Blood Metabolic Signatures of Body Mass Index: A Targeted Metabolomics Study in the EPIC Cohort.

Metabolomics is now widely used to characterize metabolic phenotypes associated with lifestyle risk factors such as obesity. The objective of the present study was to explore the associations of body mass index (BMI) with 145 metabolites measured in blood samples in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Metabolites were measured in blood from 392 men from the Oxford (UK) cohort (EPIC-Oxford) and in 327 control subjects who were part of a nested case-control study on hepatobiliary carcinomas (EPIC-Hepatobiliary). Measured metabolites included amino acids, acylcarnitines, hexoses, biogenic amines, phosphatidylcholines, and sphingomyelins. Linear regression models controlled for potential confounders and multiple testing were run to evaluate the associations of metabolite concentrations with BMI. 40 and 45 individual metabolites showed significant differences according to BMI variations, in the EPIC-Oxford and EPIC-Hepatobiliary subcohorts, respectively. Twenty two individual metabolites (kynurenine, one sphingomyelin, glutamate and 19 phosphatidylcholines) were associated with BMI in both subcohorts. The present findings provide additional knowledge on blood metabolic signatures of BMI in European adults, which may help identify mechanisms mediating the relationship of BMI with obesity-related diseases.

Investigations on collectin liver 1.

Collectins are pattern recognition molecules of the innate immune system showing binding to carbohydrate structures on microorganisms in a calcium-dependent manner. Recently, three novel collectins, collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1 and CL-11), and collectin placenta 1 (CL-P1), were discovered. The roles of these three collectins remain largely unknown. Here, we present a time-resolved immunofluorometric assay for quantification of CL-L1. The concentration of CL-L1 in donor plasma (n = 210) was distributed log-normally with a median value of 3.0 µg/ml (range 1.5-5.5 µg/ml). We observed on average 30% higher concentrations of CL-L1 in plasma as compared with serum. Size analysis by gel-permeation chromatography showed CL-L1 in serum to elute as large 700-800-kDa complexes and smaller 200-300-kDa complexes. CL-L1 showed specific binding to mannose-TSK beads in a Ca(2+)-dependent manner. This binding could be inhibited by mannose and glucose, but not galactose, indicating that CL-L1 binds via its carbohydrate-recognition domain and has ligand specificity similar to that of mannan-binding lectin. Western blot analysis of CL-L1 showed the presence of several oligomeric forms in serum. Ontogeny studies showed CL-L1 to be present at birth at near adult levels. CL-L1 levels exhibit low variation in healthy adults over a 1-year period. During acute-phase responses, the CL-L1 levels display only minor variations. In serum, CL-L1 was found in complexes with mannan-binding lectin-associated serine proteases, suggesting a role in the lectin pathway of complement activation. The presented data establish a basis for future studies on the biological role of CL-L1.

Serum metabolic profiles of pregnant women with burdened obstetrical history.

The content of low-molecular-weight components in blood serum was studied by tandem mass-spectrometry in pregnant women. Serum metabolic profiles of patients with a grave obstetrical history were detected. The most significant changes were observed for the concentrations of low-molecular-weight substances involved in glucogenesis and ß-oxidation processes and in metabolic chains involving carbohydrates, carnitines, amino acids, and lipids.

Metabolomics profiles associated with HbA1c levels in patients with type 2 diabetes.

Glycated hemoglobin (HbA1c) is an indicator of the average blood glucose concentration. Failing to control HbA1c levels can accelerate the development of complications in patients with diabetes. Although metabolite profiles associated with HbA1c level in diabetes patients have been characterized using different platforms, more studies using high-throughput technology will be helpful to identify additional metabolites related to diabetes. Type 2 diabetes (T2D) patients were divided into two groups based on the HbA1c level: normal (HbA1c ≤6%) and high (HbA1c ≥9%) in both discovery and replication sets. A targeted metabolomics approach was used to quantify serum metabolites and multivariate logistic regression was used to identify significant differences between groups. The concentrations of 22 metabolites differed significantly between the two groups in the discovery set. In the replication set, the levels of 21 metabolites, including 16 metabolites identified in the discovery set, differed between groups. Among these, concentrations of eleven amino acids and one phosphatidylcholine (PC), lysoPC a C16:1, were higher and four metabolites, including three PCs (PC ae C36:1, PC aa C26:0, PC aa C34:2) and hexose, were lower in the group with normal HbA1c group than in the group with high HbA1c. Metabolites with high concentrations in the normal HbA1c group, such as glycine, valine, and PCs, may contribute to reducing HbA1c levels in patients with T2D. The metabolite signatures identified in this study provide insight into the mechanisms underlying changes in HbA1c levels in T2D.

Synthesis of hemoglobin Aic and related minor hemoglobin by erythrocytes. In vitro study of regulation.

Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.

Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency Differentiated from Biliary Atresia.

Purpose The aim of this article is to differentiate neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) from biliary atresia (BA) by total hexose. Methods A total of 11 patients with NICCD, 29 patients with BA, and 4,898 children as controls were involved in this study. The blood concentration of amino acids, carnitine, acylcarnitines, and total hexose were measured in dry blood spots (DBS) using tandem mass spectrometry (MS/MS). Results In the patients with NICCD, the blood concentration of the total hexose (15.3 ± 9.0 mmol/L vs. 7.3 ± 2.7 mmol/L; p < 0.001), citrulline (Cit) (197.9 ± 93.7 µmol/L vs. 17.5 ± 7.4 µmol/L; p < 0.001) were higher than those of patients with BA. Using total hexose (> 10 mmol/L), Cit (> 55 µmol/L) to diagnose NICCD, the sensitivity and specificity were 66.7 and 97.8% and 90.0 and 99.1%, respectively, and all of the areas under the receiver-operating characteristic curves were greater than 0.85. Conclusion Elevated total hexose in DBS measured by MS/MS associated with elevated amino acids, especially Cit can be used to diagnose NICCD and differentiate it from BA.

Cardiovascular Risk Factors Associated With Blood Metabolite Concentrations and Their Alterations During a 4-Year Period in a Population-Based Cohort.

BACKGROUND: The effects of lifestyle risk factors considered collectively on the human metabolism are to date unknown. We aim to investigate the association of these risk factors with metabolites and their changes during 4 years. METHODS AND RESULTS: One hundred and sixty-three metabolites were measured in serum samples with the AbsoluteIDQ kit p150 (Biocrates) following a targeted metabolomics approach, in a population-based cohort of 1030 individuals, aged 45 to 83 years at baseline. We evaluated associations between metabolite concentrations (28 acylcarnitines, 14 amino acids, 9 lysophosphocholines, 72 phosphocholines, 10 sphingomyelins and sum of hexoses) and 5 lifestyle risk factors (body mass index [BMI], alcohol consumption, smoking, diet, and exercise). Multilevel or simple linear regression modeling adjusted for relevant covariates was used for the evaluation of cross-sectional or longitudinal associations, respectively; multiple testing correction was based on false discovery rate. BMI, alcohol consumption, and smoking were associated with lipid metabolism (reduced lyso- and acyl-alkyl-phosphatidylcholines and increased diacylphosphatidylcholines concentrations). Smoking showed positive associations with acylcarnitines, and BMI correlated inversely with nonessential amino acids. Fewer metabolites showed relative changes that were associated with baseline risk factors: increases in 5 different acyl-alkyl phosphatidylcholines were associated with lower alcohol consumption and BMI and with a healthier diet. Increased levels of tyrosine were associated with BMI. Sex-specific effects of smoking and BMI were found specifically related to acylcarnitine metabolism: in women higher BMI and in men more pack-years were associated with increases in acylcarnitines. CONCLUSIONS: This study showed sex-specific effects of lifestyle risks factors on human metabolism and highlighted their long-term metabolic consequences.

Glycosylation of hemoglobin S by reducing sugars and its effect on gelation.

The binding of various reducing mono- and disaccharides to hemoglobin S has been measured both before and after treatment of the sugar-protein adducts with NaBH4. Incubation of 0.3 M solutions of D-glucose, D-galactose, D-maltose, and lactose, with 2% hemoglobin for 2 h at 37 degrees C, pH 7.2, leads to the incorporation of 1.1, 1.8, 1.8, and 3.3 mol of sugar, respectively, into 1 mol of hemoglobin tetramer (either A or S). Exposure of these aldose-protein adducts to NaBH4 for an additional hour at 10 degrees C increases the binding to 2.0, 3.3, 2.5, and 4.1 mol per mol tetramer, as would be expected if Schiff base linkages were involved in this protein modification reaction. The data suggest a stereochemical requirement for enhanced binding. The dependence of the pre-reduction binding of glucose on the sugar concentration, and on the oxygenation state of hemoglobin has also been examined. Glycosylation of hemoglobin significantly increases the minimum gelling concentration of the deoxy conformation, as measured by sedimentation equilibrium ultracentrifugation. Of the sugar derivatives of hemoglobin S examined by this method, those modified by D-galactose or lactose have minimum gelling concentrations (in the absence of 2,3-diphosphoglycerate) which are comparable to, or greater than, that of fully carbamylated hemoglobinS.

Inhibitory effect of mannose on erythrocyte defense against oxidants.

The erythrocyte can phosphorylate a variety of hexoses. Since it can consume mannose and glucose equivalently in the hereditary deficiencies of hexokinase and phosphoglucose isomerase and since erythrocyte defense against oxidants is impaired in a variety of hereditary hemolytic anemias, we tested the hypothesis that mannose may be a significant alternative to glucose as a fuel for this defense system. Unexpectedly, mannose inhibited defense against oxidants as manifested by increased Heinz body formation when both normal and high-reticulocyte erythrocytes were incubated with acetylphenylhydrazine (APH). Using APH as the oxidant, mannose-incubated erythrocytes had decreased reduced glutathione stability and impaired hexose oxidation by the pentose shunt compared to glucose-incubated erythrocytes. After incubation with mannose and APH, normal erythrocytes showed a decrease in ATP content. Approximately 25% of the consumed mannose accumulated in the erythrocytes as mannose 6-phosphate. Erythrocytes incubated with mannose and APH displayed a significant loss of redox potential as manifested by decreased NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios. Since phosphomannose isomerase is the rate-limiting step for mannose metabolism, our results suggest that mannose impairs erythrocyte defense against oxidants by causing ATP depletion and by impairing the regeneration of reduced pyridine nucleotides by the Embden-Meyerhof and pentose phosphate pathways.

Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexose transfer.

(1) The Michaelis-Menten parameters for hexose transfer in erythrocytes, erythrocyte ghosts and inside-out vesicles at 20 degrees C were determined using the light scattering method of Sen and Widdas ((1962) J. Physiol. 160, 392-403). (2) The external Km for infinite-cis exit of D-glucose in cells and ghosts is 3.6 +/- 0.5 mM. (3) Dilution of cellular solute (up to X 90 dilution) by lysing and resealing cells in varying volumes of lysate is without effect on the Vm for net D-glucose exit. The Km for net exit, however, falls from 32.4 +/- 3.7 mM in intact cells to 12.9 +/- 2.3 mM in ghosts. This effect is reversible. (4) Infinite-cis net D-glucose uptake measurements in cells and ghosts reveal the presence of a low Km, high affinity internal site of 5.9 +/- 0.8 mM. The Vm for net glucose entry increases from 23.2 +/- 3.7 mmol/1 per min in intact cells to 55.4 +/- 6.3 mmol/l per min in ghosts. (5) The external Km for infinite-cis D-glucose exit in inside-out vesicles is 6.8 +/- 2.7 mM. The kinetics of zero-trans D-glucose exit from inside-out vesicles are changed markedly when cellular solute (obtained by lysis of intact cells) is applied to either surface of inside-out vesicles. When solute is present externally, the Km and Vmax for zero-trans exit are decreased by up to 10-fold. When solute is present at the interior of inside-out vesicles, Vmax for zero-trans exit is reduced; Km for exit is unaffected. In the nominal absence of cell solute, transfer is symmetric in inside-out vesicles. The orientation of transporter in the bilayer is unaffected by the vesiculation procedure. (6) External application of cellular solute to ghosts reduces Vmax for D-glucose exit but is without effect on the external Km for infinite-cis exit. (7) The inhibitory potency of cell lysate on hexose transfer is lost following dialysis indicating that the factors responsible for transfer modulation are low molecular weight species. (8) We consider the hexose transfer in human erythrocytes is intrinsically symmetric and that asymmetry of transfer is conferred by interaction of the system with low molecular weight cytosolic factors.

Isolation, characterization and chemical composition of the membrane from sheep platelets.

A procedure of the isolation of platelets from the blood of adult sheep (Ovis aries L. var domestica) is reported. This procedure is based on differential centrifugation and a specific lysis for elimination of erythrocytes. We have obtained platelets with a purity at least 99% and a relative high yield (2.3 +/- 0.4 g wet/l whole blood identical to 235 +/- 40 mg platelet proteins/l whole blood). After disruption, homogenisation and ultracentrifugation onto a discontinuous sucrose gradient (1.6 M, 1.1 M, 1.0 M and 0.6 M sucrose), four fractions were obtained. We have separated, for the first time, a particulate preparation enriched in the whole sheep plasma membrane. This fraction was characterized by: (i) the typical membrane morphology as shown by electron micrographs; (ii) the highest activities in membrane marker enzymes such as bis(p-nitrophenyl)phosphate phosphodiesterase (EC 3.1.16.1) and 5'-dTMP-p-nitrophenyl ester phosphodiesterase (EC 3.1.3.35), and the relatively low activity for marker enzymes associated to other subcellular fractions; (iii) the highest sialic acid, cholesterol and phospholipid concentrations. The chemical composition of the platelet membrane isolated is: total proteins, 49%; lipids, 47%; carbohydrates, congruent to 3.4% (the content of hexoses is twice as high as that of hexosamines and sialic acid). The similarities and differences of this preparation with others from several sources are discussed.

Influence of sialic acid groups on the retention of glycosphingolipids in blood plasma.

The removal of several glycosphingolipids from the circulation and their disposal in different tissue and fluid compartments was studied in adult rats. 3H-labeled dihydro analogs of several glycosphingolipids were injected intravenously and radioactivity was measured in arterial blood samples at subsequent time intervals, to obtain half life values for the labeled compound in the plasma. Half life values of less than 1 min were obtained for neutral glycosphingolipids whereas the half lives of labeled gangliosides were much longer and ranged from 3.8 to 21 h. The prompt removal of labeled neutral glycosphingolipids but not of the gangliosides indicates that sialic acid groups play a significant role in the retention of glycosphingolipids in the circulation. The results suggest that neutral glycosphingolipids are rapidly exchanged with their counterparts in a large extraplasma pool and that a major portion of this exchange could occur between plasma and liver. The detection of only a minute fraction of the injected glycosphingolipids in the cerebrospinal fluid indicates that a blood-cerebrospinal fluid barrier exists for these compounds in the rat.

The monosaccharide transport system of the human erythrocyte. Orientation upon reconstitution.

Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain external.

Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. I. Hexose transport inhibition and photolabeling of mutarotase.

A new phloretin derivative, phloretinyl-3'-benzylazide (PBAz), has been synthesized and compared with phloretin for its ability to inhibit the hexose transporter in human erythrocyte membranes in subdued light. Transport measurements were made using the light scattering (Orskov optical) method and a Millipore filtration technique with isotopically labeled sugars. Initial rates of sugar flux were measured under four different conditions to test for inhibition asymmetry. In each experimental condition, PBAz is from 6-20-times more potent than phloretin, making it one of the most effective reversible inhibitors known. Although both agents penetrate the cell membrane, they apparently fail to reach inhibitory levels at the inner surface over the time course of our nonequilibrated experiments, because of extensive binding to hemoglobin. The mechanism by which PBAz and its parent phloretin inhibit transport is pure competition with hexose for the carrier which faces the exterior of the membrane. If given time to equilibrate with the cells, the inhibition by both agents converts to a mixed type, i.e., both competitive and noncompetitive. The noncompetitive component could be due to inhibition of those transporter units oriented internally. Alternatively pre-equilibration with the inhibitors may cause them to attain high levels in the lipid membrane and produce nonspecific effects. PBAz and its precursor amine, phloretinyl-3'-benzylamine (PBA), compete with glucose for the sugar binding site on mutarotase at least as well as phloretin. When exposed to long wavelength ultraviolet radiation, PBAz is converted to a reactive intermediate which becomes covalently bound to the enzyme. Both irreversible ligand attachment and mutarotase inhibition are related to dose of the azide and irradiation time, but inactivation is from 5 to 6-times greater than label incorporation. We conclude that PBAz is a potentially useful photoaffinity labeling agent capable of covalently interacting with the transporter site facing the exterior of the red cell.

Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents.

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.

Effect of chemotactic factors on hexose transport in polymorphonuclear leucocytes.

Transport of the nonmetabolizable glucose analogue, 3-O-methylglucose, was assessed in human polymorphonuclear leucocytes with or without the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). The peptide increased entry of labelled 3-O-methylglucose about 5-fold and the intracellular distribution space about 70%. The half-time of equilibration was 3 s in the treated cells. Similar effects were observed with zymosan-treated serum (containing the chemotactic factor C5a), with arachidonic acid, calcium ionophore A23187 and phorbol myristate acetate. However, the chemotactic protein, thrombin, had no effect, even though binding to high-affinity receptors was demonstrated. Km for zero-trans entry of 3-O-methylglucose was about 1 mM and fMet-Leu-Phe increased Vmax from 5 to about 25 amol.s-1.cell-1. Similar values were obtained from incubations for a few seconds with glucose and 2-deoxyglucose. The rate of 2-deoxyglucose uptake (8 min incubations) was limited by the transport step at substrate concentrations lower than approx. 0.1 mM, whereas the phosphorylation step became rate-limiting at higher concentrations. Thus, 2-deoxyglucose uptake can only be taken as a measure of transport at a tracer concentration. It is concluded that chemotactic factors can, but do not necessarily, increase the maximal transport velocity of hexoses entering the polymorphonuclear leucocyte via the glucose transporter.

Experimental galactosemia produces diabetic-like retinopathy.

Six normal dogs were made galactosemic by feeding a 30% D-galactose diet, and were followed up to 5 yr. For comparison, 10 normal dogs and 10 alloxan-diabetic dogs were concurrently fed the diet less the galactose supplement. Retinopathy occurred in each of four dogs glactosemic 3 or more yr, and was absent at lesser durations of galactosemia, and from normal dogs not given the galactose supplement. The retinopathy was marked by saccular capillary aneurysms, hemorrhages, nonperfused or acellular vessels, tortuous hypertrophic capillaries, loss of capillary pericytes, and other lesions typical of diabetic patients and alloxan-diabetic dogs. In galactose-fed dogs, blood galactose varied between 0 (fasted) and 250 mg/dl (postprandial), and glycosylated hemoglobin levels became supranormal. In contrast to diabetic dogs, blood levels of glucose, free fatty acids, and branched-chain amino acids were not elevated in the galactosemic dogs, and their serum insulin seemed normal. The results suggest that the level of blood hexose is itself an important determinant of retinopathy.

Influence of Casearia esculenta root extract on glycoprotein components in streptozotocin diabetic rats.

The present study was aimed to evaluate the role of the indigenous antidiabetic medicinal plant Casearia esculenta on glycoprotein components in streptozotocin-induced diabetic rats in plasma, liver, kidney and cardiac tissues. Streptozotocin injection (50 mg/kg body weight) caused massive elevation of glycoprotein components such as hexose, hexosamine, sialic acid and fucose in plasma and tissues of diabetic control and experimental animals. Oral administration of C. esculenta root extract (200 and 300 mg/kg body weight) for 45 days significantly reverted the hexose, hexosamine, sialic acid and fucose levels to near normal values. These results suggest a normalizing effect of C. esculenta on glycoprotein components in STZ diabetic rats.

The bound carbohydrates of fractionated serum proteins in protein-calorie malnutrition.

Alterations in the carbohydrate composition of serum protein fractions have been determined in pooled sera from 35 children with protein-calorie malnutrition (PCM), 30 children treated for PCM, and 24 age- and social status-matched controls. Total protein-bound hexose (PBH), protein-bound sialic acid (PBSA), and protein-bound fucose (PBF) were elevated in PCM, but only PBF returned to control levels with treatment. Ratios of bound carbohydrate to protein in the unfractioned serum were elevated in PCM but PBH and PBF protein or globulin ratios returned to normal with treatment. The most marked change in carbohydrate composition occurred in the alpha1-globulin fraction. Molar ratios in the individual globulin fractions indicated an increase in carbohydrate moieties as follows: PBSA greater than PBH greater than PBF.