A subchronic intravenous toxicity study of magnesium fructose-1,6-diphosphate in beagle dogs.

Magnesium fructose-1,6-diphosphate is a novel agent of antimyocardial ischaemia. In the present study, the subchronic toxicity of magnesium fructose-1,6-diphosphate was investigated after 13-week repeated intravenous administration in beagle dogs. The animals received doses of 0, 75, 150 and 300 mg/kg/day (three males and three females for each dose). During the study period, clinical signs, mortality, body weights, food consumption, electrocardiogram, urinalysis, haematology, clinical biochemistry, macroscopic findings, organ weights and histopathology were examined. The administration of magnesium fructose-1,6-diphosphate resulted in increased incidence of clinical signs, including salivation and emesis. These effects were transient and were noted in almost all dogs given 300 mg/kg/day and occasionally noted in the 150 mg/kg/day dose-treated animals. Serum magnesium in the 150 mg/kg/day and 300 mg/kg/day dose-treated animals was significantly increased after 6- and 13-week administration, but recovered at the end of a 2-week recovery period. At 6 weeks, a statistically significant decrease in serum electrolytes, including sodium and potassium, was observed in the treatment groups. There were no other treatment-related findings. Under the conditions of the present study, magnesium fructose-1,6-diphosphate did not show any evidence of target organ toxicity. The no-observed-adverse-effect level for 13-week intravenous administration of magnesium fructose-1,6-diphosphate to beagle dogs was considered 75 mg/kg/day based on observations of clinical signs and serum electrolytes.

A modelling study of feedforward activation in human erythrocyte glycolysis.

Though feedforward activation (FA) is a little known principle of control in metabolic networks, there is one well-known example; namely, the activation of pyruvate kinase (PK) by fructose-1,6-biphosphate (FBP) in glycolysis. The effects of this activation on the enzyme's kinetics are well characterised, but its possible role in glycolytic control has not been determined, and, experimentally, there is as yet no direct way of modifying the enzyme to remove just the FBP activation without affecting other aspects of the enzyme's kinetics. Given this limitation, we used a detailed numerical simulation of human erythrocyte glycolysis to simulate the effects of selective removal of the activation of PK by FBP on steady-state metabolite concentrations and on the dynamic response of glycolytic flux to a sudden increase of the cell's demand for ATP. Our modelling results predict that in the absence of FA steady-state levels of metabolites within the activation loop, i.e. from FBP to phosphoenolpyruvate, would be four- to thirteen-fold higher than normal, whereas levels of ATP and metabolites outside the loop, i.e. glucose-6-phosphate, fructose-6-phosphate and pyruvate, would be lower than normal. Existing clinical evidence in a patient with haemolytic anaemia, correlated with a lack of activation of PK by FBP (Paglia D.E., Valentine W.N., Holbrook C.T., Brockway R., Blood (1983) 62 972-979), is consistent with this prediction. In response to changing demand for ATP, the model predicts that the corresponding change of glycolytic flux would entail changes of metabolite concentrations in the absence of FA, but that in its presence the levels of metabolites within the activation loop remain essentially unperturbed. Thus, our results suggest that by stabilising metabolite pools in the face of variable glycolytic flux, FA may serve to avoid perturbations of the oxygen affinity of haemoglobin (sensitive to the levels of 2,3-phosphoglycerate) and of cell osmolality that would otherwise occur during variations in the cell's demand for ATP. In addition, by significantly raising the steady-state setpoint of intermediate metabolite pools, the productivity index (ratio of glycolytic flux to total metabolites in the pathway) of glycolysis would fall almost four-fold in the absence of forward activation.

[Changes in the level of fructose-2,6-biphosphate in peripheral blood lymphocytes in patients with diabetes mellitus]. / Izmenenie soderzhaniia fruktozo-2,6-bifosfata v limfotsitakh perifericheskoi krovi bol'nykh sakharnym diabetom.

Variation of fructose 2,6-bisphosphate (F-2,6-P2) content in the peripheral blood lymphocytes of patients with diabetes mellitus (type II) was investigated. The 2.5-fold increase of F-2,6-P2 level compared with to the control group was observed in untreated patients with early stage of the disease. In patients using of peroral antidiabetic drugs that corrected diabetes F-2,6-P2 level was approached to the normal one. At the same time F-2,6-P2 content decreases in patients with severe (noncorrected) diabetes mellitus. The possible mechanisms of regulation of lymphocyte F-2,6-P2 concentration in normal conditions and diabetes mellitus are discussed.

Toxicity of fructose-1,6-bisphosphate in developing normoxic rats.

Giving 500 mg/kg of fructose-1,6-bisphosphate intraperitoneally decreases hypoxic/ischaemic CNS injury of neonatal rats. Before administering fructose-1,6-bisphosphate to human neonates, its toxicity must be determined in neonatal animals. Thus, saline or 4,000, 6,000, or 8,000 mg/kg of fructose-1,6-bisphosphate was given intraperitoneally to normoxic 7 days old rats. One, 2, and 24 hr and 7 days later, blood Ca2+, PO(4)3-, blood urea nitrogen, and creatinine concentrations, and aspartate aminotransferase activity were measured. Organ pathology was determined at necropsy. Pups receiving 4,000 mg/kg of fructose-1,6-bisphosphate survived without evidence of injury or toxicity. All animals receiving 8,000 mg/kg and 27 percent of those receiving 6,000 mg/kg of fructose-1,6-bisphosphate died. Surviving fructose-1,6-bisphosphate-treated animals grew at the same rates and had similar weights as saline-treated animals. Nineteen percent of pups given 6,000 or 8,000 mg/kg of fructose-1,6-bisphosphate had mild perivascular fluid cuffing and/or microscopic pulmonary haemorrhage, but none of the animals given 4,000 mg/kg of the compound had evidence of injury. No other organ pathology was found in any of the animals. Renal and hepatic function were normal in all animals. Fructose-1,6-bisphosphate administration was associated with a significant increase in the fructose-1,6-bisphosphate concentration of blood. Administering 4,000 to 8,000 mg/kg of fructose-1,6-bisphosphate significantly decreased Ca2+ concentrations and increased PO(4)3- concentrations 1 and 2 hrs after fructose-1,6-bisphosphate administration. Similar changes in Ca2+ and PO(4)3- concentrations occurred after the administration of 10 mmol/kg of sodium phosphate. The wide margin of safety for fructose-1,6-bisphosphate (8 times the dose needed to prevent or reduce CNS injury) may render fructose-1,6-bisphosphate safe for use in neonates.

Hereditary deficiency of lactate dehydrogenase H-subunit.

We report herein the fifth family of hereditary deficiency of lactate dehydrogenase (LDH) H-subunit with an autosomal recessive inheritance including two cases of complete deficiency. Their LDH activities were low both in the serum and in the red blood cells (RBC). Electrophoretic analysis revealed that the patients with the complete deficiency had only the LDH5 isozyme. The complete deficiency was associated with marked elevation of fructose-1, 6-diphosphate (FDP) and dihydroxyacetonephosphate (DHAP) and a less marked rise in glyceraldehyde-3-phosphate (GA3P) among glycolytic intermediates in the RBC. Furthermore, hemolysis was observed in the present cases, but this finding was not included in the other reports.

[Fructose-2,6-bisphosphate system and experimental states]. / Sistema fruktozo-2,6-bisfosfata i éksperimental'nye sostoianiia.

The molecular mechanisms of the inhibitory action of fructose- 2,6-bisphosphate (F-2,6-P2) on fructose-1,6-biphosphatase (FB-Pase-1), the key enzyme of gluconeogenesis, and the those of the activating action of F-2,6-P2 on phosphofructo-1-kinase (PFK-1), the key enzyme of glycolysis, NMR spectroscopy first provided direct evidence for the fact that F-2,6-P2 was involved in the regulation of the sedoheptulose cycle of a nonoxidative stage of the pentosephosphate pathway. Procedures were developed in measuring the levels of F-2,6-P2 in the cell of experimental animal tissues and human blood lymphocytes. Naturally different emergencies substantially affected the F-2,6-P2 system by triggering these or those mechanisms controlling the activity of enzymes of this system. Vanadium-containing compounds were demonstrated to have a positive action on carbohydrate metabolism in diabetic (streptozotocin-induced) rat hepatocytes.

Alteration of aldolase isozymes in serum and tissues of patients with cancer and other diseases.

We studied the alteration of aldolase isozymes in the serum and tissues of patients with cancer and other diseases using radioimmunoassays specific for aldolase A, B, and C subunits. Aldolase B was predominantly found in adult liver, where aldolase A and C were distinctly low. Aldolase A and B showed almost the same concentration in fetal liver, while in neonatal liver aldolase B protein concentrations were much higher than aldolase A. In contrast, aldolase A was the predominant isozyme found in hepatoma and gastric cancer tissues, whereas aldolase B was distinctly low in hepatoma tissues, and extremely low in gastric cancer tissues. These results suggest that the aldolase A is a more fetal type of liver isozyme than the aldolase B and C, and aldolase B is a more differentiated type of liver isozyme than aldolase A and C. Serum FDP aldolase activities were elevated in half of patients with liver diseases, all patients with muscle diseases and a few patients with cancer. Serum aldolase A levels were elevated in patients with muscle diseases and cancer, but not elevated in patients with liver diseases. In contrast, serum aldolase B levels were elevated in patients with liver disease, but not elevated in patients with muscle diseases and other diseases without liver injury. Serum aldolase B levels showed a trend to decrease in cancer patients with normal GPT levels. Serum aldolase A/B ratios were significantly increased in cancer patients with normal GPT levels, whereas they showed the decreased levels in patients with liver diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Fructose-1,6-bisphosphate does not ameliorate hypoxic ischemic injury to the central nervous system in the newborn pig.

BACKGROUND AND METHODS: Fructose-1,6-bisphosphate has been shown to improve the outcome of hypoxic ischemic brain injury in adult rabbits. We wished to see if these results could be extended to a newborn animal. Twenty-four 0- to 3-day-old piglets were randomized to receive 300 mg/kg of fructose-1,6-bisphosphate 5 mins before injury, followed by a continuous infusion of 15 mg/kg/min of fructose-1,6-bisphosphate for the next 90 mins, or the equivalent volume of normal saline. Hypoxic ischemic central nervous system damage was induced by ligating both carotid arteries and reducing their BP to two thirds of the normal value for 30 mins. In the last 15 mins of this 30 mins, the FIO2 was reduced to 0.6. At 30 mins, the piglets were resuscitated with an FIO2 of 1.0, the carotid ligatures were released, and the removed blood was reinfused. RESULTS: The neurologic examination scores at 1, 2, and 3 days after injury and pathologic examination scores at 3 days after injury were not different in the fructose-1,6-bisphosphate-treated and the control animals. CONCLUSION: Fructose-1,6-bisphosphate does not ameliorate hypoxic ischemic brain injury in the newborn pig.

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes.

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.

Activating effect of adenosine on rat erythrocyte glycolysis.

1. Adenosine increases the adenine nucleotide pool in rat erythrocytes. Hence, we tested the effect of the nucleoside on the glycolytic pathway in red blood cells. 2. A 2.5-fold increase in the level of fructose-1,6-bisphosphate and a 34% augmentation in lactate pool were observed in rat erythrocytes, 30 min after adenosine treatment. 3. Under conditions preventing adenosine metabolism, 1 microM nucleoside addition to isolated erythrocytes induced an 89% increase in lactate production and an increase in glucose consumption. 4. Activation of red cell phosphofructokinase (PFK) is produced by addition of microM concentrations of adenosine. Our data suggest a role for adenosine in the glycolysis flux regulation through PFK activation.

An inositol phosphoglycan stimulates glycolysis in human platelets.

Upon hydrolysis of membrane glycosyl-phosphatidylinositol (gly-PtdIns), an inositol phosphoglycan (IPG) is generated, responsible for multiple biological activities and recently proposed as mediator of the action of a variety of hormones and growth factors. The present study shows that IPG is able to significantly stimulate platelet glycolysis, which represents the major energy producing pathway in this cell system. The activation of glycolytic flux induced by IPG appears to be specific and very rapid even though the molecular mechanism involved remains to be elucidated.

Malonyl-CoA in skeletal muscle and liver of streptozotocin-diabetic rats.

Malonyl-CoA, the inhibitor of carnitine palmitoyl transferase I, has been examined in this study in the muscle and liver of diabetic rats. Male Sprague-Dawley rats were rendered diabetic with streptozotocin (6 mg/100 g body wt). The gastrocnemius/plantaris muscles and liver samples were frozen at liquid nitrogen temperature. Muscle malonyl-CoA was 1.8 +/- 0.2 pmol/mg in control rats and 1.5 +/- 0.2 pmol/mg in the diabetic rats. This difference was not statistically significant. Liver malonyl-CoA of control rats was 8.6 +/- 0.8 pmol/mg, in comparison to 4.3 +/- 0.6 pmol/mg in diabetic rats. In the liver, high concentrations of malonyl-CoA inhibit fatty acid oxidation and ketogenesis. Failure of malonyl-CoA to decline in muscle in the diabetic may be responsible in part for the diversion of fatty acids to the liver, thereby enhancing hepatic fatty acid oxidation and ketogenesis.

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.

Erythrocyte fructose 2,6-bisphosphate content in congenital hemolytic anemias.

We have investigated the levels of fructose 2,6-bisphosphate and its synthesizing enzyme 6-phosphofructo-2-kinase in red blood cells from different congenital anemias. Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity are markedly influenced by the number of reticulocytes in all the cases studied with the exception of homozygous pyruvate kinase deficiency, where no correlation was observed with the percentage of reticulocytes.

2,3-Bisphosphoglycerate, fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content.

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.

Lead poisoning: clinical, biochemical, and haematological aspects of a recent outbreak.

The clinical, biochemical, and haematological aspects of a recent outbreak of lead poisoning, in which exposure was related to the oxyacetylene cutting of red lead painted ironwork, were investigated. Initial suspicion was raised when a blood film showed punctate basophilia which remains a simple and useful method of picking up lead toxicity. Estimations of blood lead concentration and conventional laboratory data confirmed the diagnosis. Although there was prominent punctate basophilia, spectrophotometric analysis showed only negligible accumulation of pyrimidine-5'-nucleotides despite severe suppression of pyrimidine-5'-nucleotidase activity. The pattern of the red cell glycolytic intermediates, investigated for the first time, suggested that lead may also affect glycolysis at the hexokinase step. Once the diagnosis was made intravenous chelation treatment was begun with a rapid improvement in symptoms. Long term follow up is required to assess any sequelae of intoxication. These cases emphasise the classic features of lead poisoning, and despite the currently available diagnostic tests, lead intoxication may still go unrecognised unless a thorough occupational history is taken.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development.

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.

pH sensitivity of the thrombin-induced rise in fructose 2,6-bisphosphate content of human platelets.

The stimulation of human platelets with thrombin results in a rapid and sustained increase in the fructose 2,6-bisphosphate content which may play an important role in the potentiation of glycolytic flux induced by the agonist. The investigation of the effect of pH on thrombin-induced rise in platelet fructose 2,6-bisphosphate content is reported here. The results indicate that the early intracellular alkalinization which follows platelet stimulation may contribute to mediate the positive effect of thrombin on the regulatory metabolite.