Liver-type Alkaline Phosphatase Research Articles

Active-phase Plasma Alkaline Phosphatase Isozyme Activity Is a Sensitive Biomarker for Excessive Fructose Intake.

Excessive fructose intake reportedly leads to the development of nonalcoholic fatty liver disease (NAFLD). In our previous study, we reported that plasma activities of alkaline phosphatase (ALP) isozymes were markedly changed in rats with excessive fructose intake-induced hepatomegaly. In this study, we examined ALP isozyme activity prior to the occurrence of hepatomegaly, and investigated the effect of the timing of sample collection, to explore its potential as a biomarker. After 1-week intake of a 63% high-fructose diet (HFrD), blood samples were collected from male rats during sleep or active phases to analyze biochemical parameters. Body and liver weights were similar between the HFrD and control diet groups, indicating that hepatomegaly due to excessive fructose intake had not occurred. The triglyceride levels and glutamate dehydrogenase (GLDH) activity were significantly elevated to similar degrees at both time points. HFrD intake significantly increased liver-type ALP (L-ALP) activity, stimulating it by 12.7% at the sleep phase and by 124.3% at the active phase. HFrD consumption also significantly decreased intestinal-type ALP (I-ALP) at the active phase, but only showed a decreasing trend during the sleep phase. Measurements of plasma ALP isozyme and GLDH activity, and triglyceride levels are effective early biomarkers of impending NAFLD caused by excessive fructose intake. L-ALP and I-ALP activities during the active phase are particularly sensitive for detection of excessive fructose intake before the occurrence of NAFLD.

Novel Biomarker Establishment for Evaluation of Excessive Fructose Consumption Using a Rat Model.

The habitual consumption of excessive fructose is associated with the onset and progression of lifestyle-related diseases, such as nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the physiological changes observed when consuming a diet containing excessive fructose on the viewpoints of hepatotoxicity biological markers using a rat model and explored the biomarker candidates that could detect the effects of excessive fructose intake at an early stage. Male rats were fed 63% high fructose diet (HFrD) ad libitum and their blood samples were collected before and at 1, 2, 3, and 4 weeks after allocation. The plasma biochemical parameters, hepatotoxic enzyme activities including alkaline phosphatase (ALP) isozymes were analyzed. HFrD consumption for 4-weeks created NAFLD-like symptoms, including elevated plasma lipid parameters and hepatotoxicity markers, as well as fat accumulation in the liver compared with rats consuming a control diet. Alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) were increased from the 3rd and 2nd weeks, respectively, but no changes were observed on ALP activity. However, the daily consumption of the HFrD increased the plasma activities of liver-type ALP isozyme, and decreased plasma small intestinal-type ALP isozyme soon after the start of feeding. ALP isozyme analysis in combination with GLDH and ALT activities in the plasma samples could be a useful tool to detect the physiological changes induced by excessive fructose intake at an early stage of the development of NAFLD.

Alkaline phosphatase in rheumatoid arthritis patients: possible contribution of bone-type ALP to the raised activities of ALP in rheumatoid arthritis patients.

Raised serum alkaline phosphatase (ALP) activity in rheumatoid arthritis (RA) has been reported, although its aetiology is not clear. In this paper we investigate whether synovial tissue is a possible source of raised ALP activity in RA. The activities and isozymes of ALP were determined in sera and synovial fluids from 22 RA and seven osteoarthritis (OA) patients. The expression of both protein and ALP mRNA in synovial tissue was investigated immunohistochemically and by reverse transcription (RT) PCR. ALP activity was higher in serum and synovial fluid from RA patients than in those from OA patients. In addition, the ratio of levels of bone-type ALP to those of liver-type ALP was significantly higher in synovial fluid than in serum from RA patients. Bone-type ALP was positive around the perivascular area and the subepithelial cells in the synovial tissue from RA patients. In contrast, the synovial tissue from OA patients exhibited no staining. The mRNA of bone-type ALP was detected in RA synoviocytes. In conclusion, ALP levels were elevated in both serum and synovial fluid from RA patients. Bone-type ALP derived from the synovial tissue may contribute to the raised activities of ALP in RA patients.

Sodium butyrate-induced liver-type alkaline phosphatase activity in a small intestinal epithelial cell line, IEC6.

Sodium butyrate is a well-recognized differentiating agent inducing alkaline phosphatase activity, one of the epithelial differentiation markers. When IEC6 cells, a nontransformed, small intestinal epithelial cell line, were cultured with butyrate, this substrate induced alkaline phosphate activity in a time- and dose-dependent fashion. However, the type of isoenzyme involved was a liver-type, not an intestinal-type. Electron microscopy revealed that the induced activity was strictly localized in the cytosol and not on the plasma membrane. However, disaccharidase activities, another kind of differentiation marker, were also enhanced by sodium butyrate. In addition, the positive cells demonstrating the presence of alkaline phosphatase activity were preferentially observed in tubular structures. These data show that butyrate-induced alkaline phosphatase activity is closely associated with differentiation-like phenomena in IEC6 cells, although the type of isoenzyme and cellular localization of the activity are different from those observed in mucosa.

Effects of adrenalectomy on the increased serum alkaline phosphatase activity induced by DT-5061, an oral contraceptive, in rats

DT-5061, a steroid oral contraceptive which contains norethisterone (NET) and ethinyl estradiol (EE) was administered orally to adrenalectomized rats for 3 days to investigate involvement of the adrenals in the increased serum alkaline phosphatase (ALP). In addition, corticosterone or aldosterone were administered to the adrenalectomized rats to examine their effects on serum ALP. Increases in serum total ALP, liver-type and bone-type ALP isozymes were observed in rats with intact adrenals following administration of DT-5061, but these responses were not noted in adrenalectomized rats. Increases in liver weight and ALP activity in the liver after administration of the drug were reduced but not abolished in the adrenalectomized rats. The adrenalectomized rats receiving corticosterone showed increases in serum total ALP, liver-type ALP isozyme and liver weight but did not exhibit any increase in bone-type ALP isozyme. On the other hand, aldosterone did not increase and even reduced serum ALP although ALP activity in the liver was increased in the adrenalectomized rats.

Simultaneous Determinations of Liver- and Bone-Type Alkaline Phosphatase by Curve-Fitting of Inhibition Kinetic Data. II. Development and Evaluation of a Fluorescence-Based Method

We describe a kinetic method for the determination of alkaline phosphatase (ALP) isoenzymes, based on fluorescence detection of 4-methylumbelliferone. Several different buffer-inhibitor combinations and substrate concentrations were evaluated. Best results were obtained for inhibition with 2.9 mol/L urea in amino-2-methyl-1-propanol buffer. With this combination, normal concentrations of bone- and liver-type ALP could be determined from kinetic data during an 8-min measurement period. We computed initial velocities from parameters for first-order fits during 1.2 half-lives of the response for liver-type ALP. A linear least-squares fit of initial velocities (y) determined in this way vs results obtained with a comparison procedure (x) gave good correlations. We also estimated total signal changes, delta S, from first-order fits during four half-lives. Isoenzyme content correlated well with parameters computed from the first-order fits. Values for standard errors of the estimates represent 5% and 3% of median responses for activities and isoenzyme content, respectively. When compared with an absorbance-based method described previously, this method had threefold shorter measurement times, but imprecisions were 1.6- to 1.8-fold larger.

Prenatal diagnosis of infantile hypophosphatasia

Prenatal diagnosis was attempted in a pregnant Japanese woman whose son had died of infantile hypophosphatasia, using chorionic villi sampled at 10 weeks of gestation. Southern blot analysis of restriction fragment length polymorphism was used as a guide, with cDNA for the human liver-type alkaline phosphatase as a probe, and BclI as a restriction enzyme. The fetus was found to be a heterozygote; the pregnancy was allowed to continue; and the baby born was phenotypically normal.

Alkaline Phosphatase Expression in Human Cell Lines Derived from Primary Hepatomas

Isozyme patterns of alkaline phosphatase (ALP) were electrophoretically examined in human cell lines derived from one hepatoblastoma, five hepatocellular carcinomas (HCCs) and two cholangiocellular carcinomas. Most of the cell lines tested had a liver-type ALP isozyme. In addition, an abnormal ALP isozyme, which was similar to variant ALP, was detected in one hepatoblastoma and two HCC cell lines. One HCC cell line of these variant-like ALP-positive cell lines was alpha-fetoprotein (AFP)-negative. These findings suggest that variant-like ALP may be useful for the identification of human hepatoma cell lines, especially in AFP or albumin-negative cell lines.

Nucleotide sequence of the human liver-type alkaline phosphatase cDNA.

Nucleotide sequence of the human liver-type alkaline phosphatase cDNA.

Studies on alkaline phosphatase isoenzyme in gastric carcinoma tissues.

An analysis of the alkaline phosphatase (ALP) isoenzyme in 75 gastric carcinoma tissues was performed. The properties and the originating cells of ALP isoenzymes in them were clarified by using both enzyme histochemical and biochemical methods which had respective superiorities. Also the relationship between intestinal metaplasia and gastric carcinoma was discussed. Liver, placental and small intestinal type ALPs were found in the carcinoma tissues. No ALP activity was found in the carcinoma cells except in the stromal cells in 46 out of 75 cases. In all of these cases, only liver type ALP was found. While, in 29 cases, the activity was also found in carcinoma cells. In these cases, placental and small intestinal type ALPs were found in addition to liver type ALP in 26 and 3 cases, respectively These results indicated that liver type ALP came from the stromal cells and placental and small intestinal type ALPs came from carcinoma cells. The ALP found in carcinoma cells was placental (Nagao isoenzyme) type in most cases. While, the ALP in intestinal metaplastic epithelial cells was small intestinal type in all cases. No sufficient evidence indicating the relationship between intestinal metaplasia and gastric carcinoma was obtained in respect of ALP isoenzyme.

Alkaline phosphatase activity in human bladder tumor cell lines.

The cellular localization and isoenzyme pattern of alkaline phosphatase in five cell lines derived from human bladder carcinomas (T24, RT4, RT112, J82, EJ) shown not to be HeLa cells has been established. RT112 cells had a high level of alkaline phosphatase. RT4 had a moderate amount of alkaline phosphatase but in the other three lines, levels were extremely low. Prednisolone caused a small (2 to 3-fold) increase in total alkaline phosphatase in T24 and RT112 lines only. Electrophoretic separation of isoenzymes showed that RT112 and RT4 cells (derived from more highly differentiated tumor types) had three heat stable bands equivalent to placental alkaline phosphatase and three slower bands of a modified placental type. Prednisolone increased only the former. In T24 cells the enzyme resembled the liver-type alkaline phosphatase in electrophoretic mobility and sensitivity to heat denaturation. Cytochemical studies confirmed the presence of cell surface-associated extramembraneous placental type enzyme in RT112 cells. All five cell lines had small deposits of intramembraneous alkaline phosphatase in the plasma membrane and deposits associated tith the mitochondrial membranes and the endoplasmic reticulum that were not completely inhibited by phenylalanine or Levamisole.

Metabolic variants of intestinal alkaline phosphatase in relation to fat absorption: in situ demonstration with the organ-specific inhibitors L-phenylalanine and L-homoarginine.

The localization of rat intestinal alkaline phosphatase has been studied in relation to fat absorption. The observations support a theory of conversion, within the intestinal mucosa, of intestinal type to liver type alkaline phosphatase when the criterion of differential sensitivity to two amino acid inhibitors, l-phenylalanine, and l-homoarginine, is applied.

A comparison of chyle isoenzymes of alkaline phosphatase in chyle and hypophosphatasemic sera

A chylous effusion fluid was the source of “chyle” alkaline phosphatase which has been partially purified. It has been characterized with respect to migration on starch gel, effect of neuraminidase, reaction with rabbit antisera to liver and to intestinal isoenzymes and with regard to its inhibition by l-phenylalanine and l-homoarginine. The major component has been identified as intestinal-type alkaline phosphatase and a minor component has the properties of liver-type alkaline phosphatase. The identical results were obtained in the examination of the sera of two hypophosphatasemic children which suggests a major chylous source of the alkaline phosphatase in hypophosphatasemia.

Origins of serum alkaline phosphatase.

A very rapid method of agar gel electrophoresis on glass slides, together with a superior visualization technique employing simultaneous coupling of a hydrolysed naphthol substrate, have been developed for the identification of the tissues of origin of serum alkaline phosphatase. Combined with L-phenylalanine inhibition, specific for the intestinal enzyme, and heat inactivation, specific for the placental enzyme, the heterogeneity of serum alkaline phosphatase has been demonstrated. Normal adult serum contains predominantly liver-type alkaline phosphatase with a small but variable quantity of intestinal enzyme, and little or no bone enzyme. In childhood and in infancy there is in addition a bone isoenzyme present, the amount gradually falling to adult levels with age. In pregnancy, the rise in serum alkaline phosphatase is due to the placental enzyme.A study of nearly 2,000 sera has been undertaken and it is found that the bone enzyme is increased in osteoblastic bone diseases while in hepato-biliary disorders there is an increase in liver type enzyme. The main theories explaining the rise in serum alkaline phosphatase are examined.