Fructose 1-phosphate Aldolase Research Articles

Use of FDP and F1P aldolase to detect tumorous and nontumorous tissue damage by ethanol injection of hepatocellular carcinoma.

Changes in serum levels of tumor-specific fructose 1,6-diphosphate (FDP) aldolase and nontumor-specific fructose 1-phosphate (F1P) aldolase activities were analyzed in patients with hepatocellular carcinoma (HCC) to detect the damage of tumorous and nontumorous hepatic cells after percutaneous ethanol injection (PEI). Initial PEI was performed in 20 patients containing 22 HCC nodules with a diameter of < or = 4 cm. Changes in serum hepatic enzyme activities were measured before and after repeated PEI. FDP and F1P aldolase levels were measured by substrate-specific enzymatic methods. Pre- and posttreatment alpha-fetoprotein (AFP) levels were determined by radioimmunoassay. The consequent changes in the total nontumorous liver volumes after PEI were also analyzed by follow-up CT scans. Serum levels of FDP aldolase released by ethanol injection were progressively increased (P < 0.0001) until the third PEI and thereafter decreased. In contrast, serum levels of F1P aldolase were continuously elevated even after the third PEI (P < 0.0001). Serum levels of transaminases were also elevated after repeated PEI (P < 0.0001). The FDP/FIP aldolase ratio decreased significantly with increased volume (>20 ml) of injected ethanol (P = 0.01) caused by nontumorous liver damage. The elevation of FDP aldolase was markedly associated with a decrease in serum levels of AFP (P < 0.001), indicating adequate tumor necrosis. The progression of the total nontumor liver atrophy depended on the volume of injected ethanol and correlated significantly with F1P aldolase levels after PEI (P < 0.01) but not with FDP aldolase. These results demonstrated that caution is needed to avoid nontumorous liver damage caused by the large volume of ethanol injection in treating HCC. Measurement of FDP and F1P aldolase activities in serum after PEI is clinically useful to detect the degree of tumorous and nontumorous tissue damage by ethanol.

Use of fructose 1,6-diphosphate aldolase to detect tumour necrosis after transcatheter arterial embolization of hepatocellular carcinoma.

Transcatheter arterial embolization (TAE) is a popular and well-established devascularization treatment modality for hepatocellular carcinoma (HCC). The persistent retention of lipiodol on follow-up computed tomography (CT) scan and time-dependent decrease in size of the lipiodol-stained area of tumour after TAE does not reveal the biological death of tumour cells. Moreover, it is difficult to clinically evaluate the effective necrosis of tumour cells by TAE in cases of HCC that do not produce alpha-fetoprotein (AFP). We therefore studied the release of a relatively tumour-specific protein by the necrotic hepatoma cells to evaluate the effectiveness of TAE. Transcatheter arterial embolization was performed in 17 patients with the imaging diagnosis of HCC; either superselective (n = 6) or non-superselective (n = 11) techniques were used. We measured serum levels of relatively tumour-specific fructose 1,6-diphosphate (FDP) aldolase and non-tumour-specific fructose 1-phosphate (F1P) aldolase by substrate-specific enzymatic methods. Enzyme activities were performed before and after TAE. The time-dependent decrease in size of the lipiodol-stained areas was studied on follow-up CT scans after TAE. Pre- and post-treatment serum AFP levels were determined by radio-immunoassay. The six cases of superselective TAE underwent marked tumour regression by CT compared with the 11 cases of non-superselective TAE. Fructase 1,6-diphosphate aldolase output correlated well with post-necrotic tumour regression after TAE (r = 0.87, P= 0.001). The elevation of serum FDP aldolase was also significantly associated with a decrease in serum AFP (r = 0.72, P < 0.01). In contrast, serum F1P aldolase output was inversely correlated with either tumour regression or serum AFP concentrations after TAE. The serum levels of the tumour-specific enzyme FDP aldolase correlated significantly with effective tumour necrosis and consequent tumour regression after TAE. We suggest that measurement of FDP aldolase activity in serum after TAE can be used clinically to detect the degree of tumour necrosis by TAE.

Glucose fermentation to acetate and alanine in resting cell suspensions ofPyrococcus furiosus: Proposal of a novel glycolytic pathway based on13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1- 13 C]glucose or [3- 13 C]glucose, consumed glucose at a rate of about 10 nmol min −1 (mg protein) −1 . Acetate (10 mM), alanine (3 mM), CO 2 and H 2 were the fermentation products. The 13 C-labelling pattern in alamine and acetate were analyzed. With [1- 13 C]glucose the methyl group of both alanine and acetate was labelled; with [3- 13 C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg −1 , 100°C), ketohexokinase (0.23 U mg −1 , 100°C), and fructose 1-phosphate aldolase (0.06 U mg −1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [ 13 C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.

Evaluation of nontumorous tissue damage by transcatheter arterial embolization for hepatocellular carcinoma.

The serial changes in serum hepatic enzyme activities by transcatheter arterial embolization (TAE) were analyzed in 17 patients with hepatocellular carcinoma to estimate the contribution to the value by the damage of tumor or nontumorous hepatic cells. The serum levels of relatively tumor-specific fructose 1,6-diphosphate (FDP) aldolase were elevated after TAE in the cases of both superselective and nonsuperselective TAE that were performed from the segmental and the nonsegmental hepatic artery, respectively, but we found the marked elevation of FDP aldolase in the cases of the superselective TAE. In contrast, the non-tumor-specific fructose 1-phosphate (F1P) aldolase was markedly elevated only in the cases of nonsuperselective TAE. The total amount of FDP aldolase released by TAE correlated significantly with the integrated tumor tissue volume (P less than 0.005), whereas the total amount of F1P aldolase output correlated significantly with the integrated nontumorous tissue volume (P less than 0.005) as defined by lipiodol accumulation on computerized tomography scan. The consequent changes in the total nontumorous liver volumes after TAE were also analyzed by the follow-up computerized tomography scan. The nonsuperselective TAE caused the significant total nontumorous liver atrophy when compared with the superselective TAE. The progression of the total nontumorous liver atrophy correlated significantly with F1P aldolase output by TAE (P less than 0.001) but not with FDP aldolase output. These results suggest that the outputs of FDP and F1P aldolase are useful to estimate the degree of the tumorous and nontumorous tissue damage by TAE, respectively, and F1P aldolase output can be used to predict the progression of liver atrophy caused by TAE.

Effect of chronic caloric restriction on hepatic enzymes of intermediary metabolism in the male Fischer 344 rat

It is well established that caloric restriction extends life span and significantly retards the rate of occurrence of most age-associated degenerative disease processes. A paucity of data exists relative to the mechanisms by which caloric restriction accomplishes these events. We have examined the effect of caloric restriction in rats on several hepatic enzymes of intermediary metabolism. The activities of glycolytic and supporting enzymes including lactate dehydrogenase, pyruvate kinase, sorbitol dehydrogenase, and alcohol dehydrogenase were all decreased in response to caloric restriction. Fructose 1-phosphate aldolase and creatine phpshokinase were not altered. Likewise, enzymes associated with lipid metabolism (malic enzyme and glycerokinase) were reduced (fatty acid synthetase was reduced, but not to a statistically significant degree). Activities of enzymes supporting gluconeogenesis (glutamate oxaloacetate transaminase, tyrosine aminotransferase, glutamate pyruvate transaminase, glutamate dehydrogenase, amino acid oxidase, malate dehydrogenase, and glucose 6-phosphatase) were either unchanged or increased significantly by caloric restriction. Glucagon levels were decreased. Comparisons between young ad libitum fed and older calorically restricted rats revealed similar but not identical metabolic activity. These results suggest that caloric restriction produces an effect on intermediary metabolism, favoring the role of glucagon and glucose synthesis; but limiting the role of insulin and glucose catabolism in the liver. The former observation provides for the efficient support of peripheral tissues and the latter a level of energy production necessary only for self maintenance. Limited lipid metabolism suggests decreased potential for fatty acid epoxide formation and free radical damage to cellular macromolecules. Additionally, caloric restriction may delay the progressive age associated changes in the activities of some of the enzymes investigated.

Hereditary fructose intolerance: presence of a potent inhibitor of fructose 1-phosphate aldolase in the liver

Hereditary fructose intolerance: presence of a potent inhibitor of fructose 1-phosphate aldolase in the liver

Enzymic studies on the lenses of alloxan-diabetic rats.

The accumulation of sorbitol has been described as one of the causative factors of the diabetic cataract (Kinoshita, 1974; Dvornik et al., 1973), and hyperglycaemia seems to have a potentiating effect on the formation of the senile type of cataract. Pirie & van Heyningen (1964) found that, in the cataractous lenses of the diabetic patients, more glucose and fructose accumulated than in non-diabetic cataractous lenses. The observations indicate that inhibition of metabolism of the two substrates occurs in both types of cataracts. The present work describes the enzyme activities of the cataractous lenses in experimental diabetes. The method of Gomori & Goldner (1943) was adopted for producing alloxan-diabetes and hyperglycaemia. Alloxan was injected intraperitoneally (200mg/kg body wt.) to the animals starved overnight. From0 to 9 days after alloxan treatment five animals were randomly picked, and blood samples were collected after decapitation. Plasma was used for determination of glucose by the method of Raabo & Terkildsen (1960). The lenses were taken out immediately and preserved in 50% (v/v) buffered glycerol at 20°C. Some animals were left until 60 days after alloxan treatment, when the initiation of opacity was observed. In a 10% (w/v) homogenate of each lens in Tris buffer (20mM, pH7.8) protein was determined by the method of Lowry et al. (1951). A portion was centrifuged at 12000g at OIf : 1°C for 1 h and the supernatant was used for the enzyme assays. Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), lactate dehydrogenase (EC 1.1,1.27), phosphofructokinase (EC 2.7.1.1 1) and fructose 1-phosphate aldolase (EC 4.1.2.13) were assayed by the method of Burch et al. (1963). In the case of phosphofructokinase, KCI was omitted from the reagent mixture and 20m~-Na,HPo, and 5-AMP (1mM) were added as recommended by Burch et al. (1963). The assay temperature for these enzymes was 38°C. An Aminco-Bowman spectrophotofluorimeter was used for determining the fluorescence of NADH and NADPH or that of NAD+-alkali complex. The activity of ATP citrate lyase (EC 4.1.3.8) was assayed at 25°C with a Beckman DU-2 spectrophotometer by the method of Srere (1 962). Plasma glucose of the alloxan-treated animals reached a peak of 398.4f 3.2mg/100ml after 4 days of alloxan treatment, whereas the lens weights increased after 60 days and the amount of lens protein decreased significantly (P < 0.001). Glucose 6-phosphate dehydrogenase activity per g fresh wt., its specific activity (per mg of protein) and the total activity (per whole lens) decreased after 5 days of alloxan treatment. In the case of 6phosphogluconate dehydrogenase, the activity per g fresh wt. decreased after 6 days, whereas a significant decrease was observed in the specific and the total activities after 8 days. The activities of both the enzymes after 60 days of alloxan treatment were lower than in the control group. The activity per g fresh wt., the specific activity and the total activity of phosphofructokinase and lactate dehydrogenase decreased after 4 and 5 days respectively. The total activity of phosphofructokinase increased after 6 days in comparison with that observed after 8 days. The activities of both the enzymes, as measured per g fresh wt., per mg of protein and per whole lens, decreased markedly after 60 days, but in the case of phosphofructokinase the decrease was most conspicuous. Fructose 1-phosphate aldolase activity per g fresh wt. and the total activity showed a decrease after 60 days. In the case of ATP citrate lyase, the activities decreased after 3 days, but the specific and the total activities returned to normal values after 60 days. The decrease in the activities of the enzymes of the hexose monophosphate shunt seems to be an adaptive phenomenon by which the supply of the necessary coenzyme NADPH for aldose reductase is decreased. In the case of phosphofructokinase, a precipitous fall in the activity coincided with the initiation of opacities. The lens ATP citrate lyase is more susceptible to hyperglycaemia than the liver enzyme (Kornacker & Lowenstein, 1965) and also has a shorter time of recovery.

Comparison of the adaptive changes in disaccharidase, glycolytic enzyme and fructosediphosphatase activities after intravenous and oral glucose in normal men

Seven subjects were fed a 3,000 kcal defined formula diet daily for 19 days. Except for one 5-day period, 50% of the total caloric intake was provided as either oral or intravenous glucose. The study was divided into four periods as follows: period I lasted 5 days and provided 50% of calories as glucose; period II lasted 5 days and provided no carbohydrate (70% fat and 30% protein); period III lasted 4 days and provided 50% of calories as intravenous glucose and 50% of calories as oral fat plus protein; period IV lasted 5 days and provided 50% of calories as oral glucose. Intestinal biopsy specimens were taken on days 3 and 5 of each period, except period III when biopsies were done only on day 4. No change in intestinal morphology occurred during the study. The carbohydrate-free diet caused the alpha-glucosidase (maltase and sucrase) activities to decrease significantly from that seen with the glucose diet. Sucrase decreased from 14.4 +/- 1.0 to 7.1 +/- 0.9 mumoles/min per g tissue and maltase decreased from 56.1 +/- 3.4 to 30.0 +/- 2.1 mumoles/min per g tissue. Glycolytic enzyme activities decreased during the carbohydrate-free period (pyruvate kinase decreased from 236 +/- 12 to 78 +/- 8, fructose 1-phosphate aldolase decreased from 147 +/- 6 to 53 +/- 4, fructose-1,6-diphosphate aldolase decreased from 151 +/- 8 to 55 +/- 3, and hexokinase decreased from 21 +/- 3 to 7 +/- 1 nmoles/min per mg protein, respectively). Intravenous glucose caused no change in disaccharidase activities. The enzyme activities during periods I and IV were identical and significantly higher than during period II with the exception of fructose-1,6-diphosphatase which increased during period II as compared with periods I and IV. These findings provide an explanation for the transient period of decreased tolerance to dietary sugars when patients are weaned from total parenteral feedings to enteral feedings.

Regulation of fructose metabolism in the perfused rat liver: Interrelation with inorganic phosphate, glucose, ketone body and ethanol metabolism

1. The regulation of fructose metabolism was investigated in rat liver using a non-recirculating perfussion system. 2. 1. At 25 mM fructose the rate of fructose uptake was 6.6 μmoles·min −1·g −1. Calculated per whole liver the rate of fructose uptake was independent of the nutritional state, whereas the rate of fructose uptake calculated per g liver increased 25% by 48 h fasting. 3. 2. The rate of fructose uptake was diminished in the first (20 min) period after addition of fructose. Glucose up to 25 mM did not significantly decrease the rate of fructose uptake indicating a minor contribution of hexokinase (ATP: d-hexone 6-phosphotransferase, EC 2.7.1.1) in phosphorylating fructose. The rate of fructose uptake was increased 25% by increasing the P i concentration from 1 to 5 mM in the affluent medium. Ethanol caused a decreased in the rate of fructose phosphorylation. This was counterpoised by an increased rate of sorbitol production. 4. 3. 5 mM P i caused an increased rate of O 2 uptake with and without fructose, and the concentration of ATP during steady-state fructose metabolism was increased 25% which was enough to account for the increase in the rate of fructose uptake (ketohexokinase (ATP: d-fructose 1-phosphotransferase, EC 2.7.1.3) K m with ATP , 0.9 mM). 5. 4. At a high NAD redox level which was seen in the initial 10-min period after addition of fructose and after addition of ethanol, glycerol and sorbitol were released from the liver. The kinetics of sorbitol release reflected the intracellular fructose concentration. 6. 5. In livers of fed but not of fasted animals the release of glucose was inhibited in the initial 10-min period after addition of fructose. The inhibition is related to an increased inhibition of fructose 1-phosphate aldolase and fructose diphosphatase in the fed animal.

The interrelationship of the concentration of hydrogen ions, bicarbonate ions, carbon dioxide and calcium ions in the regulation of renal gluconeogenesis in the rat.

1. The interrelationship of acidosis and Ca(2+) on the stimulation of gluconeogenesis by rat kidney-cortex slices was studied. 2. Ca(2+) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate, lactate and fructose, but not from galactose. 3. The [Ca(2+)] needed for optimum gluconeogenesis was about 2mm, but at this concentration, acidosis, produced in vitro by a decrease of [HCO(3) (-)] in the medium at constant pCO(2) or by an increase in pCO(2) at constant [HCO(3) (-)], did not stimulate gluconeogenesis. 4. In the absence of Ca(2+), acidosis (low [HCO(3) (-)]) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate and lactate but not from fructose or galactose. With succinate as substrate, the stimulatory effect of acidosis (low [HCO(3) (-)]) disappeared at Ca(2+) concentrations above 1.0mm. 5. The [HCO(3) (-)] was the most important determinant of the acidosis effect since a decrease of pH caused by an increase in pCO(2) did not uniformly stimulate gluconeogenesis, whereas a decrease in [HCO(3) (-)] without a change in pH consistently stimulated glucose formation in a way similar to the stimulation produced by acidosis (low [HCO(3) (-)]) in the absence of Ca(2+). 6. Acidosis in vitro inhibited the rate of decrease of activity of phosphoenolpyruvate carboxylase in slices, and Ca(2+) caused an increase in the activity of fructose 1-phosphate aldolase. 7. Respiratory acidosis in vitro caused an increase in the activity of phosphoenolpyruvate carboxylase in kidney cortex and an increase in gluconeogenesis from glutamine. 8. Possible points of interaction between Ca(2+), H(+) and HCO(3) (-) with the gluconeogenic sequence are discussed.

High liver glycogen in hereditary fructose intolerance.

A case of hereditary fructose intolerance is reported in a girl aged 2 years at the time of her death. She had apparently progressed normally until the age of 14 months. At 19 months she was admitted to hospital with failure to thrive, hepatomegaly, and superficial infections. Investigations revealed hypoglycaemia, persistent acidosis, aminoaciduria, and a high liver glycogen level which suggested that she had glycogen storage disease. There was also some evidence of malabsorption. At necropsy the liver enzyme estimations showed that fructose 1-phosphate aldolase activity was absent and that fructose 1,6-diphosphate aldolase activity was reduced. Hereditary fructose intolerance and glycogen storage disease have been confused in the past on clinical grounds, but a high liver glycogen level has not previously been reported in hereditary fructose intolerance.