A Simple Rapid Method for Measuring Liver Steatosis Using Bioelectrical Impedance.

BACKGROUND/AIM: Easy measurement of liver steatosis without pathological diagnosis may help improve donor surgery efficiency and increase the chances of organ donations. We analyzed the correlations between bioelectrical impedance (BI) in human livers, liver fat content, and pathological findings. MATERIALS AND METHODS: Sixteen tumor-free liver specimens resected during elective oncological surgery were analyzed. All samples were stored in ice chilled saline before BI measurement. The BI measurement was performed using a device with the tetrapolar circuit method in which the current and voltage electrodes are independent. Liver cholesterol and triglyceride levels were investigated from the same specimen using the Soxhlet extraction method. Pathological findings were examined by counting the number of hepatocytes with fatty changes per high-power field. RESULTS: The median liver steatosis percentage was 0.4%. The liver steatosis percentage was significantly correlated with the intrahepatic triglyceride content (r=0.82, p<0.001). Linear regression of the measurements and predicted values yielded an r2 of 0.63 between the BI at 100 kHz and liver steatosis, indicating reasonable agreement (p<0.001). CONCLUSION: BI analysis is a simple, non-invasive method that can be easily applied to evaluate liver steatosis.

A convenient HPLC method for detection of okadaic acid analogs as 9-anthrylmethyl esters with automated sample cleanup by column switching.

A convenient HPLC-fluorometric detection (FLD) method for okadaic acid (OA) analogs as 9-anthrylmethyl esters was developed with the addition of column switching to simplify and automate cleanup. Methanol extracts of shellfish were first treated to hydrolyze OA esters and then reacted with 9-anthryldiazomethane (ADAM). ADAM derivatives of OA and dinophysistoxin-1 (DTX1) were subsequently determined by HPLC-FLD following automated column-switching cleanup. The LOD (S/N = 3) and LOQ (S/N = 10) of OA and DTX1 obtained from bivalves fortified with toxin in our method were approximately 2.6 and 8.6 ng/g whole meat, respectively. The recoveries of OA and DTX1 at all fortification levels of bivalve extracts ranged from 90 to 113%, with RSD values of 0.9-9.9%. The new method is applicable to the routine monitoring of OA analogs as an inexpensive and convenient alternative to HPLC/MS.

Microdevice for on-site fish freshness checking based on K-value measurement.

An electrochemical microfluidic device with two sensing sites in the upper and lower streams of a flow channel was fabricated to measure the K-value as a means of evaluating the freshness of fish. In this device, plugs of solutions were processed using mechanisms to place a plug at the sensing site and to merge and mix two plugs in a single flow channel. The sums of ATP-related compound concentrations used for the calculation of the K-value were measured at the first and second sensing sites. The ratio of the output currents agreed well with the K-value calculated from predetermined concentrations in standard solutions. The K-values of jack mackerel, yellow tail, and sea bream extracts were then obtained using the device and were found to agree well with those obtained by high-performance liquid chromatography (HPLC). In addition, the changes in the K-value with time were observed to depend strongly on the type of fish for these three fish extracts.

Quantitative determination of paralytic shellfish toxins in cultured toxic algae by LC-MS/MS.

We developed a sample preparation and LC-MS/MS method for the determination of saxitoxins in toxic algae. Paralytic shellfish toxins (PSTs) were successfully separated by gradient elution on an amide column with the hydrophilic interaction mode and quantified with multiple reaction monitoring (MRM) detection in the positive ion mode. This method showed good performance in the summed LODs and LOQs for all 12 toxins, 25 and 84 nM, respectively. Next, extracts of cultured strains of a toxic dinoflagellate Alexandrium tamarense and a freshwater cyanobacteria Anabaena circinalis were treated in a short column of basic alumina and the toxic fractions were analysed by our LC-MS/MS method and by HPLC with fluorescence detection. Comparison of the results obtained by the two methods demonstrated that approximately equivalent results were obtained for both the dinoflagellate and the cyanobacteria. In addition, the retention time of the toxins showed acceptable shifts. Therefore, the clean-up of the toxic algal extracts by using the basic alumina column controlled unwanted chromatographic behaviour and variable ionisation efficiency during MS detection. LC-MS/MS for saxitoxins has great potential as a rapid analytical method for determining all primary saxitoxins in cultured algae.

LC-MS/MS analysis of palytoxin analogues in blue humphead parrotfish Scarus ovifrons causing human poisoning in Japan.

Since 1953, a total of 27 human poisoning cases caused by the consumption of blue humphead parrotfish, Scarus ovifrons, have been reported in Japan. Characteristic symptoms are severe muscle pain associated with rhabdomyolysis. Although it is believed that palytoxin, which is one of the most potent non-protein marine biotoxins, is the most likely causative toxin in blue humphead parrotfish poisoning, palytoxin has not been proven conclusively as the causative toxin because of lack of a reliable and sensitive analytical method for palytoxin. In 2011, human poisoning cases caused by the consumption of blue humphead parrotfish occurred in Miyazaki and Tokyo. In our present study, an LC-MS/MS method for palytoxin and its analogues in the blue humphead parrotfish samples causing the human poisoning cases in 2011 was developed and the samples were analysed by using the newly developed LC-MS/MS method. Palytoxin and its analogues were not detected in the samples from the food poisoning cases. The LC-MS/MS findings therefore do not support the recently accepted hypothesis that palytoxin is the causative agent in blue humphead parrotfish poisoning in Japan.

Pentosan reduces osteonecrosis of femoral head in SHRSP.

Increased oxidative stress is considered one of the main causes of steroid-induced osteonecrosis of the femoral head (ONFH). The aim of this study was to evaluate the effects of a steroid hormone and pentosan polysulfate sodium (pentosan), a heparin analog, in stroke-prone spontaneously hypertensive rats (SHRSP) as a model of ONFH. One hundred twenty-three 13-week-old male SHRSP/Izm rats were divided into four groups: a control group (group C), pentosan-administered group (group P), steroid-administered group (group S), and group administered pentosan plus steroid (group PS). Methylprednisolone acetate, as the steroid hormone, at a dose of 4 mg (15 mg/kg) was administered at 15 weeks of age. Pentosan at a dose of 3 mg/day/kg was continuously administered intraperitoneally from 13 weeks of age for 4 weeks. Rats were sacrificed at 17 weeks of age, and heart blood and both femora were collected. Triglyceride levels were significantly lower in group PS than in group S, indicating that pentosan improves lipid metabolism. The incidence of histologic ONFH was significantly lower in group P, at 14.8% (10/71 femoral heads), than in group C, at 30.4% (17/56 femoral heads), and significantly lower in group PS, at 40.8% (29/71 femoral heads), than in group S, at 91.3% (42/46 femoral heads), indicating that pentosan markedly inhibits ONFH. Immunohistochemical staining for oxidative stress showed that the stainability was significantly lower in group PS than in group S. Pentosan seems to reduce the incidence of ONFH in SHRSP by improving lipid metabolism and decreasing oxidative stress.

Sodium pentosan polysulfate resulted in cartilage improvement in knee osteoarthritis--an open clinical trial.

BACKGROUND: Pentosan polysulfate sodium (pentosan) is a semi-synthetic drug manufactured from beech-wood hemicellulose by sulfate esterification of the xylopyranose hydroxyl groups. From in vitro and animal model studies, pentosan has been proposed as a disease modifying osteoarthritis drug (DMOAD). The objective of this study was to assess the efficacy, safety, and patient satisfaction in patients with mild radiographic knee osteoarthritis (OA) findings and OA-associated symptoms and signs. METHODS: Twenty patients were assessed clinically at Nagasaki University Hospital. The radiographic indications of OA were grade 1 to 3 using the Kellgren-Lawrence Grading System (K/L grade). Pentosan used in this study was manufactured and supplied in sterile injectable vials (100 mg/ml) by bene GmbH, Munich, Germany. The study was a single-center, open-label trial. Treatment consisted of 6 weekly subcutaneous injections (sc) of pentosan (2 mg/kg). Patients were clinically assessed at entry and 1 to 8, 11, 15, 24 & 52 weeks post treatment. The results were analyzed using one way ANOVA and Dunnett's method. RESULTS: Hydrarthroses were reduced quickly in all cases. The clinical assessments, i.e., knee flexion, pain while walking, pain after climbing up and down stairs, etc, were improved significantly and these clinical improvements continued for almost one year. The dose used in this study affected the blood coagulation test, but was within safe levels. Slightly abnormal findings were noted in serum triglycerides. CONCLUSIONS: Pentosan treatment in twenty patients with mild knee OA seemed to provide improvements in clinical assessments and C2C level of cartilage metabolism.

Osteonecrosis of femoral head in the stroke-prone spontaneously hypertensive rats, especially old rats.

The average life span of stroke-prone spontaneously hypertensive rats (SHRSP) is about eight months. Male SHRSPs at 40 weeks old were used to study the idiopathic osteonecrosis of the femoral head (ION). The control group showed about 40% old necrosis and 20% early necrosis. The group administered with steroid hormone showed an increasing degeneration of adipocyte in the bone marrow, and 20% fresh necrosis was recognized. Furthermore, we observed the adipocyte change as well as early necrosis occurring among old necrosis sites. The study of aged rats may provide further understanding into the pathogenesis of ION.

Mycosporine-like amino acids extracted from scallop (Patinopecten yessoensis) ovaries: UV protection and growth stimulation activities on human cells.

Scallops (Patinopecten yessoensis) are extensively cultured and landed in Japan. During the processing of scallops, large amounts of internal organs and shells are discharged as industrial wastes. To reduce the burden on the environment, effective utilization and disposal methods of the wastes are required. Therefore, we have screened for useful materials in scallop internal organs, and found ultraviolet (UV) absorbing compounds from scallop ovaries. Based on UV absorption, electrospray ionization-mass spectrometry (ESI-MS), ESI-MS/MS, and nuclear magnetic resonance (NMR) spectra, three UV absorbing compounds were identified as mycosporine-like amino acids (MAAs): shinorine, porphyra-334 (P-334), and mycosporine-glycine. To investigate whether MAAs can act as a UV protector for human cells, we examined the protective effects of the three MAAs on human fibroblast cells from UV irradiation. All of the three examined MAAs protected the cells from UV-induced cell death. In particular, mycosporine-glycine had the strongest effect. Further, we found a promotion effect of MAAs on the proliferation of human skin fibroblast cells. From these results, it was found that the three MAAs isolated from scallop ovaries have a protective effect on human cells against UV light. MAAs have potential applications in cosmetics and toiletries as a UV protectors and activators of cell proliferation.

Hip rotationplasty with antibiotic-loaded bone cement spacer for severe infection following limb-sparing surgery.

Knee rotationplasty was initially proposed for the reconstruction of the knee joint in the congenital hypoplasia of the femur. Its application was extended to functional reconstruction of the knee joint after wide resection of malignant bone or soft tissue tumor around the knee. It has also been shown to salvage a failed knee-sparing surgery due to infection or the aseptic loosening of the prosthesis. Hip rotationplasty has been described as a method for the reconstuction of hip function, as well as in the knee joint, in the case of a primary malignant tumor of the proximal part of the femur in children. It has also been described as having a surgical application for the severe congenital deficiency of the proximal part of the femur to reconstruct hip and knee joints, as well as for the massive bone defect of the proximal part of the femur due to infection to mimic a functional femoral shaft. This article reports a case where the hip joint was secondarily reconstructed with hip rotationplasty after subtotal resection of the femur due to infection of the hip hemiarthroplastic prosthesis and osteomyelitis of the hip joint and femur.

Osteonecrosis in stroke-prone spontaneously hypertensive rats: effect of glucocorticoid.

BACKGROUND: High-dose administration of a steroid hormone has been associated with a major risk of osteonecrosis. In this study we investigated the effects of a steroid hormone on the incidence of osteonecrosis of the femoral head in stroke-prone spontaneously hypertensive rats/Nagasaki (SHRSP/Ngsks). METHODS: A total of 71 SHRSP/Ngsks were divided into two groups: a control group (C group, n = 40) and a steroid hormone group (S group, n = 31) given 5 mg (about 20 mg/kg) of methylprednisolone acetate during the 17th week of age. We compared the groups' laboratory data, histological appearance, incidence of osteonecrosis, and expression of oxidative stress on immunohistochemical analysis using the monoclonal antibodies anti-4HNE and anti-8OHdG. RESULTS: The S group showed an increase in total cholesterol, with the amounts of high-density lipoprotein, low-density lipoprotein, and triglycerides all significantly higher than in the C group. Histological examination showed that the frequency of necrosis of the femoral head was significantly higher in the S group (95.2%) than in the C group (51.2%). Most of the histological features of the osteonecrosis demonstrated typical features of a similar sort in the two groups. However, the S group showed bone marrow spaces in the femoral head that were occupied by an increased number of adipocytes and that were swollen, partially degenerative, and necrotic. On immunohistochemical analysis, the stains of anti-4HNE and anti-8OHdG antibody were stronger in the S group than in the C group. CONCLUSIONS: This study confirmed, to a remarkable degree, the suspicion that the administration of steroid hormone increases the number of adipocytes in marrow. Fat degeneration and necrosis, considered early signs of osteonecrosis, were also observed. It has been hypothesized that osteonecrosis is produced by the ischemic change accompanying compartment pressure load in marrow, where fat degeneration, necrosis, and endothelial cell injury might occur together with oxidative stress.

Inhibitory effect of porphyran, prepared from dried "Nori", on contact hypersensitivity in mice.

Porphyran is a major component of the red algae, Porphyra tenera and P. yezoensis, which are processed into a sheet type of dried food, "Nori". Porphyran has been reported to activate murine macrophages by in vitro and i.p. injection studies. The contact hypersensitivity (CHS) reaction in mice is commonly used as a model to evaluate the anti-allergic activity of food and food components. We therefore studied the effect of porphyran on the CHS reaction in Balb/c mice to evaluate anti-allergic activity of porphyran. We found that an oral administration of porphyran (2% in drinking water) suppressed the CHS reaction (ear edema) induced by 2,4,6-trinitrochlorobenzene. We also found that porphyran suppressed the serum level of IgE and the production of interferon-gamma (IFN-gamma) in the challenged ear lobe. We conclude from these results that the CHS reaction was suppressed by oral porphyran due to the decreased serum level of IgE and the production of IFN-gamma in the challenged ear lobe.

Fish protein stimulated the fibrinolysis in rats.

OBJECTIVE: We hypothesized that fish protein affects blood coagulation and/or fibrinolysis, and compared the activity and amounts of factors involved in blood coagulation and fibrinolysis in rats fed the fish protein, which was treated to remove water-soluble and ethanol-soluble elements, from sardine (sardine protein). METHODS: In the first experiment, rats were fed for 21 days an AIN-93G-based control diet, and diets in which the casein of the control diet was exchanged for sardine protein at 5, 10 and 20% levels. In the second experiment, rats were fed an AIN-93G control diet and diets containing 5% fish oil, 10% sardine protein or both (5% fish oil + 10% sardine protein) for 21 days. At the end of the experiments, blood coagulation time, hemostatic parameters and fibrinolysis parameters were measured. RESULTS: The activated partial thromboplastin time (APTT), which is an assay for blood coagulation time in the intrinsic blood coagulation pathway, of rats fed the 20% sardine protein diet was significantly prolonged compared to that of rats fed the control diet. The prolonged APTT by dietary sardine protein was due to a significant decrease of the activities of plasma blood coagulation factors VIII, IX, XI and XII. On the other hand, dietary sardine protein significantly increased the activity of tissue-type plasminogen activator, and the amount of plasma plasmin-alpha(2)-plasmin inhibitor complex, which are markers of activated plasmin. Moreover, we observed that the 20% sardine protein diet increased the amount of plasma D-dimer, which is a degraded product of the fibrin polymer by plasmin. In the second experiment, the APTT and PT of rats fed the F diet were prolonged compared to those of rats fed the control diet, however the concentration and amount of fibrinolytic parameters in the plasma were almost the same as those of rats fed the control diet. In contrast, the F+S diet not only prolonged APTT and PT, but also increased the concentration and amount of fibrinolytic parameters in plasma. CONCLUSIONS: We consider that the beneficial effects to health and amelioration of cardiovascular and cerebrovascular diseases by fish consumption are caused by a combination of the suppressing effect on blood coagulation of n-3 polyunsaturated fatty acids and the promoting effect on fibrinolysis of fish protein.

Warfarin reduces the incidence of osteonecrosis of the femoral head in spontaneously hypertensive rats.

In this study, we investigated the effects of warfarin potassium on the incidence of the femoral head osteonecrosis in spontaneously hypertensive rats (SHR). Twenty-four SHRs were divided into two equal groups, one given normal water (water group) and another provided with water containing warfarin (warfarin group). We compared the two groups histologically and observed the incidence of osteonecrosis. We also studied 17 Wistar Kyoto rats (WKY) to compare with SHR. Coagulation time, platelet count, and protein C activity were measured. Immunohistochemistry was also performed using endothelial nitric oxide synthetase (eNOS) antibody to investigate the function of endothelial cells. The incidence of osteonecrosis was significantly less in the warfarin group (10.5%) than in the water group (52.6%). Coagulation time was significantly longer in the warfarin group than the water group. Platelet count and protein C activity were not statistically different between the warfarin group and the water group. Results of immunohistochemistry revealed that endothelial cells in the femoral head were positive for eNOS in WKY but not in SHR. Our results indicated that warfarin reduced the incidence of femoral head necrosis in SHR.

Blood coagulation and fibrinolysis of rats fed fish oil: reduced coagulation factors especially involved in intrinsic pathway and increased activity of plasminogen activator inhibitor.

Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.

Docosahexaenoic acid suppresses nitric oxide production and inducible nitric oxide synthase expression in interferon-gamma plus lipopolysaccharide-stimulated murine macrophages by inhibiting the oxidative stress.

N-3 polyunsaturated fatty acids (PUFAs) are known to have anti-inflammatory effects. Excess production of nitric oxide (NO) is associated with inflammation. Therefore, we examined the effects of PUFAs on NO production and inducible NO synthase (iNOS) expression by stimulated murine macrophages. One typical n-3 PUFA docosahexaenoic acid (DHA) strongly inhibited NO production and iNOS expression in RAW264 macrophages and mouse peritoneal macrophages in a dose-dependent manner. This inhibition was accompanied by inhibiting the oxidative stress-sensitive transcription factor nuclear factor (NF)-kappaB activation. In stimulated macrophages, intracellular peroxides level was enhanced, but pretreatment of DHA dose-dependently inhibited this enhancement. These results suggest that DHA has an antioxidative effect based on the inhibition of the accumulation of intracellular peroxides, and this inhibition caused the suppression of the activation of NF-kappaB, resulting in the inhibition of NO production and iNOS expression. On the other hand, DHA treatment enhanced the level of intracellular glutathione (GSH), and this enhancement is thought to mediate the activity of DHA because lowering the GSH level by inhibiting GSH biosynthesis reversed the DHA-induced suppression of NO production, NF-kappaB activation, and the accumulation of intracellular peroxides. Our results demonstrate that DHA inhibits NO production in macrophages and this inhibition is, in part, mediated by upregulation of GSH.

Dietary fish oil and Undaria pinnatifida (wakame) synergistically decrease rat serum and liver triacylglycerol.

Japanese eating habits are characterized by the consumption of various food materials such as cereals, vegetables, fish, shellfish, marine algae and meat. Therefore, properties of functional substances in food materials may be enhanced or lessened by the combination of various food materials. In the present study, we examined how the combination of wakame and fish containing polyunsaturated fatty acids, which are typical Japanese food materials, affected rat lipid metabolism. Rats were fed one of four diets [control diet (C), AIN-76 diet with 5 g/100 g rapeseed oil; wakame diet (W) containing 19.1 g/100 g Undaria pinnatifida (wakame) dried powder in the C diet; fish oil diet (FO), AIN-76 diet with 4.1 g/100 g fish oil; wakame-fish oil diet (W + FO), the FO diet containing 19.1 g/100 g dried wakame powder] for 4 wk. We measured the concentration of lipids in serum and liver and hepatic activities of enzymes involved in fatty acid metabolism. The W diet, FO diet and W + FO diet significantly reduced the concentration of triacylglycerols in the serum and liver compared with the C diet. This decrease in the concentration of hepatic triacylglycerol was greatest in rats fed the W + FO diet. The activity of glucose-6-phosphate dehydrogenase, which is involved in fatty acid synthesis in the liver, of rats fed the W, FO and W + FO diets was lower than that in rats fed the C diet. However, the activities of malic enzyme and fatty acid synthetase did not differ among the four groups. In contrast, the W diet and W + FO diet increased the serum concentration of beta-hydroxybutyrate. Further, the activity of 3-hydroxyacyl-CoA dehydrogenase, which is involved in fatty acid beta-oxidation in the liver, was greater in rats fed the W diet (42%), the FO diet (154%) and the W + FO diet (381%) than in those fed the C diet. Because the decrease in the concentration of triacylglycerol in the liver was greatest when rats were fed wakame and fish oil at the same time (W + FO diet), we conclude that there was a synergistic process affecting fatty acid beta-oxidation in the liver. These results suggest that the simultaneous consumption of fish (fish oil) and wakame decreases the concentration of triacylglycerol in the serum and liver.