 2015 and 2016. High-performance liquid chromatographic analyses of the pigments extracted from the colored snow showed that their composition varied within the study area and were classified into four types: Type A (astaxanthin-monoester dominant), Type B (medium astaxanthin-monoester content), Type C (abundant primary carotenoids and free-astaxanthin), and Type D (abundant primary carotenoids and astaxanthin diesters). Types A and B were most commonly observed in the study area, whereas Types C and D appeared only at specific sites. Analysis of the 18S ribosomal RNA (18S rRNA) gene revealed six major amplicon sequence variants (ASVs) of snow algae, belonging to the Sanguina, Chloromonas, and Chlainomonas groups. The relative abundance of the algal ASVs showed that Sanguina was dominant (>48%) in both Types A and B, suggesting that the difference in astaxanthin abundance between the two types was caused by the production of pigments in the algal cells. The algal community structures of Types C and D differed from those of Types A and B, indicating that the primary carotenoids and astaxanthin diesters were derived from certain algal species in these types. Therefore, astaxanthin-rich Sanguina algae mostly induced the red snow that appeared widely in this alpine area; however, they were partially dominated by Chloromonas or Chlainomonas algae, causing different pigment compositions.

To understand the geological origins of minerals in cryoconite and the nutrients sources for microbes on glaciers, we analyzed the Sr–Nd isotopic ratios of the four mineral fractions in cryoconites including saline, carbonate, phosphate, silicate and the organic fraction obtained from Gulkana Glacier in Alaska. The isotopes in the silicate mineral fraction exhibited spatial variation within the glacier (87Sr/86Sr: 0.704533–0.709563, εNd (0): −16.0 to 0.5), which can be explained by the different mixing ratios of the two distinct sources: one of the sources is lateral and terminal moraines or soil, and the other is the medial moraine of the glacier. The minerals in the cryoconite at the lower sites in the glacier are likely derived from the former source, whereas those at the upper sites are from latter sources. The mineralogical and elemental compositions also support mixing of the silicate minerals from the two local sources. The Sr isotopic ratios of the organic fraction also showed spatial variation on the glacier in the middle sites – a trend similar to those of the phosphate fraction. The results suggest that the organic matter is mostly the byproducts of microbes using the phosphate minerals as a nutrient source.

Snow algae are photosynthetic microbes growing in thawing snow. They usually show various morphological cell types. The aim of this study was to carry out microscopic and spectroscopic analysis of different forms of cells of snow algae collected on glaciers in Alaska. Four different shapes of algal cells were observed with the use of bright field LM (Light Microscopy), DIC (Differential Interference Contrast), EDF (Extended Depth Focus), fluorescence microscopy, and SEM (Scanning Electron Microscopy). The cells exhibited the strongest autofluorescence after the exposure to 365-nm excitation light, and the intensity differed among the cell types. Zygotes (cysts) showed the most intense fluorescence. Acridine orange staining revealed the acid nature of the algal cells. The use of Congo red and Calcofluor white fluorochromes indicated differences in the structure of polysaccharides in the cell wall in the individual types of algal cells. FTIR (Fourier-Transform Infrared Spectroscopy) analyses showed the presence of polysaccharides not only in the algal cells but also in the fixative solution. The presence of polysaccharides in the extracellular algal fraction was confirmed by X-ray dispersion spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy imaging (SEM). The differences observed in the structure of the cell wall of the different forms of red snow algae prompt further analysis of this structure.

Cryoconite granules are dark-colored biological aggregates on glaciers. Bacterial community varies with granule size, however, community change in space and their susceptibility to environmental factors has not been described yet. Therefore, we focused on bacterial community from four different granule sizes (30–249 μm, 250–750 μm, 750–1599 μm, more than 1600 μm diameter) in 10 glaciers in northwestern Greenland and their susceptibility to exogenous nutrients in cryoconite hole. A filamentous cyanobacterium Phormidesmis priestleyi, which has been frequently reported from glaciers in Arctic was abundant (10%–26%) across any size of granules on most of glaciers. Bacterial community across glaciers became similar with size increase, and whence smallest size fractions contain more unique genera in each glacier. Multivariate analysis revealed that effect of nutrients to beta diversity is larger in smaller granules (30–249 μm and 250–750 μm diameter), suggesting that bacterial susceptibility to nutrients changes with growth of granule (i.e. P. priestleyi was affected by nitrate in early growth stage).

Cryoconite granules, which are dark-colored biological aggregates on glaciers, effectively accelerate the melting of glacier ice. Bacterial community varies with granule size, however, community change in space and their susceptibility to environmental factors has not been described yet. Therefore, we focused on bacterial community from 4 different granule sizes (30-249 μm, 250- 750 μm, 750-1599 μm, more than 1600 μm diameter) in 10 glaciers in northwestern Greenland and their susceptibility for exogenous nutrients in cryoconite hole. A filamentous cyanobacterium Phormidesmis priestleyi, which has been frequently reported from glaciers in Arctic was abundant (10-26%) across any size of granules on most of glaciers. Bacterial community across glaciers became similar with size increase, and whence smallest size fractions contain more unique genera in each glacier. Multivariate analysis suggests that phosphate, which is significantly higher in one glacier (Scarlet Heart Glacier), is primary associated with bacterial beta diversity. Correlation coefficients between abundance of major genera and nutrients largely changed with granule size, suggesting that nutrients susceptibility to genera changes with growth process of granule (e.g. P. priestleyi was affected by nitrate in early growth stage).

Glacial meltwater samples were collected from 15 supraglacial ponds at elevations ranging between 3989 and 4292 m above sea level in the debris-covered area of Lirung glacier in central Nepal Himalayas, from November 2010 to October 2011 on a bimonthly basis. We examined elevation trends, and their control by seasonality, dominant geochemical processes, and seasonal dynamics in these supraglacial ponds. Concentration of some parameters showed a decreasing trend with elevation but with high variability and modulation by seasons, being more prevalent in the post-monsoon. Sulfate is the only parameter showing significant elevation trends regardless of the season, although the trend is also affected by the monsoon. The concentrations of major chemical solutes were highest during post-monsoon season and lower in the pre-monsoon and monsoon seasons, indicating influence of seasonality on dissolution, dilution, and hydrological processes. Contribution to chemical loads from marine aerosols to these pond water appeared to be minor except for sodium and magnesium. Chemical weathering of carbonate and silicate minerals fueled by dissolution of pyrite appeared as the dominant geochemical processes.

The mineralogical composition of cryoconite on a glacial surface was investigated on six glaciers in northwest Greenland (Qaanaaq Qaqortaq Tugto Bowdoin Sun and Scarlet Heart. The X‑ray diffraction analysis showed that the cryoconites mainly contained seven silicate minerals: hornblende quartz potassium feldspar plagioclase illite kaolinite and chlorite. Semi‑quantitative mineralogical analysis of the silicate mineral composition on the Qaanaaq Glacier showed little variation among the samples collected from five different elevations. This indicates that the minerals on the glacier were probably dominated by dust from a unique source which is recently transported from local sediments including soil and moraine. On the other hand the mineral composition varied significantly among the glaciers. Based on the clay mineral content the glaciers could be classified into three groups. Type A: high clay mineral content composed of illite and kaolinite found on Qaanaaq Qaqortaq and Tugto; Type B: high clay mineral content composed of only kaolinite found on Sun; and Type C: lower clay mineral content composed of only kaolinite found on Bowdoin and Scarlet Heart. The geographical distribution of the types of glaciers did not correspond with the geology in this area indicating that the mineralogical composition is not determined just by the geologi‑ cal conditions around glaciers but also by other factors such as the redistribution of sediments by glacial fluvial or coastal processes.

Stable-isotopic ratios of strontium (Sr), neodymium (Nd) and lead (Pb) provide a means of identifying a geological source of substances and are used as tracers of elements in biological and geochemical processes. We analyzed these isotopic ratios of surface dust (cryoconite) collected on Ürümqi glacier No. 1 , Tien Shan, China. The dust was separated chemically into five fractions (four minerals and organic matter), and the isotopic ratios of each fraction were measured. The Sr and Nd isotopic ratios in the fractions extracted with ultrapure water (saline minerals), hydrogen peroxide solution (organic matter) and acetic acid (carbonate minerals) were low and invariable, whereas those extracted by hydrochloric acid (phosphate minerals) and the residual fraction (silicate minerals) were higher. The difference was likely due to the original source of each fraction. The isotopic ratios of the surface dust collected from different sites showed no significant difference, suggesting that they were spatially uniform across the glacier. The isotopic ratios of the silicate fraction were closer to those of desert sand reported in China than those of the soil and bedrock around the glacier. This suggests that the silicate minerals on the glacier were derived from distant deserts. The isotopic ratios in saline, carbonate and phosphate fractions were also close to those of evaporites and apatite in that desert region, suggesting that these minerals were also derived from that source. The Sr isotopic ratios in the organic fraction were closer to ratios in the saline and carbonate fractions rather than the silicate or phosphate fractions and may therefore reflect the isotopic ratios of the elements when they are incorporated into living microbes on the glacier.

We have developed a new ELISA for quantifying N-acetyl-β-d-glucosaminidase (NAG) isoenzyme B in human urine after raising monoclonal antibodies against the isoenzyme from human placenta. Though the obtained antibodies reacted not only to isoenzyme B but also to A, we could detect isoenzyme B selectively by a two-step sandwich ELISA with a pair of selected antibodies at low pH in the first reaction. The detected limit was 0.5 μg/L for a sample volume of 25 μL. Within-run CVs ranged from 2.5% to 5.4% and between-run CVs ranged from 6.2% to 9.1%. Recoveries of NAG isoenzyme B added to each of three urine samples ranged from 91% to 114%. The dilution curves of urine samples showed good linearity. The cross-reactivity of NAG isoenzyme A was practically negligible (2–3%). The mean value for NAG isoenzyme B in spot urines from healthy adults was 2.9 μg/g creatinine. This ELISA method is rapid and precise enough for routine determination of NAG isoenzyme B in human urine.

Mouse complement component C7 was purified from serum by a sequential procedure of fractionation precipitation by ammonium sulfate, followed by DE-52 anion exchange chromatography, Protein G affinity column chromatography, Mono S cation exchange chromatography and Superdex 200 gel filtration. The final product contained a highly purified mouse C7 component showing a single band on SDS-PAGE at the apparent Mrs of 90 kDa and 100 kDa under non-reduced and reduced conditions respectively. The yield of C7, which was measured by the biological activity, was 7.0%.

Rats with alloxan-induced diabetes developed severe atherosclerotic lesions when they were maintained on a 0.25% cholesterol diet for one year. The atheromatous changes developed at the aortic arch, appeared as early as 3 months after the start of the experiment, and increased thereafter. The diabetic rats also developed atherosclerosis when they were fed standard rat chow, but the area of the atheromatous lesion was about one tenth of that in rats fed the high-cholesterol diet. Normal rats did not develop atherosclerosis even when fed the high-cholesterol diet for one year. The alloxan diabetic rats showed no increase in body weight, but developed serum glucose levels as high as 600-800 mg/dl as well as high serum cholesterol levels and lower serum HDL-cholesterol levels. The development of atherosclerosis in these rats was significantly related to an increase in the serum cholesterol/phospholipid ratio, the atherogenic index (TC-HDLC/HDLC), and the serum total cholesterol level, but was not related to the serum glucose, HDL-cholesterol, triglyceride, or lipid peroxide levels. These relationships were found as early as B-16 weeks after the start of the experiment. These data suggest that the serum cholesterol/phospholipid ratio, the atherogenic index, and the total cholesterol level are important risk factors for the development of atherosclerosis in rats with alloxan diabetes.

In vitro glycation of very low density lipoprotein (VLDL) reduced the susceptibility to lipoprotein lipase (LPL) as the level of glycation increased. Addition of reduced glutathione to an incubation medium of serum and glucose interfered with glycation of serum proteins when the concentration of reduced glutathione was higher than 10 mM. At concentrations higher than 25 mM, it also significantly prevented the glycation induced reduction of fatty acid releases from VLDL by LPL. There were no such effects on glycation of serum protein and the fatty acid release from the addition of aminoguanidine. By contrast, addition of D-lysine enhanced glycation of serum proteins by glucose and further decreased fatty acid release from VLDL by LPL. From these results, it is suggested that glycation of VLDL decreases the susceptibility of VLDL to LPL. Delayed catabolism of VLDL in diabetic patients is considered partly caused by glycation of apoproteins, which renders VLDL less sensitive to LPL, in addition to the decreased LPL activity in diabetes mellitus.

Alloxan-diabetic rats were kept on an ordinary or 0.25% cholesterol supplemented diet for up to 12 months, and changes in the serum lipid and lipoprotein levels were examined. The diabetic rats showed IRI levels of 3-5 IU/ml, which were 1/5-1/10 that of the normal rats, and about 10 times higher serum glucose levels than the normal rats. The diabetic rats consumed 1.5-2 fold amounts of diet but their body weights were almost constant. The diabetic rats showed marked hyperlipidemia, especially in the cholesterol and triglyceride levels. The phospholipid level also increased but for a lesser extent and the c/p ratio increased. The serum lipoprotein levels increased in the diabetic rats, 5-10 times in the ordinary diet fed groups and 10-20 times in the cholesterol diet fed groups. Chylomicron, VLDL, IDL and LDL increased but HDL decreased. These changes were more significant in the diabetic rats fed the cholesterol diet. The triglyceride levels in the lipoprotein fractions increased in the diabetic rats, but the increase in cholesterol was more significant and the relative composition ratios of triglyceride decreased. The composition ratios of phospholipids, however, remained almost constant in all the lipoprotein fractions. The increase in IDL, especially cholesterol in the IDL fraction, was remarkable in the diabetic rats. The responsiveness of the diabetic rat VLDL to lipoprotein lipase (release of free fatty acids) was less than a half of that of the normal rat VLDL. In conclusion, the diabetic rats showed an increase in triglyceride but more significant increase in cholesterol in the lipoprotein fractions. These changes in serum lipid and lipoprotein levels and compositions appeared as early as 3 months after the alloxan injection and lasted for 12 months without any progress in the disturbance.

In order to clarify the relationship between composition and lipolytic responses to lipoprotein lipase (LPL), very low density lipoproteine (VLDL) from rats or humans were incubated with a commercially available LPL or with a partially purified LPL from postheparin human plasma and fatty acids released from VLDL were determined in vitro. VLDL from rats fed a diet containing 0.25 cholesterol for 6 months were rich in cholesterol and poor in triglycerides, and released less fatty acids from incubation with LPL than those from control rats. VLDL from normo- and hypertriglyceridemic human subjects were incubated with LPL. The fatty acid release poorly correlated with the apoprotein ratios of VLDL, apo C-III/C-II, B/E, and C/E with the exception of apo B/C, but it correlated well with the ratio of triglyceride/either one of the surface components including total apoproteins, free cholesterol and phospholipids in VLDL or the ratio of the triglyceride/total sum of the surface components. The correlation coefficients between fatty acid release and a ratio of triglyceride/total surface components were 0.774 (using the commercially available LPL) and 0.786 (using the partially purified human LPL). The fatty acid release increased after pretreatment of VLDL with phospholipase A2. The phospholipid content of VLDL was reduced without significant changes in other VLDL components. Thus, the responses of VLDL to LPL treatment may depend mainly upon the surface: core relationship of VLDL rather than its apoprotein composition except in rare clinical cases such as apo C-II deficiency.

Les durees de vie des emissions de F A1 + et F A2 + perturbees par les impuretes de magnesium, sont tres similaires a celles de F + dans du CaO pur. De plus, elles sont tres courtes par rapport a celles de halogenures alcalins

The effect of ascorbic acid deficiency on serum and liver cholesterol, phospholipid and triglyceride levels, serum lipoprotein levels and serum lipoprotein cholesterol levels were examined in male rats with a hereditary defect in ascorbic acid synthesis (ODS rats). Male homozygotes (od/od) and male rats of their parent strain (+/+) were each divided into four treatment groups and were fed vitamin C-deficient or vitamin C-replete diets containing either 0 or 0.5% cholesterol. During the 3-wk feeding-period the ODS (od/od) rats fed the vitamin C-deficient diet gradually decreased food intake, resulting in a lower body weight than that of od/od rats given ascorbic acid. The serum cholesterol level was significantly higher in the vitamin C-deficient od/od rats fed the cholesterol diet, and it tended to be higher in those fed the control (0% cholesterol) diet, whereas the liver lipid levels remained unchanged relative to those in od/od rats fed the vitamin C-replete diet. The serum very low density lipoprotein and high density lipoprotein (HDL) cholesterol levels were lower in od/od rats fed the vitamin C-deficient diet without cholesterol, but intermediate density lipoprotein and low density lipoprotein cholesterol levels were markedly higher in the vitamin C-deficient od/od rats than in od/od rats given ascorbic acid, regardless of dietary cholesterol level. The ratio of HDL2 cholesterol to HDL3 cholesterol was also higher in the vitamin C-deficient od/od rats. The parent strain of the od/od rats (+/+) showed no change due to vitamin C deficiency. These results suggest that vitamin C deficiency delays low density lipoprotein metabolism and produces hypercholesterolemia in male od/od rats.

Cholesterol and bile acid leves were examined in young (8 weeks), middle-aged (12 months) and old (24 months) germ-free male rats, and young (8 weeks) and middle-aged (12 months) conventional male rats. The plasma cholesterol levels were higher in the aged rats, being more marked in the conventional rats. The liver cholesterol levels also increased with age and the increases were almost identical for both groups. No age-related changes were found in the biliary bile acid secretion, the pool size and distribution of bile acids in the bile, small intestine and large intestine, nor in the turnover frequency of bile acids, but the pool size in the young and middle-aged germ-free rats was much larger than that in the conventional rats. The turnover frequency was less in the germ-free rats. The bile acid synthesis presumed from the fecal bile acid excretion decreased in the aged germ-free rats but not in the conventional rats. A most remarkable age-related change was found in the bile acid composition; cholic acid increased and beta-muricholic acid derived from chenodeoxycholic acid in the rat decreased by aging, resulting in an increase of the CA/CDCA ratio (bile acids belonging to the cholic acid group/bile acids to the chenodeoxycholic acid group) in the bile, feces and pool. These results suggest that cholic acid synthesis increases while chenodeoxycholic acid synthesis is impaired by aging in rats.

Cryoconite is a dark-coloured surface dust deposited on glaciers that consists of wind-blown mineral particles, as well as organic matter derived from microbes living on glaciers. In this paper, we analyse the Sr and Nd isotopic ratios of four mineral fractions (i.e., the saline, carbonate, phosphate, and silicate mineral fractions), as well as the organic fraction, of cryoconite samples obtained from six Asian glaciers (the Altai, Pamir, Tien Shan, Qillian Shan, and Himalayan regions), and discuss their geographical variations in terms of the geological origins of the mineral particles and the biogeochemical processes on the glaciers. The silicate mineral fraction showed lower Sr and higher Nd ratios for the glaciers located to the north (Altai, 87Sr/86Sr: 0.713?490?0.715?284, ?Nd(0): ?6.4 to ?5.6), while higher Sr and lower Nd ratios for the glaciers located to the south (Himalayas, 87Sr/86Sr: 0.740?121?0.742?088, ?Nd(0): ?16.4 to ?15.7); the ratios were similar to those of desert sand, loess, and river sediments in the respective regions of the glaciers. This result suggests that the silicate minerals within the cryoconites were derived from different sources depending on the geographical locations of the glaciers. The isotopic ratios of the saline, carbonate, and phosphate mineral fractions were distinct from those of the silicate fraction, and were similar to those of evaporites and apatite deposits from the Asian deserts, but also varied geographically, indicating that they are likely to reflect their geological origin. The Sr isotopic ratios of the organic fraction were similar to those of the saline and carbonate fractions from glaciers in the central area (Tien Shan and Qillian Shan), but were higher than those of the saline and carbonate fractions, and lower than the phosphate mineral fraction, in the northern and southern areas. The ratios of organic fraction may be determined from the mixing ratio of calcium sources incorporated by microbes on the glaciers.

Serum cholesterol, triglyceride and phospholipid levels, liver cholesterol concentration, bile flow, biliary cholesterol, phospholipid and bile acid secretion rates, fecal sterol and bile acid levels and their bile acid compositions were examined in young-old parabiotic rats and compared with those in young and old control rats and young-young parabiotic rats. Bile acid composition was expressed in terms of the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio. Body weight (BW) gain decreased after parabiosis especially in old rats, but the liver weight (g/100 g BW), diet-intake, feces dry weight, liver cholesterol concentration and fecal sterol level were almost the same in all the groups. The biliary bile acid secretion rate was higher and the fecal bile acid level was lower in old rats than those in young rats but both the levels became comparable with those in young rats after parabiosis of old rats with young rats. Young rats, however, showed no changes in these levels after parabiosis. The serum cholesterol level and the biliary and fecal CA/CDCA ratios in old rats were higher than those in young rats but decreased after parabiosis with young rats, although they were still higher than those in young rats. The serum cholesterol level in young rats increased after parabiosis with old rats, but not after parabiosis with young rats, and the fecal bile acid level and the CA/CDCA ratio were not changed in either case. It is concluded from these findings that the serum cholesterol level and the CA/CDCA ratio increased with age and that these increases were prevented after parabiosis with young rats, while young rats, although their serum cholesterol level was increased, showed no increase in the CA/CDCA ratio after parabiosis with old rats.

The effects of cholesterol feeding on serum and liver cholesterol levels, fecal and biliary bile acid levels, bile acid pool size and bile acid composition were examined in 2-, 12- and 24-month-old male germ-free rats. The major bile acids in these animals were cholic and β-muricholic acids. Cholesterol feeding increased synthesis of bile acids by 3- to 4-fold, especially that of chenodeoxycholic acid (mainly β-muricholic acid in the rat), decreasing the cholic acid/chenodeoxycholic acid (CA/CDCA) ratio in all rats regardless of age, even though the CA/CDCA ratio increased as a linear function of age in both diet groups. Cholesterol feeding increased the serum cholesterol level markedly in aged rats. This hypercholesterolemia may be produced by the increase in CA/CDCA ratio in aged rats.

Wistar male rats were treated for six days with broad spectrum beta-lactam antibiotics, latamoxef, and cefotaxime. On the seventh day, the number of fecal anaerobic microbes decreased, total fecal bile acids decreased, and bile acid pools increased. Secondary bile acids such as beta-hyocholic, hyodeoxycholic, lithocholic, and deoxycholic acids decreased in the feces while the primary bile acids, cholic, beta-muricholic, and chenodeoxycholic acids, became predominant. Coprostanol, a microbial metabolite of cholesterol, also disappeared from the feces during the treatment. The cecum enlarged to almost twice the size of that in control rats, whereas the liver weight was not significantly changed. After treatment was stopped, the number of fecal microbes returned to the initial counts within a week, but restoration of bile acid and cholesterol metabolism required at least three weeks.

Normal and alloxan diabetic rats were kept on a 0.25% cholesterol diet for 12 months and the changes in serum cholesterol levels, and fecal excretion of sterols and bile acids were examined to elucidate the influence of changes in bile acid metabolism on manifestations of hypercholesterolemia and development of atheromatous lesions. Diabetic rats fed the cholesterol diet showed increases in bile acid synthesis and in the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio, and developed significant hypercholesterolemia and atheromatous lesions. In contrast, normal rats showed increased bile acids synthesis but a decreased CA/CDCA ratio after feeding with the cholesterol diet, and developed neither hypercholesterolemia nor atheromatous lesions. Fecal sterol excretion and the cholesterol/sitosterol ratio decreased in diabetic rats. Positive correlations were found between the cumulative serum cholesterol level and the atheromatous lesion area, and between the fecal CA/CDCA ratio and the serum cholesterol level, in the latter of which the correlation was higher in rats on the cholesterol diet than in those on the standard diet. These findings suggest that alteration of bile acid metabolism with increases in cholic acid synthesis and CA/CDCA ratio in diabetic rats enhances cholesterol absorption to produce significant hypercholesterolemia, which in turn leads to development of atheromatous lesions.

Bile acid synthesis from orally administered (14)C-cholesterol or from (14)C-mevalonic acid administered intravenously were examined in male and female 2-, 12- and 24-month-old rats. About 5-9% of the '4C-cholesterol administered was recovered as bile acids from the small intestine 3 days after administration. The value in the case of (14)C-mevalonic acid was about 1/10 that of (14)C-cholesterol. Major bile acids were cholic acid and beta-muricholic acid, and the formation ratio of cholic acid increased in aged rats, although no further difference was found between 12- and 24-month-old animals of either sex. Male rats formed more beta-muricholic acid than females at all ages examined. In addition, no significant difference was found in the bile acid composition between both precursors, (14)C-cholesterol and (14)C-mevalonic acid.

In some hyperlipidemic patients, low density lipoprotein (LDL) shows several peaks (polydisperse) on polyacrylamide gel disc electrophoreses, though LDL usually shows a single peak (monodisperse). In order to clarify the relationship between the LDL polydispersion and VLDL heterogeneity, LDL and VLDL were prepared from hyperlipidemic patients sera with mono- and polydisperse LDL by sequential ultracentrifugation and fractionated by gradient ultracentrifugation and their compositions were analyzed. Polydisperse LDL was rich in triacylglycerol (TG) and poor in esterified cholesterol (CE) as compared with monodisperse LDL and consisted of the lowest and the medium density subfractions when the LDL was separated into six subfractions. The monodisperse LDL was composed of a single major subfraction of a medium density. VLDL from the patients with polydisperse LDL was relatively rich in the dense and poor in the buoyant subfractions as compared with that from the patients with monodisperse LDL. The subfractions in the former contained more CE and less TG than the corresponding subfractions in the latter. There were no significant differences in the apolipoprotein compositions between those VLDLs. The results suggest that polydisperse LDL might be originated from VLDL that differs in particle sizes, densities and compositions from ordinary VLDL.

Water samples were analyzed from ponds developed within the debris-covered area of Lirung Glacier (28º 12.9’N, 86º 39.9’E; 4000 m a.s.l.) in the Himalayas of Nepal during the pre-monsoon to post-monsoon period of 1996. Major chemical species were classified into three groups based on their relationships relative to the sum of cations: conservative (SiO2, Ca2+, K+, and Alkalinity), semiconservative (Na+, Mg2+, and SO4 2-) and non-conservative (NH4 +, NO3 - and Cl-). The dominant processes determining the chemical composition of glacier pond water were sulfide oxidation coupled with carbonate dissolution and chemical weathering of aluminosilicate as indicated by the conservative and semi-conservative species. Calcium and alkalinity appeared as the dominant cation and anion, respectively, among all samples within the basin. Compared to the discharge waters at the outlet of the glacier, most of these pond waters have lower major solutes as well as alkalinity. The availability of fresh reactive minerals at the base of the glacier, coupled with higher temperature in discharge waters than in the ponds, may be the prime factors resulting in higher concentrations of most solutes in the discharge waters than in the ponds. In the ponds, higher concentrations of major solutes as well as alkalinity were observed in the monsoon than the pre-monsoon and post-monsoon seasons, suggesting the role of hydrolysis condition in chemical weathering rates. Ponds within the debris area of Lirung glacier in central Nepal Himalaya are likely to increase in importance if global warming accelerates the rate of glacial melting.

Activation and inactivation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is a key enzyme of cholesterol synthesis, were examined in young and aged rats in vitro . The reductase activity in the liver microsomes of aged rats was always lower than that of young rats at various experimental conditions in vitro . But activation and inactivation of the reductase could operate on liver microsomes in aged rats as well as in young rats. Function of hepatic cytosol activator in aged rats seemed to be identical to that in young rats.

Four erythroleukemia cell lines were established in vitro from rats with primary leukemias induced by a set of intravenous injections of 7,8,12-trimethylbenz[a] anthracene (TMBA). These cell lines were grown in Dulbecco's medium with 20% inactivated fetal calf serum; they were characterized by specific consistent chromosome abnormalities, related to No. 2 chromosome, which were maintained in culture for more than 1 year. Addition of dexamethasone prevented the cell growth of 2 of the cell lines in this medium but failed to alter the growth rate of the other 2 cell lines. These findings are correlated with karyotype abnormalities involving chromosome No. 2.

Aged erythrocytes obtained by fractionation using gradient centrifugation with Dextran 40, showed lower glutathione reductase activity and riboflavin content than young cells. However, both young and old cells displayed almost the same increase in enzyme activity upon addition of flavin adenine dinucleotide to the test hemolysate in vitro. The lipid peroxide content in the cell membranes showed no consistent changes with aging.

Incorporations of squalene and mevalonate into cholesterol in hepatic microsomes and cytosol from growing or young rats were higher than those from adult rats. When the microsomes from growing or young rats were combined with the cytosol from adult rats instead of young cytosol, the incorporation rates of squalene and mevalonate into cholesterol were decreased. When adult microsomes were combined with young cytosol, cholesterol synthesis from mevalonate was increased but that from squalene was not. Cytosol fractions from cholesterol fed rats did not inhibit the cholesterol synthesis from squalene in the microsomes from non-treated rats, and the normal cytosol did not influence the cholesterol synthesis in the microsomes from cholesterol fed rats, suggesting the elevation of serum cholesterol in aged animals did not affect the activity of cytosol fraction. It is concluded that the function of hepatic sterol carrier protein is probably impaired by ageing process. The decrease of hepatic cholesterol synthesis in old microsomes in vitro is difficult to be recovered by the substitution of young sterol carrier protein for adult one. The decreased function of sterol carrier protein is not derived from hypercholesterolemia which is often observed in the aged.

Effects of sodium cholate, deoxycholate, chenodeoxycholate, and lithocholate on serum and liver cholesterol levels, bile flow, biliary cholesterol, phospholipids, and bile acids, and fecal sterols and bile acids were examined in Wistar strain male rats fed either an ordinary diet or a 2% cholesterol diet. Cholate and deoxycholate increased serum and liver cholesterol levels, serum pre beta-lipoprotein, bile flow, and biliary secretion of cholesterol, phospholipids, and bile acids, but chenodeoxycholate and lithocholate did not. The total amounts of sterols and of bile acids in the feces did not differ between the cholate and the chenodeoxycholate groups. All the bile acids except lithocholate decreased fecal coprostanol when the diet included cholesterol. Cholate and deoxycholate produced similar bile acid compositions in the bile and feces, as was the case between chenodeoxycholate and lithocholate, though chenodeoxycholate slightly increased the amount of muricholic acids, and lithocholate that of hyodeoxycholic acid, in the feces. The effects of cholate and deoxycholate are similar to each other but different from that of chenodeoxycholate or lithocholate in rats. Cholate causes marked accumulation of cholesterol in tissues, increased bile flow and biliary lipid secretion but chenodeoxycholate does not. Cholate is absorbed much more efficiently than chenodeoxycholate.

Stereological analysis at the electron microscope level was performed on zona fasciculata and zona reticularis of the adrenal cortex of male rats maintained on normal rat chow, clofibrate (hypolipidemic drug), cholesterol-rich diet or cholesterol-rich diet plus clofibrate. The purpose of this study is to know whether the alteration in the serum cholesterol level is accompanied by an alteration in the ultrastructure and steroidogenic activity of the adrenal cortex, and to compare these changes with those of Leydig cells reported previously. The corticosterone level in both serum and the adrenal tissue of cholesterol-fed rats remained unchanged. Treatment of the cholesterol-fed rats with clofibrate caused a significant rise in the serum corticosterone level but the concentration in the adrenal remained unchanged. Lipid droplets increased in number and volume significantly in both cortical zones in all experimental groups as compared with the control group. But neither cholesterol feeding nor clofibrate treatment of normal rats caused a change in the volume or surface area of smooth endoplasmic reticulum (ER). Treatment with clofibrate on the cholesterol-fed rats resulted in a decrease in the volume of smooth ER in both cortical zones. Mitochondria showed no change except for a decrease in volume and inner membrane surface area of zona reticularis of cholesterol-fed rats. Peroxisomes did not proliferate but rather decreased in clofibrate-treated rats. These changes in the adrenal cortex were different from those in Leydig cells. Possible reasons for these differences were discussed.

Bronchopulmonary washings from a patient with pulmonary alveolar proteinosis who underwent therapeutic pulmonary lavage were examined. There were large amounts of protein and lipids of which main classes were phospholipid, choleterol and free fatty acid. The major component of phospholipids recovered from the washing was lecithin rich in palmitic acid. We also found a high IgG level in the washings. Both the findings of free cholesterol in high concentration in the washings and the electrophoretic pattern of protein in the washings suggested that the alveoli-filling material of our patient was not wholely derived from passive transudation of plasma constituents.

Age-related changes in serum and liver cholesterol, phospholipid and triglyceride levels, serum lipoproteins, biliary secretion of cholesterol, phospholipid and bile acids, and fecal excretion of sterols and bile acids were examined in Sprague-Dawley and Wistar strain male rats, 7 to 106 weeks in age. Serum and liver lipid levels increased with age in both strains but the liver phospholipid level remained unchanged. The proportion of serum alpha-lipoprotein increases and that of beta- and prebeta-lipoproteins slightly decreased. Cholesterol and phospholipids in low density lipoprotein and cholesterol in high density lipoprotein fractions also increased with age. Bile flow and biliary secretion of cholesterol and bile acids decreased in aged rats, but when they were expressed in terms of units per rat they were almost constant without regard to age. Pool size, synthesis, secretion, and turnover frequency of bile acids also did not change when they were expressed per rat, though 7-week-old rats showed a low value for turnover frequency. Biliary secretion of phospholipid, however, increased in aged rats. Biliary secretion of chenodeoxycholic and alpha-muricholic acids decreased but that of cholic and hyodeoxycholic acids increased. Daily excretion of feces and fecal neutral sterols decreases in aged rats but the excretion of bile acids remained constant regardless of age. The ratio of coprostanol and cholesterol in the total sterols was not affected. Fecal lithocholic, beta-muricholic and P10 (probably omega-muricholic) acids were decreased with age but the other bile acid components were not changed or were slightly increased.

In order to investigate the effect of hepatic cholesterol flux on biliary bile acids, Triton WR 1339 and orotic acid were administered to rats, and the biliary cholesterol, phospholipids and bile acids were analyzed together with serum lipoproteins and hepatic lipids. Triton, which raised serum very low density lipoprotein and lipid levels and decreased serum high density lipoprotein liver lipid levels, increase the biliary cholic acid group/chenodeoxycholic acid group ratio (CA/CDCA) in the bile without affecting the total amount of bile acids and the other biliary lipids. Orotic acid, which decreased serum lipid and lipoprotein concentrations and increased liver lipid levels, increased the biliary excretion of cholesterol and phospholipids, but produced no significant change in the total amount of bile acids and in the CA/CDCA ratio in bile.

The effects of aging on serum lipids, lipoproteins and apolipoprotein C subclasses in very low density lipoprotein (VLDL) were investigated in healthy male subjects aged from the 1st to the 9th decade. The serum cholesterol, phospholipid and triglyceride concentrations, the serum beta-lipoprotein concentration determined immunologically, and the beta-lipoprotein percentage determined by electrophoresis showed the lowest levels in the 2nd decade, increased gradually with age, attained the highest level in the 6th to 7th decade and slightly declined in the 9th decade. The VLDL-low density lipoprotein (LDL) cholesterol level changed almost in parallel with the serum total cholesterol level, but the HDL cholesterol level and the apolipoprotein A concentration remained almost constant showing no age-related change. The free cholesterol percentages in every lipoprotein fraction and the apolipoprotein content in LDL were higher in the subjects in the 6th and 7th decade than those in the 2nd to 3rd decade. The apo C II/C III ratio in VLDL increased with age. These data suggest that the ability to active lipoprotein lipase may not be impaired but the lecithin:cholesterol acyltransferase (LCAT) activity declines with age.

Monoclonal antibodies (2-3E2, 6-3G11, and 7-3H6) against gap junction plaques purified from rat liver were prepared and characterized. Immunoblot analysis of liver gap junctions revealed that all three antibodies reacted with the 27-kDa protein, but not with the 22-kDa one. The 2-3E2 and 6-3G11 antibodies both reacted with the 27-kDa protein in gap junctions purified from livers of the rat, mouse, rabbit, and guinea pig; the 7-3H6 antibody, however, failed to react with the 27-kDa protein from guinea pig liver. The 7-3H6 antibody reacted strongly with the 24- to 26-kDa degradation products of the 27-kDa protein. Indirect immunofluorescence showed that the 6-3G11 and 7-3H6 antibodies both gave the same specific fluorescence labeling on rat liver cryosections, suggesting that these two antibodies recognized the cytoplasmic sites of the 27-kDa protein. Immunoblot analysis of protease-digested fragments from the 27-kDa protein revealed that the 7-3H6 antibody reacted with the 24- and 17-kDa fragments (including portions of the carboxyl-terminal domain of the 27-kDa protein) produced with endoproteinases Arg-C and Lys-C, respectively. Immunoblot analysis of CNBr fragments of the 27-kDa protein revealed that all three antibodies reacted with the 10-kDa fragment, which is thought to be the carboxyl-terminal domain of the 27-kDa protein. These results demonstrate that three monoclonal antibodies recognize different epitopes of the cytoplasmic sites (probably the carboxyl-terminal domain) of the 27-kDa liver gap junction protein.

Changes of the metabolism of lung phospholipids and alveolar wash lipids were studied in guinea pigs and rats with experimental pneumonitis produced by the injection of complete Freund's adjuvant. The amount of total lipid obtained by lung lavage decreased significantly in contrast to a marked increase in the content of total protein in treated animals. Among the lipids in the pulmonary washings, only phospholipids were strikingly reduced, and free fatty acids were increased somewhat. However, the composition of phospholipids was not affected by the treatment. The in vitro incorporation of 1-14C-acetate into the total lipid and phospholipids in the lung with pneumonitis was slightly lower compared to controls, but the difference was not significant. The in vitro 1-14C-palmitic acid incorporation into phospholipids of the lesioned lung, was also similar to that in controls. Thus, phospholipid biosynthesis in the lung was not affected by pneumonitis. On the other hand, the in vitro activity of total phospholipase in rat lung with experimental pneumonitis was enhanced significantly. These results suggest that phospholipase activity is increased in the diseased lung and this may participate in the process of the inflammatory reaction. The possible role of activated macrophages in the inflammatory response of the lung is discussed.

Clinofibrate was given to 15 patients with hyperlipidemia, for 6–8 weeks at the daily daily dose of 600 mg, and its effect on 3 biliary lipid components (cholesterol, bile acids and phospholipids) and on the lithogenic index was investigated. After clinofibrate treatment, 6 of the patients were given 1.5 g/day clofibrate for 6–8 weeks to compare the effect of clofibrate with that of clinofibrate. The molar percentages of biliary cholesterol and phospholipids to the total. mol number of the 3 biliary lipid components decreased, and that of bile acids increased during clinofibrate administration. In this way, the molar ratio of bile acids to cholesterol increased during the treatment. Neither the lithogenic index calculated by the formula of Admirand and Small nor that of Hegardt, Dam and Holzbach was altered significantly by the treatment. There was no apparent relationship between the effect of the drug on the lithogenic index and any of the factors initial lithogenic index, rate of decrease of serum lipids, or type of hyperlipidemia. Although clofibrate had no significant effect on the maximum solubility of cholesterol in the bile, the molar percentage of biliary cholesterol was elevated and the lithogenic index increased as compared with the control and clinofibrate period. No significant influence on bile acid composition in the bile was observed, with either clinofibrate or clofibrate.

Hepatic cholesterol 7α-hydroxylation and cholesterogenesis were determined in vitro at various time intervals 6 hr after oral or intravenous administration to fasted rats. Glucose administration to fasted rats enhanced cholesterol 7α-hydroxylation as well as fatty acid or cholesterol synthesis in the liver, though significant changes were not demonstrated in the cholesterol content of liver and serum throughout the observed periods. Hepatic cholesterol synthesis increased 1 hr after oral glucose ingestion. On the other hand, cholesterol 7α-hydroxylation was stimulated 2 hr after oral glucose administration, with a lag phase of 1 hr following the induction of cholesterol synthesis. From the results of the experiments on the effect of glucose ingestion, it appeared that cholesterol 7α-hydroxylation was correlated with hepatic cholesterogenesis. Hepatic cholesterogenesis was decreased in triparanol-treated rats and was not stimulated by glucose administration. Triparanol did not affect cholesterol hydroxylation in the fasted state, but did abolish the stimulatory effect of glucose. Short-term feeding of a high-cholesterol diet, which suppressed hepatic cholesterol synthesis, stimulated cholesterol 7α-hydroxylation. Oral administration of glucose to cholesterol-fed rats still further accelerated hydroxylation. In rats with ligated bile ducts, hepatic cholesterogenesis increased 24 hr after bileduct ligation while hydroxylation decreased 24 hr after the operation but was stimulated after 72 hr. Glucose ingestion by these rats stimulated both cholesterol synthesis and hydroxylation. It is concluded that increased hepatic cholesterogenesis enhances cholesterol 7α-hydroxylase activity; this activity does not depend upon increased fatty acid or other lipid synthesis, altered bile flow or gastrointestinal factors. Increased dietary cholesterol intake also accelerates hydroxylase activity. These mechanisms may preserve homeostasis in the serum cholesterol level, and an altered balance between these two enzyme systems — anabolic and catabolic — probably induces a change in the size of the body cholesterol pool.

Age-related changes in serum and liver cholesterol, phospholipid and triglyceride levels, serum lipoproteins, biliary secretion of cholesterol, phospholipids and bile acids, fecal excretion of sterols and bile acids, and the pool size of bile acids were examined in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR). SHR showed distinct age- and sex-related changes when young and marked aged-rat hypercholesterolemia after 1 yr of age. (1) Cholesterol shifted from blood to the liver between 10 and 20 wk only in male SHR and not at all in WKR. (2) Serum lipoprotein percentages changes; alpha-lipoprotein decreased, pre-beta-lipoprotein increased, but beta-lipoprotein did not change. These changes appeared only in male SHR and after 13 to 15 wk of age. (3) Liver enlargement in SHR, although not detected at 5 to 6 wk, progressed more rapidly than in WKR, giving values almost double those in WKR after 13 to 15 wk. Liver enlargement in female SHR was much less than in the male. (4) Bile flow, biliary secretion, and the pool size of bile acids increased. However, when expressed on the basis of liver weight, these values were similar to those in WKR, suggesting that the increases were caused by the hepatomegaly. (5) Differences appeared in the bile acid composition. A large amount of beta-muricholic acid was present in SHR of both sexes and the cholic acid percentage was low in male SHR. (6) Changes were observed in fecal bile acid excretion. Since the daily amounts in male SHR were similar to those in WKR, the hepatic synthesis activity (mg per day per 10 g liver) in male SHR was almost half that in WKR at all ages.

Thiobarbituric acid reactive substances (TBARS) in serum and hepatic subcellular organelles of rats were increased markedly after the feeding of a tocopherol deficient diet for 2–16 weeks. Although serum lipid concentrations from the rats fed the deficient diet for 2 weeks were significantly higher than those from the controls, there was no difference between the two groups after feeding for more than 4 weeks. However, a supplement of 2% cholesterol in these diets increased the serum cholesterol and phospholipid concentration in such tocopherol deficient rats. Refeeding of a tocopherol-rich diet to the deficient animals reduced the serum lipid concentrations concomitantly with the decrease of TBARS in serum and hepatic subcellular fractions. The tocopherol deficient animals showed lower synthetic activities of hepatic cholesterol and fatty acid as well as glucose-6-phosphatase in microsomes. These enzyme activities of cholesterol and fatty acid synthesis in the deficient animals tended to be restored by the tocopherol administration.

Changes of cholesterol, phospholipid, triglyceride or bile acid levels in serum liver, bile and feces after the treatment with alloxan were examined in Wistar strain male rats. Serum cholesterol, phospholipid and triglyceride levels and liver cholesterol level markedly increased but liver phospholipid and triglyceride levels remained unchanged. The lipid levels in serum very low density and low density lipoproteins were elevated but those in high density lipoprotein were not. Bile flow was not changed but biliary secretion of cholesterol, phospholipid and bile acids markedly increased. Among the biliary bile acid components, cholic acid markedly increased but the amount of chenodeoxycholic acid was similar to that of normal rats. Fecal excretion of deoxycholic acid increased but that of lithocholic and hyodeoxycholic acids decreased, and alpha, beta- and omega-muricholic acids did not change, thus, the total amount of fecal bile acids remained unchanged. Hepatic cholesterol synthesis was markedly depressed, while cholesterol 7 alpha-hydroxylase activity did not change and cytochrome P-450 content was elevated by about 40%. From such evidence, it was apparent that synthesis of cholic acid increased while that of chenodeoxycholic acid decreased and the total amount of bile acids synthesized did not change in the diabetic rats. Furthermore, marked increase of the pool size of cholic acid and hepatic secretion of cholic acid stimulated the absorption of lipids and produced a hyperlipidemia in the diabetic rats.