Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose.

Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.

Role of Ca2+ and Mg2+ in neutrophil hexose transport.

The influence of extracellular Ca2+ and Mg2+ on the transport of 2-deoxy-[3H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[3H]glucose uptake stimulated by two chemotactic factors (C5a and N-formylmethionylleucylphenylalanine) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca2+ and Mg2+ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca2+, but not Mg2+, supported optimal enhanced hexose transport induced by stimuli. Activation of 2-deoxy-D-[3H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca2+ into the cell (as exemplified by the action of the two ionophores), such Ca2+ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca2+, may be involved in mediating the activation of hexose transport induced by the latter stimuli.

Marked depletion of plasma 1,5-anhydroglucitol, a major polyol, in streptozocin-induced diabetes in rats and the effect of insulin treatment.

Plasma levels of 1,5-anhydroglucitol (1,5AG), a major polyol resembling glucose in structure, fell rapidly and dramatically in streptozocin (STZ)-treated rats. 1,5AG fell immediately after STZ injection, reaching a plasma level 6 h after administration of the drug that was one-third that in the plasma of control rats. Reduction of 1,5AG was independent of the profile of blood glucose induced by STZ. After intravenous injection of [14C]-1,5AG, its plasma half-life was determined to be between 120 and 180 min. After a phase of acute decrease, the reduction of 1,5AG became gradual, stopping within 6 days after treatment. However, in some cases, the drop in 1,5AG was partially reversible by insulin treatment. The extent to which 1,5AG fell did not strictly correspond to the dose of STZ. The particular organ(s) consuming or accumulating 1,5AG was not identified. However, aside from the large amount of 1,5AG in plasma and the small amount of 1,5AG in the urine, the liver appears to be a significant organ for metabolism of 1,5AG.

Reduction and recovery of plasma 1,5-anhydro-D-glucitol level in diabetes mellitus.

The plasma concentration of 1,5-anhydro-D-glucitol (AG) was measured in 135 newly diagnosed patients who were referred for oral glucose tolerance tests. AG concentrations in the nondiabetic patients indicated that the mean value of normal AG concentration was 21.8 micrograms/ml (SD = 5.9 micrograms/ml, range 9.6-38.8 micrograms/ml). This distribution of AG concentration was significantly different from that in patients with impaired glucose tolerance (IGT) (13.3 +/- 5.4 micrograms/ml) and definitely different from that in diabetic patients (2.1 +/- 1.8 micrograms/ml). In a standard glucagon test, it was suggested that the decrease of plasma AG was affected not only by glycemic control of the patients but also by pancreatic cell secretory activity. The reduction of AG concentration was more marked in IDDM patients than in NIDDM patients. In longitudinal studies, AG concentration was shown to be sensitive to glycemic control. However, its recovery showed a tendency toward much delay after the improvement of fasting blood glucose or HbA1 concentrations. On the other hand, AG concentration showed negligible diurnal change and no immediate change as a result of diet, oral glucose load, or acute shift of the insulin level in both normal and diabetic subjects.

Plasma 1,5-anhydro-D-glucitol as new clinical marker of glycemic control in NIDDM patients.

To elucidate the value of using plasma 1,5-anhydro-D-glucitol (AG) as a marker of glycemic control in diabetic patients, the relationship between the plasma concentration of AG and glucosuria was examined in 152 patients with non-insulin-dependent diabetes mellitus (NIDDM). After recovery from the deterioration of glycemic control in NIDDM patients had started, AG began to increase day by day. The recovery of plasma AG showed a constant linear increase curve when excellent glycemic control was attained. The ordinary daily recovery rate of plasma AG was estimated to be 0.3 microgram/ml, which was independent of body weight, sex, age, the difference in treatment, the duration of diabetes, or the level of plasma AG among NIDDM patients. This rate decreased according to the increase in urinary glucose. When we calculated the decrease rate of plasma AG (delta AG), assuming 0.3 microgram/day to be the maximum increase rate in a day, we found a high correlation between delta AG and urinary glucose at almost all AG levels except the normal range and observed that plasma AG (A) times urinary glucose (G) was relatively constant. The formula A x G = 16 is a simple equation for rough estimation of urinary glucose from the plasma AG concentration in a stable glycemic-controlled NIDDM patient, and we call it the A.G index. The plasma AG also correlated significantly with fasting plasma glucose (r = -.810) and glycosylated hemoglobin (r = -.856) in the same stable glycemic-controlled NIDDM patients. Based on these observations, we propose that plasma AG can serve as a new marker that may provide sensitive and analytical information about glycemic control.

A possible hemoglobin marker of alcoholism.

Human erythrocytes were found to synthesize 5-deoxy-D-xylulose-1-phosphate from added acetaldehyde and endogenous dihydroxyacetone phosphate. In separate experiments 5-deoxy-D-xylulose-1-phosphate was observed to react with hemoglobin Ao, forming a stable adduct. Formation and accumulation of this unique form of hemoglobin in the blood of alcohol users, by integrating the blood acetaldehyde concentration, would provide a dose-time record of alcohol consumption.

Automation of a fluorometric method for the determination of 2-deoxy-D-glucose.

A continuous-flow fluorometric procedure for the determination of 2-deoxy-D-glucose (2DG) is described. The method utilizes Technicon Autoanalyzer equipment and modules, and is based on the acid-catalyzed condensation of 3,5-diaminobenzoic acid with 2DG. The procedure permits analysis of 20 samples/h, is sensitive to concentrations of 2DG as low as 0.2 mg/100 ml, and requires sample volumes of only 0.25 ml. 2DG can be quantitatively measured in serum samples or tissue extracts without requiring deproteinization. Glucose does not interfere with the assay while 2-deoxy-D-ribose develops a fluorescence which is about 15% of that produced by the same amount of 2DG and is additive when both deoxy sugars are present together. The procedure is accurate, reproducible, and fast, and can be run continuously.

Simple enzymatic method for determining 1,5-anhydro-D-glucitol in plasma for diagnosis of diabetes mellitus.

We have developed a simple method for determination of 1,5-anhydro-D-glucitol in plasma, based on use of pyranose oxidase (EC 1.1.3.10), an enzyme with specificity toward pyranoid compounds such as 1,5-anhydro-D-glucitol and glucose. Plasma samples deproteinized with trichloroacetic acid are passed through a two-layer mini-column packed with strongly basic anion (OH- form, the upper layer) and strongly acidic cation (H+ form, the lower layer) exchange resins. 1,5-Anhydro-D-glucitol is efficiently recovered in the flow-through fraction, which is almost devoid of other sugars that are sensitive to pyranose oxidase. The hydrogen peroxide formed in the enzymatic oxidation of 1,5-anhydro-D-glucitol is detected by a standard method utilizing an enzymatic color-developing system. The overall assay system is highly specific for 1,5-anhydro-D-glucitol. The correlation between results obtained in the present method (x) and in the gas-liquid chromatographic (GLC) method (y) was: y = 1.062x-0.293 mg/L (r = 0.997, n = 49, Sxy = 10.78 mg/L). Compared with GLC, our method is simpler in the sample treatment step and quicker in the measuring step. The precisions of the two methods are comparable.

Simultaneous determination of 2-deoxy-D-glucose and D-glucose in rat serum by high-performance liquid chromatography with post-column fluorescence derivatization.

A simple high-performance liquid chromatographic method for the determination of 2-deoxy-D-glucose and D-glucose in rat serum is described; this method is based on a post-column fluorescence derivatization. The sugars are automatically converted into fluorescent derivatives by reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine in an alkaline medium after their separation on a strong anion exchanger column (TSK gel Sugar AXG). The detection limits (S/N = 3) for 2-deoxy-D-glucose and D-glucose in rat serum are 0.52 and 0.56 nmol/ml, respectively.

Serum 1,5-anhydroglucitol in normal subjects and in patients with insulin-dependent diabetes mellitus.

The serum levels of 1,5-anhydroglucitol were measured by gas chromatography in normal subjects and in patients with type 1 (insulin-dependent) diabetes mellitus and compared with those found in some other common diseases. The identity of the compound was checked by thin-layer chromatography and by means of mass fragmentography. The mean level was 81 mumol/l (range 10-146 mumol/l, n = 139) in normal subjects and comparable levels were found in patients with rheumatic disease (n = 20) and in several patients with circulatory diseases. The level was less than 10 mumol/l in 44 patients with insulin-dependent diabetes mellitus, both in newly diagnosed cases and in patients with a long history of the disease with or without nephropathy. The compound did not appear in serum during near normoglycaemic periods elicited by continuous subcutaneous insulin infusion therapy, nor after successful kidney transplantation.

Studies on the energy metabolism of opossum Didelphis virginiana erythrocytes--II. Comparative aspects of 2-deoxy-D-glucose catabolism in opossum and human red cells in vitro.

1. G-6-P, F-6-P, F-1, 6-P, DHAP and GA-3-P in opossum erythrocytes were found at levels above those reported in human red cells. 2. About 1% of the radioactivity provided as [1-14C] DOG to red cells of both species was recovered as 14CO2 in 1 hr. 3. Unlike [1-14C] DOG, radiochromatography of extracts of cells incubated DOG revealed two diffusible radiolabelled compounds in the supernatant of cell suspensions. 4. The catabolism of DOG was quantitatively and qualitatively similar in opossum and human erythrocytes under the conditions of this study.

Changes in relationship between blood glucose level and plasma 1,5-anhydroglucitol level in KK mice.

We compared the relationship of the blood glucose level to the plasma 1,5-anhydroglucitol (1,5AG) level between KK mice with abnormal glucose metabolism and ICR mice as controls. Although the plasma 1,5AG level did not show any significant correlation with the blood glucose level in the controls, it tended to logarithmically decrease with the rise in the blood glucose level in KK mice. Thus it is possible that the plasma 1,5AG level is specifically related to the abnormal glucose metabolism in this model of diabetes mellitus and that its routine examination in diabetic patients may help delineate the metabolic derangement in the disease.

Impaired glucose transport in polymorphonuclear leukocytes in glycogen storage disease Ib.

A study of 2-deoxyglucose transport into polymorphonuclear leukocytes (PMN) was performed in three patients with glycogen storage disease (GSD) type Ib. The rate of 2-deoxyglucose transport into GSD Ib PMN was 30% of that of cells of normal controls. In GSD Ib lymphocytes, transport was normal. Km for 2-deoxyglucose in the PMN of one patient was within the normal range. The reduced transport was not due to the elevation in Km for 2-deoxyglucose nor to the decreased rate of phosphorylation of 2-deoxyglucose. The striking limitation of glucose transport across the cell membrane may account for the impairment of leukocyte function which is characteristic of GSD Ib.

2-Deoxyglucose as a substrate for glutathione regeneration in human and ruminant red blood cells.

2-Deoxyglucose was found to be a substrate for the regeneration of GSH from GSSG in the red blood cells of humans, sheep, goats and cattle. The regeneration rate with 2-deoxyglucose varied from 15 to 56% of the rate with glucose. It is likely that 2-deoxyglucose is phosphorylated to 2-deoxyglucose-6-phosphate by HK and is then oxidized to 2-deoxygluconate-6-phosphate by G6PD. The latter reaction would produce the NADPH required for GSH regeneration.

Identification and metabolic implication of 1-deoxyglucose (1,5-anhydroglucitol) in human plasma.

1-Deoxyglucose (1,5-anhydroglucitol), a metabolite related to diabetes mellitus, was identified in human plasma by gas-liquid chromatography and by gas-liquid chromatography/mass spectrometry. Plasma polyols were accurately determined with a gas-liquid chromatograph equipped with an all-glass capillary column. The plasma content of 1-deoxyglucose in healthy persons varies with age. Although the precise physiological role of 1-deoxyglucose remains obscure, the method described here for determining the minor polyol components of plasma, as well as the findings of 1-deoxyglucose in the plasma of healthy subjects, may be useful for investigating the metabolic roles of 1-deoxyglucose.

Reduction of plasma 1,5-anhydroglucitol (1-deoxyglucose) concentration in diabetic patients.

The plasma concentration of 1,5-anhydro-D-glucitol(AG)(1-deoxyglucose) is known to decrease in diabetic patients. In order to evaluate the usefulness of this polyol as a diabetic marker, we examined the specificity of the plasma AG reduction in various diseases: the plasma AG level was determined in 108 newly diagnosed diabetic patients, 229 normal subjects and 200 patients with various other disorders. The mean plasma AG concentration in diabetes mellitus was 1.9 +/- 1.8 micrograms/ml (mean +/- SD), which was definitely lower than that in healthy subjects and patients with other diseases including some metabolic and hormonal diseases (mean value range: 13.4-28.3 micrograms/ml). Only the "malignancies" group showed statistically different mean values from that in normal subjects; however, these values were much higher than those of diabetic patients. The AG concentration seemed to be relatively low in some severe by uraemic patients, but is likely to be little influenced by the glomerular filtration rate. Upon adjustment for sex and age, AG concentration was not found to be correlated with the degree of obesity in both healthy subjects and diabetic patients. The plasma AG concentration showed a tendency to be higher in healthy males than in healthy females in all age-matched groups; however, statistically significant differences were not seen. Also, no significant influence of age was observed.