The purpose of the present study was to compare the therapeutic effects of alendronate and alfacalcidol on the cancellous and cortical bone mass and mechanical properties in ovariectomized osteopenic rats in a head-to-head fashion. Twenty-five female Sprague-Dawley rats, 7 mo of age, were randomly divided by the stratified weight method into four groups: the sham-operated control (Sham) group and three ovariectomized groups treated with vehicle, alendronate (2.5 mg/kg, p.o., daily), or alfacalcidol (0.5 μg/kg, p.o., daily). Treatment was started 6 wk after the surgery and continued for 6 wk. At the end of the experiment, urinary deoxypyridinoline (DPD) and serum osteocalcin (OC) levels were evaluated, and cancellous and cortical bone histomorphometric analyses were performed for the proximal tibial metaphysis and tibial diaphysis, respectively. Alendronate prevented the elevation of the urinary DPD level induced by ovariectomy (OVX), and markedly decreased the serum OC level to below the value observed in the Sham group, while alfacalcidol prevented the elevation of the urinary DPD and serum OC levels induced by OVX. Alendronate increased the cancellous bone volume/total tissue volume (BV/TV) relative to the values observed in the OVX-Vehicle group by preventing the increases in the eroded surface/bone surface (ES/BS), osteoclast surface (OcS)/BS, and bone formation rate (BFR)/BS induced by OVX. However, it decreased the mineral apposition rate (MAR) in the ovariectomized osteopenic rats to below the value observed in the Sham group. It also prevented the increase in the marrow area (Ma Ar) caused by OVX. Alfacalcidol increased the BV/TV relative to the values observed in the OVX-Vehicle group by decreasing the ES/BS and OcS/BS, but maintaining the BFR/BS. The effect of alfacalcidol on the BV/TV was more pronounced than that of alendronate, despite the less pronounced suppression of OcS/BS by this drug in the ovariectomized osteopenic rats. In addition, this drug increased the cortical area (Ct Ar) and prevented the increase in the Ma Ar in the ovariectomized osteopenic rats by decreasing the endocortical ES/BS, and even increasing the endocortical BFR/BS. Furthermore, it also prevented the reduction in the maximum load of the femoral distal metaphysis in the ovariectomized osteopenic rats. Thus, the present study clearly showed that alendronate and alfacalcidol had differential therapeutic effects on the cancellous and cortical bone mass and mechanical properties in ovariectomized osteopenic rats.

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It has been reported that coenzyme Q₁₀ (CoQ₁₀) functions as an electron transfer carrier in mitochondria, and can produce an improvement in heart diseases such as congestive heart failure. Its (2Z)-isomer contains a cis-double bond at the 2-position of the decaprenyl side chain. As the original organic industrial synthesis of CoQ₁₀ resulted in a product that contained a small amount of this isomer, the efficacy and safety of CoQ₁₀ was determined using CoQ₁₀ containing this isomer; however, no toxicity data have been reported for the (2Z)-isomer itself. Thus, we conducted single (2,000 mg/kg) and 4-wk repeated (1,000 mg/kg) oral dose toxicity studies in rats to compare the toxicological profiles of CoQ₁₀ and its (2Z)-isomer. The two compounds displayed similar toxicological profiles, and it was concluded that neither CoQ₁₀ nor its (2Z)-isomer produce toxic effects in rats in single or repeated doses.

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Objectives: To compared the effects of risedronate (Ris) and calcitriol (Cal) on cancellous bone in rats with glucocorticoid (GC)-induced osteopenia. Materials and Methods: Thirty female Sprague-Dawley rats, 4 mo of age, were randomly divided by the stratified weight method into three groups of 10 rats each according to the following treatment schedule: 8-wk GC administration with 4-wk vehicle (control), Ris, and Cal as therapeutic treatment initiated after 4-wk GC administration. The GC (methylprednisolone sodium succinate, 5.0 mg/kg, s.c.), Ris (10 μg/kg, s.c.), and Cal (0.1 μg/kg, p.o.) were administered 3 times a week. At the end of the 8-wk treatment period, two-dimensional (2D) bone histomorphometric analysis was performed for cancellous bone of the proximal tibial metaphysis, 3D micro-computed tomographic analysis was performed for the distal femoral metaphysis, and the mechanical strength of the distal femoral metaphysis was evaluated by a compression test. Results: Ris and Cal treatment increased both 2D and 3D cancellous bone mass. However, Ris treatment exhibited more pronounced effects on 2D and 3D cancellous bone mass than Cal treatment, and the effects of both Ris and Cal treatment were greater on 3D cancellous bone mass than on 2D cancellous bone mass. The response of 2D and 3D cancellous bone mass to Ris treatment was characterized by its effect on trabecular number and thickness, which was associated with markedly suppressed bone resorption and bone formation in terms of suppressed bone turnover. On the other hand, the response of 2D cancellous bone mass to Cal treatment was attributed to the effect of Cal on 2D trabecular thickness, and the response of 3D cancellous bone mass to Cal treatment might be characterized by the effect of Cal on 3D trabecular number and thickness, with a more marked effect of trabecular thickness. These effects were primarily due to mildly suppressed bone resorption and maintained or even increased bone formation. Despite the differential effect of Ris and Cal treatment on the cancellous bone structure and bone metabolism, both treatment increased the maximum load and braking energy of the distal femoral metaphysis to a similar extent, suggesting different mechanisms for improving bone strength. Conclusions: This study showed the differential effects of Ris and Cal on cancellous bone in rats with GC-induced osteopenia.

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ODS rat has a hereditary defect in ascorbic acid biosynthesis and is a useful animal model for elucidating the physiological role of ascorbic acid. We previously demonstrated by using ODS rats that ascorbic acid deficiency changes the hepatic gene expression of acute phase proteins, as seen in acute inflammation. In this study, we investigated the effects of ascorbic acid deficiency on the production of inflammatory chemokine, cytokine-induced neutrophil chemoattractant-1 (CINC-1), in ODS rats. Male ODS rats (6 wk of age) were fed a basal diet containing ascorbic acid (300 mg/kg diet) or a diet without ascorbic acid for 14 d. Obvious symptoms of scurvy were not observed in the ascorbic acid-deficient rats. Ascorbic acid deficiency significantly elevated the serum concentration of CINC-1 on d 14. The liver and spleen CINC-1 concentrations in the ascorbic acid-deficient rats were significantly elevated to 600% and 180% of the respective values in the control rats. However, the lung concentration of CINC-1 was not affected by ascorbic acid deficiency. Ascorbic acid deficiency significantly elevated the hepatic mRNA level of CINC-1 (to 480% of the value in the control rats), but not the lung mRNA level. These results demonstrate that ascorbic acid deficiency elevates the serum, liver and spleen concentrations of CINC-1 as seen in acute inflammation, and suggest that ascorbic acid deficiency stimulate the hepatic CINC-1 gene expression.

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In this study, the freeze-dried powders from whole grapes, pomace and juice of Campbell Early (Vitis labruscana Bailey) were prepared to determine the amount of total flavonoids, vitamins A, C, and E, and dietary fiber. Effects of whole grape, pomace, or juice intakes on their antioxidative capacity and DNA damage were investigated in Sprague-Dawley male rats. A total of 120 rats at 13 mo old and weighing 549±4 g were blocked into 8 groups according to body weight and raised for 3, 5, or 7 mo with diets containing 2% (w/w) dry powder of three different parts of grapes and 0.02% (w/w) CdCl₂. The contents of flavonoids, antioxidant vitamins A and E, and dietary fiber in freeze-dried powder were the highest in grape pomace, but the vitamin C contents were similar among the three powders. In all the 16, 18, and 20-mo-old animals, plasma and liver thiobarbituric acid reactive substances levels of grape-ingesting groups were lower than those of the controls and that of the grape pomace group was the lowest among the groups. Cd administration increased plasma and liver thiobarbituric acid reactive substances levels remarkably; however, Cd+grape groups were lower than the Cd-control group. Red blood cell superoxide dismutase activity of 18- and 20-mo-old rats was higher than that of 16-mo-olds, showing an age-related increase; however, red blood cell catalase and glutathione peroxidase activities decreased with age. Grape diets promoted superoxide dismutase, catalase, glutathione peroxidase activities, and the grape pomace increased the activities most significantly among three different parts of the grape. Cd decreased superoxide dismutase, catalase, glutathione peroxidase activities; however Cd+grape groups showed similar activities to the non-Cd control group. Liver superoxide dismutase activity was decreased with age but catalase activity of 18-mo-old rats was higher than those of 16- and 20-mo-old groups, and glutathione peroxidase activities of 16- and 18-mo-old groups were similar but that of 20-mo-old groups decreased markedly. Grape intake increased these three antioxidative enzyme activities while Cd administration decreased catalase and glutathione peroxidase activities except superoxide dismutase activity. The concentration in the kidney of 8-hydroxy-2'-deoxyguanosine in the 18- and 20-mo-old rats was higher than that in the 16-mo-old groups, and grape intake showed a protecting effect on DNA from age-related or Cd-induced oxidative damage. In conclusion, grape intakes, especially grape pomace with the highest content of flavonoids, β-carotene, tocopherols and dietary fiber among the three parts, showed the prominent antioxidative capacity of inhibiting age-related or Cd-induced increase of lipid peroxidation and DNA damage effectively, promoting liver and red blood cell antioxidant enzyme activities.

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The level of free fatty acid (FFA) in plasma is increased by diabetes. The increase in plasma FFA levels accompanied the stimulation of basal lipolysis (i.e. lipolysis in the absence of lipolytic agents) in fat cells. Injection of streptozotocin with rats resulted in a significant increase in basal FFA production (5.5 fold) in fat cells. However, basal glycerol production in fat cells was increased only 1.5 fold by streptozotocin-induced diabetes, implying that FFA re-esterification in fat cells was decreased by streptozotocin-induced diabetes. The FFA re-esterification in fat cells was also decreased by 1 d of fasting. Although basal lipolysis was increased by streptozotocin-induced diabetes or 1-d fasting, neutral triacylglycerol lipase activity and the immunoreactive HSL protein content in fat cells from streptozotocin-induced diabetic rats or 1-d fasting rats were not significantly changed. Although β-blockers inhibited lipolysis induced by norepinephrine at a concentration of 10^-4 M, it failed to inhibit the basal lipolysis and FFA re-esterification in fat cells from streptozotocin-induced diabetic rats. Nor did insulin or H-89, another antilipolytic agent, affect basal lipolysis or FFA re-esterification in fat cells from streptozotocin-induced diabetic rats. These results indicate that basal FFA production may be induced by a decrease of re-esterification of FFA in diabetic rats and is not affected by antilipolytic agents such as insulin, β-blockers or H-89.

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The concentrations of metabolites in human plasma after ingestion of flavanone glycosides (FG) and their aglycones (FA) in lemon were examined. FG consisting abundantly of eriocitrin were prepared from lemon peel and FA consisting abundantly of eriodictyol were prepared from FG by treatment with β-glucosidase. Eriodictyol, homoeriodictyol, and hesperetin in plasma up to 4 h after ingestion of FG with water or FA with water by subjects were not detected in plasma of non-enzyme treatment but in plasma after treatment with β-glucronidase and sulfatase. Metabolites in plasma after ingestion of FG and FA in humans were shown to exist as the glucuro- and/or sulfo-conjugates of eriodictyol, homoeriodictyol, and hesperetin. After ingestion of FA, the concentration of metabolites in plasma exhibited a high maximum peak at 1 h. The AUC (area under the blood concentration time curve) level of metabolites of FA was higher than that of FG. FA were suggested to be absorbed faster and in higher amounts than FG. The AUC of metabolites in subject plasma after ingestion of FG with flavonoid-depleted lemon juice was shown to change to a low level in comparison with that of FG with water. The maximum concentration peak of metabolites in plasma was faster at 0.5 h than FA with water but the AUC level was similar to FA with water, when subjects ingested FA with vodka (40% ethanol). The absorption hour of FG and FA was shown to be affected by the co-existing solution.

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Acute homocysteinemia mice were prepared by forcibly oral administration of homocysteine (4 mM, 2 mL). The amounts of plasma homocysteine were estimated by a fluorescence method with HPLC. Folic acid (0.6 mM, 2 mL), DMSP, or betaine (20 mM, 2 mL each) was intraperitoneally administrated into the mice suffered from the acute homocysteinemia on the 20th, 40th or 60th min after the oral supplementation of homocysteine, then amounts of plasma homocysteine were determined by the HPLC method 40 min after each addition, respectively. The results indicated that the intraperitoneal addition of folic acid or DMSP in this order of the 40th, 60th and 20th min after the oral supplementation of homocysteine significantly reduce the quantities of plasma homocysteine, but betaine exerted the fairly lesser effects. The amounts of homocysteine without any additive linearly and rapidly appeared to increase up to 60 min, at which those were about 8-12 fold the normal levels of homocysteine, and thereafter decreased in these experiments. Accordingly, folic acid which is known to effectively improve homocysteinemia was proven to be completely replaced by DMSP under the experimental conditions.

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We studied the effect of incubation temperature on the production of short-chain fatty acids (SCFA) by pig cecal bacteria in vitro in order to assess short-term influences of body temperature on bacterial metabolism in the large intestine. We employed a 200 mL scale continuous culture system using cecal bacteria from commercially slaughtered pigs as innoculum. The culture was maintained at 30, 37, 40 or 42°C and continuously diluted by continuous feeding of bicarbonate buffer (pH 7.4) added with lactose (10 g/L) and by simultaneous continuous efflux both at 4.17 mL/h. We monitored SCFA concentration of the culture for 12 h, which represents their production rate. Concentrations of SCFA increased during the first several hours and plateaued at around 11 h of incubation. Incubation temperature significantly affected mean concentrations from 1 to 12 h of acetic (40°C>42°C=37°C>30°C), propionic (40°C>42°C=30°C), n-butyric (42°C>37°C>30°C, 40°C>30°C) and n-valeric (42°C=40°C>37°C>30°C) acids, and total SCFA (40°C>42°C=37°C>30°C) (p<0.05). These results indicate that both hyperthermia and hypothermia depress the microbial breakdown of carbohydrates.

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The effect of dimethylsulfoniopropionate (DMSP) on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease (PD) of mice was examined for 5 d. The distilled water (the control group) and the DMSP solution at 5×10^-4 M (the DMSP group) were supplemented ad libitum to six mice each in two groups for 2 wk. An appropriate amount of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) solution (20 mg/kg body wt) was then intraperitoneally injected into all the test mice once a day initially for 3 d, which definitely made the control mice similar to the PD-model mice. The moving ability (running power) of the mice in both groups was measured using an automatic Wheel Running Instrument. The immobility duration of the upside-down mice in both groups was estimated by a newly developed polygraph (RMP-6008M, Nihon Koden Co., Ltd., Japan). The results indicated that the mice in the DMSP group showed a stronger moving ability and a shorter immobility duration compared to the mice in the control group during the experimental period. Furthermore, the amounts of catecholamines (dopamine and norepinephrine) in the brains except for the cerebellums of all the test mice were estimated 2 d after the last MPTP injection, which demonstrated that the brains of the mice in the DMSP group accumulate larger amounts of catecholamines, especially dopamine, than them in the control group. Accordingly, the administration of low concentrations of DMSP proved to prevent and/or ameliorate the decreased mobility and the typical immobility (Akinesia) of the MPTP-induced PD-model mice probably due to increased amounts of dopamine in the brains of the DMSP group mice.

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Peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor superfamily and participates in the regulation of key proteins involved in lipid metabolism, fatty acid oxidation, homeostasis, and inflammation. Several polymorphisms of the human PPARα gene, such as Leu162Val polymorphism and Val227Ala polymorphism, have been described in different races. Recently, another PPARα polymorphism Gly395Arg polymorphism has been reported in Caucasian and African subjects. Using the Invader assay, we searched for this polymorphism in 729 Japanese adults randomly selected in a rural population. Although the synthesized oligonucleotides of each polymorphism could be distinguished clearly, all 729 individuals had the Gly (G) allele and none had the Arg (C) allele. These data suggest that there is racial variability in the frequencies of PPARα gene polymorphisms.

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We developed a novel method that conveniently measures dietary choline-phospholipid content. Crude lipids extracted from dietary samples were reacted with phospholipase D from Streptomyces chromofuscus. The choline liberated from this reaction was then reacted with potassium triiodide, yielding choline periodide, which could be measured spectrophotometrically at 365 nm. This method proved to be more convenient than conventional assays, such as thin layer chromatography and high performance liquid chromatography. Our novel method is suitable for measuring many samples in single experiments.

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