[Increase in Alkaline Phosphatase Activity after High-Fat Meal Ingestion is Correlated to the Amount of ABH Substances in Saliva].

Intestinal alkaline phosphatase (IAP) appears in the circulation more frequently in blood group B or O secretors than in blood group A or AB secretors and non-secretors, and serum IAP activity rises following the ingestion of a high-fat meal. In a previous study, the occurrence of two IAP isoforms, with high (HIAP) and normal molecular mass (NIAP), in healthy sera was demonstrated by 6.0% polyacrylamide gel electrophoresis in the presence of 1% Triton X-100. NIAP was present in the fasting serum of only healthy blood group B or O secretors, but was present in all subjects following ingestion of a high-fat meal. We classified 56 healthy subjects into 3 blood groups: B (n = 19), O (n = 17), and A (n = 20), and measured their serum ALP activity in a fasting state and 6 h after a high-fat meal. The amount of ABH substances in the saliva of each subject was determined by the hemagglutination inhibition test. Correlation coefficients between the change in ALP activity after high-fat meal ingestion and the hemagglutination inhibition values in saliva were 0.925 in blood group B, 0.879 in blood group O, and 0.906 in blood group A. These results suggest that increases in ALP activity in the circulation following the ingestion of a high-fat meal are closely related to the amount of ABH substances in saliva.

Clostridium butyricum MIYAIRI 588 improves high-fat diet-induced non-alcoholic fatty liver disease in rats.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) has become a common liver disease, as its prevalence has increased markedly in recent decades. The aim of the present study was to examine the improving effect of Clostridium butyricum MIYAIRI 588 (CBM588), a probiotic in clinical use for antibiotic-associated diarrhea, against high-fat diet (HFD)-induced fatty liver in rats. METHODS: After feeding HFD or HFD coated with CBM588 (HFD-CBM) for 12 weeks, we evaluated the hepatic mRNA levels related to lipid metabolism, and then assessed the hepatic protein levels of several transcription factors regulating these lipogenic gene expressions. RESULTS: The HFD-CBM group had decreased accumulation of lipid droplets in the liver compared with the HFD group. The HFD-CBM group had significantly decreased diacylglycerol acyltransferase (DGAT) 2 mRNA in the liver compared with the HFD group, whereas DGAT1 mRNA did not change between the HFD group and the HFD-CBM group. Moreover, the HFD-CBM group had significantly increased hepatic mRNA regulating cholesterol catabolism enzymes and excretion transporters. Correspondingly, the HFD-CBM588 groups had increased hepatic protein levels of peroxisome proliferator-activated receptor α/γ and liver X receptor α compared with the HFD group. The HFD-CBM group had accelerated excretion of total bile acid and non-esterified fatty acid in the feces. CONCLUSIONS: CBM588 intake may have novel potential for improving NAFLD.

[Alkaline phosphatase activity in blood group B or O secretors is fluctuated by the dinner intake of previous night].

We previously reported that two intestinal alkaline phosphatase (IAP) isoforms, high molecular mass IAP (HIAP) and normal molecular mass IAP (NIAP), appear in healthy serum with our Triton-PAGE method for determination of ALP isozymes. In addition, HIAP is chiefly present in blood group B or O secretors, and a large amount of NIAP is secreted into the circulation after high-fat meal in blood group B or O secretors. In the present paper, we investigated the relationship between alkaline phosphatase (ALP) activity in early morning with the patient in a fasted state and the dinner intake of previous night. Two types of dinner were prepared; a low-fat meal (520 kcal), and a high-fat meal (1,040 kcal). Subjects ate the 2 types of dinner on different days. The mean ALP activities at 14 h after high-fat meal ingestion in blood group B or O secretors (n=14) from JSCC and IFCC methods were 8.8% and 5.2% higher than those at 14 h after low-fat meal ingestion in blood group B or O secretors, respectively. The increases in ALP activity between after high-fat meal and low-fat meal were nearly identical to the increases in NIAP activity. These results suggest that a high-fat meal is more likely to affect ALP activity at the early morning with the patient in a fasted state in blood group B or O secretors.

A highly sensitive and specific chemiluminescent enzyme immunoassay for placental alkaline phosphatase in the cerebrospinal fluid of patients with intracranial germinomas.

BACKGROUND: Placental alkaline phosphatase (PLAP) in cerebrospinal fluid (CSF) has been proposed as a tumor marker for intracranial germinomas. The purpose of the present study was to develop a sensitive assay for measuring CSF PLAP and to evaluate the clinical significance of PLAP in patients with germinomas. METHODS: A chemiluminescent enzyme assay for PLAP was developed using an anti-human-PLAP monoclonal antibody. PLAP concentrations were determined in 37 controls, 36 germinomas, 3 nongerminomatous germ cell tumors, 21 gliomas and 12 other brain tumors. RESULTS: The assay detection limit was 5 pg/ml. The median PLAP concentration in the control group was below the detection limit. Significantly higher PLAP levels were detected in all 36 germinoma patients, with values ranging from 16 to 3,700 pg/ml. The high PLAP concentrations of 17 germinoma patients decreased to below the detection limit after complete remission had been achieved with radiochemotherapy. The sensitivity and specificity of PLAP for germinomas were 94 and 97%, respectively, with a cutoff value of 30 pg/ml. CONCLUSIONS: The results of this study suggest that the determination of CSF PLAP by the chemiluminescent method described here provides a clinically useful tumor marker for the diagnosis and monitoring of intracranial germinomas.

[Relationship between the effects of a high-fat meal and blood group in determination of alkaline phosphatase activity].

We previously reported that two isoforms of intestinal alkaline phosphatase (IAP) are present in the serum, a high-molecular-weight isoform(HIAP) and a normal-molecular-weight isoform (NIAP), and that both are present at high levels in blood group B or O secretors. In the present paper, we investigated the relationship between effects of high-fat meal and blood groups on ALP activity. Subjects fasted for 14 hours after dinner the previous evening and ate a high-fat meal the following morning. Two types of meals were prepared; a low-calorie meal (470 kcal), and a high-calorie meal (950 kcal). Subjects ate the 2 types of meal on different days. Blood was collected 3 times; once preprandially, and at 3 and 6 h postprandially. Among B or O secretors (n = 24), the mean +/- SD for increase in ALP activity after the high-fat meal was 26.4 +/- 10.2 U/L and 23.3 +/- 9.0 U/L at 3 and 6 h postprandially, respectively, following the low-calorie meal, and 47.9 +/- 19.9 U/L and 55.1 +/- 21.9 U/L at 3 and 6 h postprandially, respectively, after the high-calorie meal. Thus, ALP activity increased 2-fold after the high-calorie meal. Similarly, among subjects with other blood groups (n = 28), the increase in ALP activity was 5.7 +/- 3.7 U/L and 4.2 +/- 3.1 U/L at 3 and 6 h postprandially, respectively, with the low-calorie meal and 8.5 +/- 5.2 U/L and 10.6 +/- 6.0 U/L at 3 and 6 h postprandially, respectively, with the high-calorie meal. Thus, significant differences were seen between the blood groups (p < 0.001). The increases in ALP activity after the high-fat meal were nearly identical to the increases in NIAP activity. These results suggest that a high-fat meal is more likely to affect ALP activity in blood group B or O secretors, and that this effect peaks between 3 and 6 h after the high-fat meal. Taken together, the present results indicate that, as a rule, blood samples for determining ALP activity should be collected in the early morning with the patient in a fasted state.

Geometrical separation method for lipoproteins using bioformulated-fiber matrix electrophoresis: size of high-density lipoprotein does not reflect its density.

The increasing number of patients with metabolic syndrome is a critical global problem. In this study, we describe a novel geometrical electrophoretic separation method using a bioformulated-fiber matrix to analyze high-density lipoprotein (HDL) particles. HDL particles are generally considered to be a beneficial component of the cholesterol fraction. Conventional electrophoresis is widely used but is not necessarily suitable for analyzing HDL particles. Furthermore, a higher HDL density is generally believed to correlate with a smaller particle size. Here, we use a novel geometrical separation technique incorporating recently developed nanotechnology (Nata de Coco) to contradict this belief. A dyslipidemia patient given a 1-month treatment of fenofibrate showed an inverse relationship between HDL density and size. Direct microscopic observation and morphological observation of fractionated HDL particles confirmed a lack of relationship between particle density and size. This new technique may improve diagnostic accuracy and medical treatment for lipid related diseases.

Direct and simple fluorescence detection method for oxidized lipoproteins.

The quantification of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) is currently one of the most important clinical measurements for characterizing metabolic syndrome. However, recent studies have revealed additional factors that may be more strongly associated with the coronary heart disease than simple measurement of LDL or HDL levels, such as small dense (sd) LDL particles and oxidized LDL or HDL particles. Although several methods using enzyme-antibody detection systems or fluorescent probes have been devised to characterize these factors, such methods are expensive to implement for clinical measurements. Here, we present a straightforward analytical method for direct quantitation of oxidized lipoproteins by fluorescence spectrometry, with excitation in the UV (365 +/- 10 nm) or visible (470 +/- 10 nm) range and emission detected at 450 +/- 30 nm or 535 +/- 15 nm. This method can be readily applied for clinical measurement in patients with dyslipidemia using only 1 microL of 1 mg/mL of lipoprotein and without the need for any expensive detection antibodies. Using this new technique, biological samples from patients with dyslipidemia showed higher fluorescence intensities than samples from normal subjects when detecting oxidized LDL and light HDL (d = 1.063-1.125 g/mL), whereas samples from patients with dyslipidemia showed lower fluorescence intensities than samples from normal subjects when measuring oxidized heavy HDL (d = 1.125-1.210 g/mL) levels.

A promoter in the novel exon of hPPARgamma directs the circadian expression of PPARgamma.

AIM: PPARgamma (peroxisome proliferator-activated receptor gamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. Herein, we show that expression levels of the novel PPARgamma transcript exhibit circadian oscillation. To study the mechanisms controlling PPARgamma expression, a novel PPARgamma gene promoter was cloned and characterized. METHODS: We analyzed the novel PPARgamma promoter by luciferase reporter assays and gel shift analysis. RESULTS: Surprisingly, it was not an intron but rather the novel first exon of PPARgamma that was found to have functional minimal promoter activity. Luciferase reporter assays and gel shift assays revealed that the novel first exon is essential for novel PPARgamma promoter activation and that DBP (albumin gene D-site binding protein) and E4BP4 (E4 promoter A binding protein 4) bind directly to D-sites in the novel first exon. CONCLUSION: Our results demonstrate that the PAR-bZIP (bZIP, basic leucine zipper) family and E4BP4 are the main regulatory factors involved in oscillation of novel PPARgamma expression. This regulatory mechanism clearly differs from that of the circadian expression of PPARalpha.

Toxicity against gastric cancer cells by combined treatment with 5-fluorouracil and mitomycin c: implication in oxidative stress.

PURPOSE: Combined chemotherapy of 5-fluorouracil (5FU) and mitomycin c (MMC) is clinically used for gastric cancer, but the precise conditions and molecular mechanism of these agents when used together remain unclear. We examined the administration sequence of combining 5FU with MMC to maximize toxicity against a human gastric cancer cell line, and then investigated the possible molecular mechanisms underlying the observed toxic effects. METHODS: Human gastric cancer MKN45 cells were treated with a combination of 5FU and MMC, and the changes in cell viability and apoptosis-related proteins were determined by a tetrazolium dye-based cytotoxicity assay and Western blot analysis, respectively. The intracellular levels of reactive oxygen species (ROS) were monitored using a fluorescent probe or by a cytochrome c reduction assay. RESULTS: Pretreatment for 24 h with 5FU augmented the toxic effect of MMC in MKN45 cells. The synergic effect was mediated mainly via ROS formation and the p53-dependent apoptotic pathway, leading to mitochondrial dysfunction and caspase activation. In vitro experiments using extracts of the treated cells showed superoxide anion generation in a redox cycle of MMC, involving alterations in superoxide dismutase. CONCLUSIONS: Pretreatment with 5FU enhanced the MMC-induced toxicity against gastric cancer cells via alterations in antioxidant enzymes with resulting ROS generation. This observation will need confirmation in the clinical setting.

Activating effect of momordin, extract of bitter melon (Momordica Charantia L.), on the promoter of human PPARdelta.

AIM: Bitter melon (Momordica charantia L.) is a common vegetable grown in Okinawa that has also been used recently in medicine for the treatment of diseases such as diabetes, hypertension, and dyslipidemia. Among Bitter melon extracts compounds, we focused on an extract known as momordin in the present study, to examine its effect on peroxisome-proliferator activated-receptor (PPAR) delta (also called PPARdelta in rodents) expression and promoter activity of the human PPARdelta gene. METHODS: A human PPARdelta promoter-reporter plasmid was made as a template from a BAC CLONE (RPCI-11C) containing a -3076 bp (BglI site) +74 bp (EcoRI site) sequence. Luciferase assay of PPARdelta promoter activity was performed using HepG2 cells. RESULTS: 10 and 25 nM Momordin significantly increased the expression of PPARdelta mRNA 1.5-fold (relative to the control). Moreover, 10 and 25 nM Momordin significantly increased PPARdelta promoter activity in a dose-dependent manner, reaching more than 1.5-fold relative to the control. CONCLUSION: Our present data obtained through successful cloning of the PPARdelta promoter demonstrate that PPARdelta production and activation are upregulated through PPARdelta promoter activity following momordin treatment.

Lysophosphatidylcholine for efficient intestinal lipid absorption and lipoprotein secretion in caco-2 cells.

Phosphatidylcholine (PC) and its hydrolysates are considered to stimulate intestinal lipid absorption, however, their exact effects on lipoproteins and apolipoprotein (apo) metabolism remain ambiguous. This study aimed to further differentiate the effects of them using fully differentiated enterocyte-like Caco-2 cells. Lipid micelles (oleic acid 0.6, cholesterol 0.05, monooleylglycerol 0.2, taurocholate 2 in mmol/l) with or without choline, PC, and lysoPC (0.2 mmol/l each) were applied apically to Caco-2 cells. (3)H-oleic acid and (14)C-cholesterol were added to the micelles when necessary. Secreted lipoproteins were analyzed by a HPLC method. LysoPC had the most potent promoting effect on lipid uptake, and lipoprotein and apolipoprotein B-48 secretion among the molecules tested. LysoPC doubled the output of cholesterol and triglyceride as the lipoprotein component, but PC did not. On the other hand, PC only increased the secretion of apoA-IV in the presence of lipid micelles. These findings confirm that the alteration of PC by PLA(2) hydrolysis is intrinsically involved in the intestinal lipid absorption process and suggest that PC and its hydrolysis are coordinately associated with not only lipid absorption efficiency but also lipoprotein output and metabolism.

A possible role of lysophospholipids produced by calcium-independent phospholipase A(2) in membrane-raft budding and fission.

Phospholipase A(2) (PLA(2)) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-beta-cyclodextrin. Activation of calcium-independent PLA(2) (iPLA(2)) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p<0.01), which was blocked by PLA(2) inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (<10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA(2) to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA(2) may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA(2) may provide a therapeutic target for viral infections.

Role of lysophosphatidylcholine in brush-border intestinal alkaline phosphatase release and restoration.

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased approximately 10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.

Rapid and simple profiling of lipoproteins by polyacrylamide-gel disc electrophoresis to determine the heterogeneity of low-density lipoproteins (LDLs) including small, dense LDL.

This study aimed to explore the potential of polyacrylamide-gel disc electrophoresis (PAGE) for lipoprotein profiling in clinical practice. Blood samples were collected from 146 patients with type 2 diabetes mellitus and lipid parameters were assayed by PAGE, including small, dense low-density lipoprotein (LDL) (n = 41), and triglyceride-rich lipoprotein remnant cholesterol (n = 37). We also used a commercial kit to measure small, dense LDL (n = 41). By PAGE, we obtained the percentage of the area under the curve (AUC %) of each peaks and calculated respective AUC% x total cholesterol (AUC%xTC) values. The calculated values of LDL-AUC%xTC, small LDL-AUC%xTC, and HDL-AUC%xTC values were correlated well with values from homogeneous assay for LDL-cholesterol, small, dense LDL-cholesterol, and HDL-cholesterol assays (r = 0.94, 0.81, and 0.89, respectively). PAGE combined with measurement of total cholesterol and triglycerides provides a rapid evaluation of anti- or pro-atherogenic lipoproteins and a simple profiling system for both the "quantity" and "quality" of lipoproteins, allowing a better assessment of the risk of coronary artery diseases. This article discusses several methods for simple and rapid lipid profiling and outlines some recent patents relevant to the methods.

Tartrate-resistant acid phosphatase 5a and 5b contain distinct sugar moieties.

Tartrate-resistant acid phosphatase (TRAP) consists of 2 structurally related isoforms, 5a and 5b, and the latter has been characterized as containing a sugar moiety devoid of sialic acid(s). We provide evidence of a greater difference between the sugar moieties of the two isoforms than the presence of sialic acid(s) in TRAP 5a. TRAP 5a and 5b were highly purified from human blood and femur extracts, respectively, and after purifying the TRAPs by successive chromatography on a series of lectin affinity columns, we used the chromatographs obtained to estimate the composition of their sugar chain structure and found that both TRAP 5a and 5b had contained the heterogenous sugar chains. More specifically, TRAP 5a mainly contained a high-mannose-type sugar chain, while TRAP 5b had multi-antennary complex-type sugar chain. These results indicate that the differences between TRAP 5a and 5b consist not only of a difference in modification by sialic acid but differences in other sugar chain structures. These differences are involved in an extraordinary enzyme maturation process since the carbohydrate chains cover the repressive loop where the proteolytic site is located.

Cryptochrome and Period Proteins Are Regulated by the CLOCK/BMAL1 Gene: Crosstalk between the PPARs/RXRalpha-Regulated and CLOCK/BMAL1-Regulated Systems.

Feeding and the circadian system regulate lipid absorption and metabolism, and the expression of enzymes involved in lipid metabolism is believed to be directly controlled by the clock system. To investigate the interaction between the lipid metabolism system and the circadian system, we analyzed the effect of a CLOCK/BMAL1 heterodimer on the transcriptional regulation of PPAR-controlled genes through PPAR response elements (PPREs). Transcription of acyl-CoA oxidase, cellular retinol binding protein II (CRBPII), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was altered by CLOCK/BMAL1, and transcriptional activity via PPRE by PPARs/RXRalpha was enhanced by CLOCK/BMAL1 and/or by PPARs ligand/activators. We also found that CLOCK/BMAL1-mediated transcription of period (PER) and cryptochrome (CRY) was modulated by PPARalpha/RXRalpha. These results suggest that there may be crosstalk between the PPARs/RXRalpha-regulated system and the CLOCK/BMAL1-regulated system.

Statins Activate Human PPARalpha Promoter and Increase PPARalpha mRNA Expression and Activation in HepG2 Cells.

Statins increase peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA expression, but the mechanism of this increased PPARalpha production remains elusive. To examine the regulation of PPARalpha production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) on human PPARalpha promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1) Majority of statins enhanced PPARalpha promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPARalpha promoter. This enhancement may be mediated by statin-induced HNF-4alpha. (2) PPARalpha mRNA expression was increased by statin treatment. (3) The PPARalpha levels in nuclear fractions were increased by statin treatment. (4) Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPARalpha/RXRalpha expression vectors. In summary, these data demonstrate that PPARalpha production and activation are upregulated through the PPARalpha promoter activity by statin treatment.