Inverse relationship between human erythrocyte fructose-6-phosphate and short-chain fatty acid levels.

In muscle cells, fructose is initially metabolised to fructose-6-phosphate. In the liver, fructose is metabolised to fructose-1-phosphate and thence to glyceraldehydes, which in turn can either enter glycogenolysis via pyruvate or gluconeogenesis via fructose-1,6-bisphosphate and fructose-6-phosphate. High levels of fructose-1-phosphate inhibit both glycogenolysis and gluconeogenesis. We hypothesised that, if systemically absorbed short-chain fatty acids constitute a major metabolic fate of unabsorbed dietary fructose, then levels of erythrocyte fructose-6-phosphate would be inversely correlated with plasma levels of short-chain fatty acids. The aim of this study was to test this hypothesis in respect of the three main short-chain fatty acids acetate, propionate and butyrate. Venous blood samples from 39 patients (16 male, 23 female, mean (standard error) age 42.4 (3.3) years) were analysed. Erythrocyte fructose-6-phosphate was measured using quantitative Fourier transform infrared spectrometry following gel electrophoresis, while plasma acetate, propionate and butyrate levels were measured using gas-liquid chromatography. The erythrocyte fructose-6-phosphate level was inversely correlated with the plasma acetate (r = -0.30, p = 0.06), propionate (r = -0.31, p = 0.05) and butyrate (r = -0.40, p = 0.01). These results support our hypothesis. The conversion of unabsorbed dietary fructose into short-chain fatty acids may represent a protective mechanism against the adverse effects of hypoglycaemia.

Ketohexokinase knockout mice, a model for essential fructosuria, exhibit altered fructose metabolism and are protected from diet-induced metabolic defects.

Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.

Metabonomic study on women of reproductive age treated with nutritional intervention: screening potential biomarkers related to neural tube defects occurrence.

Nutritional intervention is effective in reducing the risk of neural tube defects (NTDs). To determine the effects of nutritional supplementation on human metabolism, a metabonomic study was carried out on 96 women of reproductive age. Subjects with nutritional intervention were given fortified wheat flour (containing folic acid, vitamin B1, vitamin B2, ferric sodium edetate and zinc oxide) for 8 months. Serum metabolic fingerprinting was detected via ultraperformance liquid chromatography in tandem with time-of-flight mass spectrometry (UPLC-TOF MS), and data acquired was processed by multivariate statistical analysis. The result revealed a significant difference between the control and intervention group. Twenty potential biomarkers, including fructose 6-phosphate, sphingosine 1-phosphate, docosahexaenoic acid and hexadecanoic acid, were located and identified by the accurate mass measurement of TOF MS. These compounds are believed to be functionally related to anti-oxidative competence in vivo. In conclusion, metabonomics study is a valuable approach in exploring the effect mechanism of nutritional intervention on NTD prevention.

Effects of hyperglycemia on hepatic gluconeogenic flux during glycogen phosphorylase inhibition in the conscious dog.

The aim of these studies was to investigate the effect of hyperglycemia with or without hyperinsulinemia on hepatic gluconeogenic flux, with the hypothesis that inhibition would be greatest with combined hyperglycemia/hyperinsulinemia. A glycogen phosphorylase inhibitor (BAY R3401) was used to inhibit glycogen breakdown in the conscious overnight-fasted dog, and the effects of a twofold rise in plasma glucose level (HI group) accompanied by 1) euinsulinemia (HG group) or 2) a fourfold rise in plasma insulin were assessed over a 5-h experimental period. Hormone levels were controlled using somatostatin with portal insulin and glucagon infusion. In the HG group, net hepatic glucose uptake and net hepatic lactate output substantially increased. There was little or no effect on the net hepatic uptake of gluconeogenic precursors other than lactate (amino acids and glycerol) or on the net hepatic uptake of free fatty acids compared with the control group. Consequently, whereas hyperglycemia had little effect on gluconeogenic flux to glucose 6-phosphate (G-6-P), net hepatic gluconeogenic flux was reduced because of increased hepatic glycolytic flux during hyperglycemia. Net hepatic glycogen synthesis was increased by hyperglycemia. The effect of hyperglycemia on gluconeogenic flux to G-6-P and net hepatic gluconeogenic flux was similar. We conclude that, in the absence of appreciable glycogen breakdown, the increase in glycolytic flux that accompanies hyperglycemia results in decreased net carbon flux to G-6-P but no effect on gluconeogenic flux to G-6-P.

Fructosamine 3-kinase, an enzyme involved in protein deglycation.

The in vivo formation of fructosamines following non-enzymatic reaction of proteins with glucose (i.e. glycation) was first described almost 30 years ago. Until recently, the only known fate of fructosamines in mammalian cells was their spontaneous conversion into advanced glycation end products. The identification in human erythrocytes of a new enzyme, fructosamine 3-kinase, disclosed the existence of a so-far unsuspected intracellular metabolism of these compounds. Fructosamine 3-kinase phosphorylates with high affinity both low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, leading to the formation of fructosamine 3-phosphates. The latter are unstable and spontaneously decompose into inorganic phosphate and 3-deoxyglucosone, with concomitant regeneration of the unglycated amine. The presence of proteins related to fructosamine 3-kinase in many prokaryotic and eukaryotic genomes suggests that this 'deglycation' process is not restricted to mammals.

Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency.

BACKGROUND: Recently, several patients with abnormal polyol profiles in body fluids have been reported, but the origins of these polyols are unknown. We hypothesized that they are derived from sugar phosphate intermediates of the pentose phosphate pathway (PPP), and we developed a semiquantitative method for profiling of pentose phosphate pathway intermediates. METHODS: Sugar phosphates in blood spots were simultaneously analyzed by liquid chromatography-tandem mass spectrometry using an ion-pair-loaded C(18) HPLC column. The tandem mass spectrometer was operated in the multiple-reaction monitoring mode. Enzymatically prepared D-[(13)C(6)]glucose 6-phosphate was used as internal standard. The method was used to study sugar phosphates abnormalities in a patient affected with a deficiency of transaldolase (TALDO1; EC 2.2.1.2). RESULTS: In control blood spots, dihydroxyacetone phosphate, pentulose 5-phosphates, pentose 5-phosphates, hexose 6-phosphates, and sedoheptulose 7-phosphate were detected. Detection limits ranged from approximately 100 to approximately 500 nmol/L. Glyceraldehyde 3-phosphate and erythrose 4-phosphate were undetectable. Intra- and interassay imprecision (CVs) were 10-17% and 12-21%, respectively. In blood from the TALDO1-deficient patient, sedoheptulose 7-phosphate was increased. CONCLUSIONS: The new method allows investigation of patients in whom a defect in the PPP is suspected. Measurements of sugar phosphate intermediates of the PPP may provide new insights into metabolic defects underlying the accumulating polyols.

Role of polyol pathway in nonenzymatic glycation.

In order to confirm the link between nonenzymatic glycation and the polyol pathway, we observed the effect of treatment with epalrestat (Ep), an aldose reductase inhibitor, on the concentration of advanced glycation end-products (AGEs) in erythrocytes from diabetic patients. We also examined the effect of the drug on erythrocyte fructose 3-phosphate (F3P), a novel metabolite that has been reported to relate to the polyol pathway, and ascertained the glycation capability of F3P and its possible breakdown product, 3-deoxyglucosone (3DG), by incubating the metabolites with bovine serum albumin (BSA). Incubation of BSA with F3P or 3DG resulted in a greater production of AGEs in comparison with the incubation with glucose or fructose. F3P was significantly increased in erythrocytes from diabetic patients compared with those from nondiabetic individuals and was lower in patients who had been treated with Ep than in those who were free from the compound. A treatment of patients with Ep for 1 month resulted in a significant decrease in F3P. Erythrocyte AGEs were significantly elevated in diabetic patients compared with nondiabetic individuals and tended to be lower in patients taking Ep than in those without Ep. Administration of Ep for 2 months decreased AGEs. These results show that the polyol pathway is likely to play a substantial role in the nonenzymatic glycation of proteins and the suppression of E3P as well as AGEs by an aldose reductase inhibitor may explain in part the preventive effect of the drug on diabetic complications.

Effects of an aldose reductase inhibitor on erythrocyte fructose 3-phosphate and sorbitol 3-phosphate levels in diabetic patients.

Fructose 3-phosphate and sorbitol 3-phosphate are novel metabolites that have been shown to associate with the polyol pathway in animal experiments. Fructose 3-phosphate is of particular interest because of its potent glycation capability as compared with other glycolytic intermediates, e.g., fructose. We observed the effects of treatment with epalrestat, an aldose reductase inhibitor, on their concentrations in erythrocytes from diabetic patients. The levels of both metabolites were significantly higher in diabetic patients than in non-diabetic subjects. A group of patients who had been treated with epalrestat showed significantly lower levels of both metabolites as compared with those untreated. A treatment of three patients with epalrestat for one month resulted in obvious decreases in their concentrations. The results suggest a possible explanation for the preventive effect of an aldose reductase inhibitor on nonenzymatic glycation.

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)

Enzyme-to-enzyme tunnelling between phosphoglucoisomerase and phosphofructokinase.

1. Cross-linked and permeabilized rat erythrocytes were incubated for 2-5 min at 37 degrees C in the presence of ATP and either D-[U-14C]glucose 6-phosphate (3 mM) mixed with unlabelled D-fructose 6-phosphate (1 mM) or D-[U-14C]fructose 6-phosphate (1 mM) mixed with unlabelled D-glucose 6-phosphate (3 mM). 2. The contribution of molecules derived from the radioactive ketohexose ester relative to the total amount of newly formed D-fructose 1,6-bisphosphate was lower than the time-related average value for such a relative contribution in the pool of D-fructose 6-phosphate. 3. From such a difference, it was calculated that, under the present experimental conditions, 13.1 +/- 2.0% of the molecules of D-fructose 1,6-bisphosphate formed during incubation are directly derived from D-glucose 6-phosphate by a process of enzyme-to-enzyme channelling between phosphoglucoisomerase and phosphofructokinase, rather than originating from the free pool of D-fructose 6-phosphate. 4. A comparable value of 13.2 +/- 3.2% was reached when the process of enzyme-to-enzyme tunnelling was judged from the 3H/14C ratio in D-fructose 1,6-bisphosphate formed by permeabilized erythrocytes exposed for 5-15 min to D-glucose 6-phosphate (3 or 5 mM) mixed with tracer amounts of both D-[1-14C]glucose 6-phosphate and D-[2-3H]glucose 6-phosphate.

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.

Essential fructosuria: increased levels of fructose 3-phosphate in erythrocytes.

Erythrocytes of 3 adult siblings with essential fructosuria contained 45-200 mumol/l fructose 3-phosphate (Fru-3-P), i.e. 3-15 times the concentration in normal controls. Sorbitol 3-phosphate was also increased, but to a lesser degree. An oral load with 50 g of fructose produced an additional 40 mumol/l increase of erythrocyte Fru-3-P after 5 h. The rate of Fru-3-P formation by red cells in vitro was normal. HbA1 and HbA1c were normal. The suspected pathogenetic role of Fru-3-P in diabetic complications is questioned.

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.

Identification of sorbitol 3-phosphate and fructose 3-phosphate in normal and diabetic human erythrocytes.

Using 31P NMR spectroscopy, we have identified sorbitol 3-phosphate and fructose 3-phosphate in normal human erythrocytes wherein their concentrations are estimated to be 13 mumol/liter cells. Incubation of hemolysates with sorbitol, fructose and ATP suggest that both sorbitol and fructose are phosphorylated separately and directly at the 3-hydroxyl position suggesting the presence in these cells of a novel and specific kinase(s). In addition to sorbitol 3-phosphate and fructose 3-phosphate which were previously identified in the mammalian lens and sciatic nerve, erythrocytes have two extra metabolites resonating at 6.7 and 6.8 ppm in the 31P NMR spectrum. Although not identified in this study, the unusual chemical shifts of these compounds, their low pKa values and the fact that they appear as doublet in proton-coupled 31P NMR spectra, suggest that these phosphomonoesters belong to the same class of metabolites as sorbitol 3-phosphate and fructose 3-phosphate. Preliminary studies of erythrocytes from an unselected group of diabetic subjects showed an overall increase in the concentration of all four metabolites, although an overlap with normal values was noted.

Reduced anaerobic glycolysis in oral contraceptive users.

Oral contraceptives containing combinations of estrogens and progestogens are known to impair glucose tolerance. The biochemical mechanisms underlying this lesion are speculative. In the present study women treated with OC for periods exceeding 10 cycles showed significant reduction in the activity of the key glycolytic enzyme phosphofructokinase (40%) and the levels of lactate (42%) in the erythrocytes compared to controls. These observations in women are analogous to those made earlier in female rats.

PIP: Combined oral contraceptives (30 mg ethinyl estradiol, 150 mg d-norgestrel) are known to reduce glucose tolerance. To study the possible mechanisms of this impairment, 20 women were divided into 3 groups based on length of contraceptive use -- 3-5 months, 6-11 months, and 12-36 months. Blood samples were analyzed for phosphofructokinase activity and levels of glycolytic metabolites. Changes in glucose tolerance are seen within 3-6 months of oral contraceptive use, but only the blood taken from the 3rd group (12-36 months) showed significantly lower levels of phosphofructokinase activity and lowered levels of fructose-1,6-diphosphate, lactate, and pyruvate. It is suggested that impaired glucose tolerance is due to reduced glycolysis due to lower levels of phosphofructokinase synthesis, which cannot be detected for at least a year in erythrocytes, since they do not synthesize the enzyme.

Changes in glycolytic intermediates in rat erythrocytes during exposure to simulated high altitude.

Regulatory mechanisms of erythrocyte glycolysis and 2,3-diphosphoglycerate (2,3DPG) metabolism under hypoxia were studied in rats exposed to a simulated altitude of 18,000 ft (5,486 m) for 5 d. Changes in erythrocyte glycolytic intermediates were determined by enzymatic analysis. Marked alterations of glycolytic intermediates were found during 1 d of exposure which were quite different from those observed during exposure for 2, 3, and 5 d. Alterations of intermediates seem to be highly correlated with blood pH changes; however, pH alone cannot explain the overall changes in intermediates. Results suggested that overall intermediate changes are the results of the combined effect of alterations of cellular pH and hemoglobin desaturation. Increased 2,3DPG at initial stages of exposure (within 1 d) may be caused mainly by the increased cellular pH; sustained elevation of 2,3DPG at later stages could be attributed to the relief of product inhibition of diphosphoglycerate mutase by deoxygenation.