Ketoamine Linkage Research Articles

COF-based nanofiltration membrane for effective treatment of wastewater containing pharmaceutical residues

Nanofiltration has been identified as an effective method for removing emerging pharmaceuticals from wastewater with potential to mitigate environmental impacts and improve water quality. However, the low separation efficiency from the current nanofiltration membranes impeded their development. Here, using the vacuum-assisted self-assembly method, we prepared a thin film composite membrane by stacking covalent organic framework (COF) nanosheets on a predesigned ceramic hollow fibre. By adding methanol as the co-solvent for the assembly, a continuous and defect-free COF TpPa-SO3H layer was formed on top of the YSZ hollow fibre. The resultant COF composite membrane showed high rejection for five environmentally persistent pharmaceuticals (i.e., diclofenac, sulfamethoxazole, ketoprofen, naproxen, and ibuprofen). The hydrophilic pore surface and strong keto-amine linkages of the COF ensured high and stable permeation during operation with separation governed by electrostatic repulsion and steric exclusion. Due to our design using ceramic hollow fibres, these membranes have a small footprint and can be easily integrated into existing water treatment systems. These features make COF-based nanofiltration membranes a promising option for mitigating the environmental impacts of emerging pharmaceuticals in wastewater.

MON-LB025 An Artificially Low HbA1c as a Result of Dapsone Therapy

Since the 1970s, hemoglobin A1c (HbA1c) has been incorporated in clinical practice to monitor glycemic control. Furthermore, in 2010, the American Diabetes Association included HbA1c as a diagnostic criterion for diabetes. According to Zion Market Research, the global HbA1c market is expected to reach 2.2 billion USD by 2021. HbA1c is useful to clinicians because it provides a broader perspective of a patient’s glycemic control compared to the snapshot granted by blood glucose measurement. The erythrocyte membrane is permeable to glucose, allowing the sugar to react nonenzymatically with the N-terminal amino group of the beta chain via a ketoamine linkage. This process occurs continuously over the erythrocyte lifetime, which results in HbA1c representing a three-month window which reflects patients’ glycemic control. However, instances exist in which the utility of HbA1c diminishes in diabetic monitoring. We present the 8th case report in which dapsone interfered with the ability of HbA1c to accurately depict the glycemic status of a patient. The patient is a Caucasian male who was started on dapsone therapy on December 9th, 2013 for lichen planus. HbA1c values ranged from 7.2-8.9% during the four years before initiation of dapsone therapy. Two months after dapsone was started, HbA1c dropped to 6.9% and subsequently remained at 4.8%-5.8% until the drug was discontinued on May 11th, 2016. Three months later, HbA1c levels increased to 7.6% and ranged from 7.3%-7.8% until June the following year. In addition, fingerstick glucose values during this period also suggested poor glycemic control. Because HbA1c levels decreased in the presence of elevated fingerstick glucose, fructosamine levels were analyzed. Fructosamine refers to proteins that are nonenzymatically glycated via ketoamine linkages at the N-amino terminal (Danese et al 2015). Because albumin is the major serum protein, fructosamine primarily reflects glycated albumin. As opposed to erythrocytes, albumin exists in the blood for about two weeks. Therefore, fructosamine, indicates recent glycemic status for a narrower window than HbA1c. On July 23rd, 2015 the patient’s fructosamine levels were 299 mcmol/L (reference range: 200-285 mcmol/L) while HbA1c was 4.9%. As a result, the fructosamine results suggest that the patient’s glycemic control had not improved as suggested by the HbA1c values. A side effect of dapsone is hemolytic anemia. During the time that the patient was on dapsone, his hemoglobin levels dropped from about 14 g/dL to 11.7 g/dL. Simultaneously, total bilirubin levels increased from approximately 0.5 mg/dL to 0.9 mg/dL, suggesting a hemolytic anemia. We propose that dapsone generated a hemolytic anemia in this patient, which caused his HbA1c values to be unreliable in monitoring his glycemic control. Clinicians should be wary when monitoring the course of diabetic disease in patients on dapsone therapy. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

What can we learn from variation of red cell size distribution?

What can we learn from variation of red cell size distribution?

Temporal association between lens protein glycation and cataract development in diabetic rats.

In an attempt to shed more light on the relation between the glycation process and structural protein alterations, we followed the formation of glycated products in the lenses of hyperglycaemic Wistar rats during a period of 5 months following alloxan diabetes inducement. The study groups included non-diabetic (control), untreated diabetic rats (D), and diabetic rats receiving insulin alone or in combination with phlorizin, an inhibitor of renal tubular glucose transport. Lenses were removed at 4 and 20 weeks, and advanced glycation products in alkalisoluble lens proteins were determined by their characteristic spectrofluorescence (emission at 385 nm with excitation of 335 nm). In 20-week untreated diabetic rats as compared to control rats, a significant increase was observed in the fluorescence level (3.25 +/- 1.02 vs 1.61 +/- 0.17 FU/mg, p < 0.001), while in 4-week animals the increase was from 1.26 +/- 0.11 FU/mg in controls to 1.80 +/- 0.25 FU/mg in diabetics (P < 0.001). Daily treatment of 20-week diabetic rats with insulin alone (2.46 +/- 0.48 FU/mg) or in combination with phlorizin (2.30 +/- 0.26 FU/mg) did not significantly influence lens fluorescence level. The amount of glucosebound ketoamine linkage was estimated after acid hydrolysis as released 5-hydroxymethylfurfural (HMF). In 20-week controls, it was slightly higher than in 4-week controls (0.57 +/- 0.31 vs 0.41 +/- 0.20 nmol HMF/mg, respectively). The diabetic group showed a significant increase, however. In 4-week diabetics, a level of 1.07 +/- 0.36 nmol HMF/mg was found, while in 20-week animals the glycated protein amount rose to 2.46 +/- 0.79 nmol HMF/mg. In addition to the increases in glycated content with continuing diabetic hyperglycaemia, significant changes in protein composition of alkali-soluble lenses developed. The SDS-PAGE pattern showed an appearance of protein polymers of heterogeneous size (C 4 weeks 3.0 +/- 1.1% vs C 20 weeks 17.9 +/- 2.9%, D 4 weeks 7.3 +/- 2.1% vs D 20 weeks 19.8 +/- 3.6%) and the proteins of high molecular weight (HMW) failed to penetrate into the gel. Only a small amount of these HMW proteins was present in controls (C 20 weeks 2.5 +/- 1.2%) and short-term diabetes (D 4 weeks 0.8 +/- 0.2%), whereas in long-term untreated diabetes there was a dramatic increase (D 20 weeks 30.5 +/- 3.2%) with a corresponding decrease in other peaks. All diabetic animals from this group had macroscopically detectable cataractous lenses. Treatment with insulin or insulin/phlorizin followed the HMW protein level of the untreated animals (28.2 +/- 4.0% or 27.08 +/- 3.3% vs 30.52 +/- 3.32%).

Fixation of various aldehydic dextrans onto human hemoglobin: Study of conjugate stability

Formation and stability of different aldehydic dextran-hemoglobin conjugates were studied. Two types of polymers were used: sulfated or unsulfated oxidized dextrans and 4-carboxamidobenzaldehyde dextran. Periodate-oxidized dextran forms imine and ketoamine linkages by reaction with hemoglobin and the obtained conjugates are not completely stable, as their molecular size increases with time or decreases after incubation with lysine. The sulfated conjugates are more sensitive to lysine action than the unsulfated ones, which is consistent with the decreased possibilities of Amadori rearrangement. Therefore, this proves the importance of ketoamines for ensuring the cohesion of oxidized dextran-based conjugates. Carboxamidobenzaldehyde dextran forms only imine linkages with hemoglobin and the corresponding conjugates possess a marked instability in the absence of reductive treatment. The different types of conjugates could be stabilized by a sodium borohydride treatment in a satisfying manner.

Determination of nonenzymatically glycated albumin and IgG by affinity chromatography and colorimetry

A colorimetric procedure was developed for the sequential determination of nonenzymatically glycated IgG (GIgG) and albumin (GA). IgG and then albumin were separated from serum or plasma using a single DEAE Cibacron blue F3GA Affi-Gel column. The stable ketoamine linkages present in GIgG and GA reduced a tetrazolium salt to its colored formazan. The method was linear over the range 2.0–25.0% GIgG and 4.0–18.0% GA. For both GIgG and GA, the within- and between-run CV's were <4.5 and 8.5%, respectively, and recoveries were quantitative. The labile aldimine fraction, free glucose, and ascorbate did not affect the results. Nondiabetic reference intervals were 13.2–16.8% GIgG and 6.2–9.7% GA. Nondiabetic and diabetic populations can be clearly discriminated ( P < 0.005). When compared with the thiobarbituric acid assay, the correlation coefficients for GIgG and GA were r = 0.98 and 0.97, respectively. In a mixed group of diabetic and nondiabetic subjects, glycated hemoglobin levels were compared with those of GIgG and GA, and r = 0.68 and 0.35, respectively.

Estimation and characterization of glycosylated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus

Glycosylated form of carbonic anhydrase isozyme I was found in human erythrocytes. The percent of glycosylated enzyme of the total erythrocyte carbonic anhydrase I of patients with diabetes mellitus was significantly higher than that from normal controls. Characterization of the glycosylated carbonic anhydrase I was studied using an enzyme purified from diabetic patients. The glycosylated enzyme showed a slightly acidic isoelectric point in comparison with that of a nonglycosylated enzyme. The specific activity of the glycosylated enzyme was approximately 40% of that of the normal enzyme, and the immunological activity decreased to 52% of that of the normal enzyme. Estimation of carbohydrates which may form a ketoamine linkage with the enzyme was studied using [ 3H]-labelled glycosylated enzyme synthesized by incubation of the enzyme with [ 3H]- d-glucose in vitro, and it was found that one mol of glucose binds to one mol of enzyme. Exposure of red cells to a higher concentration of glucose in diabetics brought about glycosylation of carbonic anhydrase, which is associated with its low activity enzymatically and immunologically.

Age-related acceleration of glycation of tissue proteins in rats.

The Maillard reaction on proteins is a nonenzymatic glycosylation that is now termed glycation and produces a ketoamine linkage on the protein. The extent of this glycation was estimated by determining furosine (epsilon-N-(2-furoylmethyl)-L-lysine), which is derived from glycated lysine residues. Glycation of rat sciatic nerve and aorta increased with advancing age for up to 50 weeks and the high degree of glycation was maintained thereafter. The level of hemoglobin did not change significantly after 14 weeks. These results suggest that tissue glycation may increase with aging and may be involved in the mechanism of aging.

Kinetic analysis of glycation as a tool for assessing the half-life of proteins

Glycation of proteins, a common postribosomal modification, proceeds via Amadori rearrangement to yield a stable ketoamine linkage of glucose with the protein. Kinetic analysis of the reaction shows that the amount of glycation at steady state is proportional to the glucose concentration, to protein half-life and to tje rate of glycation. Thus, when the rate of glycation is determined in vitro and the extent of glycation of a given protein isolated from euglycemic subjects is measured, the half-life may be calculated. As the in vivo situation may not be simulated accurately in vitro, the calculated values may be considered as approximation. When the calculated values were compared with values reported in the literature fairly good agreement was found except for hemoglobin. Studies on stability of glycated albumin show that ketoamine decreases by about 20% when incubated under physiological conditions for 20 days. The method described by us is especially valuable when turnover of proteins in normal and pathophysiological states are compared. The half-life of plasma low-density lipoprotein is longer in patients with hypothyroidism or a high plasma low-density lipoprotein level than in normal subjects. Extending our studies to tissue proteins we did not find a significant increase in half-like of tendon collagen with age. Basement membrane collagen turnover is faster in diabetic patients in bad metabolic control. Thus, the procedure using fructosylamine as endogenous label of protein offers a method of great potential to study the turnover of human body proteins.

Colorimetric determination of non-enzymatically glycated albumin

A colorimetric method was developed for the determination of nonenzymatically glycated albumin and adapted to a Flexigem centrifugal analyzer. Albumin was separated from serum or plasma using Sepharose-blue dextran affinity chromatography. The stable ketoamine linkage in glycated albumin reduced a tetrazolium salt to its colored formazan. Glycated human serum albumin was used as the standard and optimum conditions for the assay were established. Recovery of glycated albumin was quantitative. The coefficients of variation for within-run and day-to-day precision were 4.6% and 8.5%, respectively. The labile aldimine fraction, lipemia, icterus, hemolysis and type of anticoagulant used did not affect the results. The non-diabetic reference interval for this method was 7.9-11.6% glycated albumin, and normal and diabetic populations can be clearly discriminated (p less than 0.005). Values obtained with this method correlated well with a thiobarbituric acid assay (r = 0.974) but less so with those for glycated hemoglobin (r = 0.35).

Structural and functional characterization of a canine minor glycosylated hemoglobin, Hb Can AIc.

Structural and functional studies of minor glycosylated hemoglobins in non-human mammals have been very few. Therefore, we attempted further characterization of a canine glycosylated hemoglobin (tentatively designated as Hb Can AIc) which had been isolated by Bio-Rex 70 chromatography (Enoki, Y. et al. (1982) Hemoglobin 6, 143-151). After isolation of the constituent subunits by hybridization-mercuration technique, we found by peptide mapping that the structural modification was confined in amino-termini of the beta subunits in which glycosyl ketoamine linkage was also shown to be localized by thiobarbituric acid reaction. Compared with the major hemoglobin (Hb Can A0), Hb Can AIc was characterized by such functional properties as a slightly lower oxygen affinity and a markedly reduced response to the allosteric effects of carbon dioxide and organic phosphates. The anion and H+ Bohr effects were not different from those in Hb Can A0. All these facts support that this hemoglobin is a canine counterpart of Hb AIc.

50 Changes in performance in clinical chemistrylaboratories in Ontario, 1978–1983

This study shows that the thiobarbituric acid (TBA) reaction as applied to the measurement of non-enzymatically glycosylated serum protein(s) [1], yields erroneous results unless strictly standardized conditions are followed. Critical points are in particular (a) the concentrations of NaBH4 required for the preparation of the blanks by reduction of the ketoamine linkages; (b) the amounts of protein which should be identical in all serum samples to be compared; (c) dialysis for removal of glucose and NaBH4 prior to hydrolysis; (d) increase in the yield of 5-hydroxymethylfurfural (HMF) by appropriate conditions of hydrolysis. From our data obtained with an improved TBA-assay it appears that about 90% of total glycosylated serum protein is accounted for by glycosylated albumin. Thus changes in the latter used for diagnostic purposes in diabetes, may well be reflected by total serum glycosylprotein measurement.

49 Precision of clinical chemistry testing in Ontario

This study shows that the thiobarbituric acid (TBA) reaction as applied to the measurement of non-enzymatically glycosylated serum protein(s) [1], yields erroneous results unless strictly standardized conditions are followed. Critical points are in particular (a) the concentrations of NaBH4 required for the preparation of the blanks by reduction of the ketoamine linkages; (b) the amounts of protein which should be identical in all serum samples to be compared; (c) dialysis for removal of glucose and NaBH4 prior to hydrolysis; (d) increase in the yield of 5-hydroxymethylfurfural (HMF) by appropriate conditions of hydrolysis. From our data obtained with an improved TBA-assay it appears that about 90% of total glycosylated serum protein is accounted for by glycosylated albumin. Thus changes in the latter used for diagnostic purposes in diabetes, may well be reflected by total serum glycosylprotein measurement.

The reversibility of the ketoamine linkages of aldoses with proteins.

The reaction of glyceraldehyde (aldotriose) with hemoglobin A is analogous to the nonenzymic glycosylation of the protein with glucose in that the initial reversible Schiff base adduct (aldimine) of aldotriose undergoes Amadori rearrangement as does that of aldohexose to form the more stable ketoamine adduct. The modification of the alpha-amino group on Val-1(beta) of hemoglobin A as a ketoamine (2-oxo-3-hydroxypropyl group) apparently lowers the pKa of the alpha-amino group of the protein, since this derivative of hemoglobin elutes earlier on carboxymethylcellulose columns than the derivatives containing 2-oxo-3-hydroxypropyl groups on the epsilon-amino groups, and unmodified hemoglobin A. Similar chromatographic behavior has been reported for hemoglobin A1c which contains glucose at its Val-1(beta) as the ketoamine adduct. This suggests the similarity in the chemical consequences of having the ketoamine adduct of an aldose at Val-1(beta) of hemoglobin A under physiological conditions. The formation of the 2-oxo-3-hydroxypropyl groups on Val-1(beta) is nearly irreversible as has been suggested for similar adducts of glucose. On the other hand, the 2-oxo-3-hydroxypropyl groups on the epsilon-amino groups appear to be labile. The buffer conditions considerably influence the reversibility of the ketoamine adducts of aldotriose on the epsilon-amino groups; the reversibility is significantly higher in Tris buffers as compared with that in phosphate buffers. It is suggested that under physiological conditions the ketoamine adducts of aldotriose on the epsilon-amino groups exist in equilibrium with the aldimine, the equilibrium being favored toward the ketoamine. The enhanced release of the 2-oxo-3-hydroxypropyl groups in Tris buffers is probably a reflection of the trans-Schiff base reaction of aldimine with Tris. In support of this hypothesis, sodium cyanoborohydride, a reagent selective for the reduction of the aldimine linkages, inhibited the labilizing influence of Tris. A similar reversibility appears to be true of the ketoamine adducts of aldohexoses at the epsilon-amino groups of hemoglobin A, and ribonuclease A as well. This information on the reversibility of the ketoamine adducts of glucose on the epsilon-amino groups of proteins should be useful in understanding the details of the mechanism of nonenzymic glycosylation. It is also expected to influence the interpretation of the measurements of nonenzymically glucosylated hemoglobin A in diabetic patients.

Age-related changes in non-enzymatic glycosylation of human basement membranes

Age and diabetes-related changes in the extent of non-enzymatic glycosylation of two anatomically distinct human basement membranes were examined. The amount of glucose in ketoamine linkage to glomerular basement membrane, measured by the thiobarbituric acid reaction, correlated positively and significantly with age. For analysis of lens capsule basement membrane, adsorption to agarose-linked phenylboronate was used. The ability of this resin to completely discriminate non-glycosylated from glycosylated residues was first documented by separating synthetic preparations of radioactive glucitol-lysine and glucitol-hydroxylysine, the glucosylamines formed with non-enzymatic glycosylation of collagen, from the respectivel free amino acids by affinity chromatography on and batch adsorption to phenylboronate. The extent of non-enzymatic glycosylation of lens capsule basement membranes correlated with age in both non-diabetic and diabetic samples, and the slopes of the lines for age versus glycosylation were similar in both groups. The calculated line for diabetic specimens, however, was displaced about 15 years to the left, compatible with premature aging. The results indicate that non-enzymatic glycosylation can be accurately assessed in minute amounts of tissue by phenylboronate adsorption, and provide evidence that human basement membrane is subject to increased glycosylation with aging and diabetes.

Glycosylation of human glomerular basement membrane collagen: increased content of hexose in ketoamine linkage and unaltered hydroxylysine-O-glycosides in patients with diabetes.

To study the glycosylation of glomerular basement membrane collagen (GBMC) in diabetes, kidneys were obtained at autopsy from 5 patients with insulin-requiring diabetes of long duration and diabetic complications, and from 5 control subjects. Glomeruli were prepared by sieving and collagen was isolated by limited pepsin proteolysis followed by salt precipitations. Amino acid analyses of the collagen preparations, after acid hydrolysis, indicated a composition consistent with that of type IV collagen. No differences in the relative contents of various amino acids, and in particular, 3-hydroxyproline, 4-hydroxyproline and hydroxylysine, were noted between diabetic and control samples. Non-enzymatic glucosylation was assessed by measuring hexose in ketoamine linkage with thiobarbituric acid after conversion to 5-hydroxymethylfurfural. In 4 of the 5 patients studied, glucosylation values exceeded the mean +2 S.D. of the controls; in the fifth subject glucosylation was in the high normal range. No correlation between the severity of diabetes and hexose content of GBMC was noted, however. In further studies, enzymatic glycosylation of GBMC was assayed after alkaline hydrolysis by separation of glucosylgalactosyl-O-hydroxylysine, galactosyl-O-hydroxylysine, and unsubstituted hydroxylysine in an amino acid analyzer. No differences in the relative contents of hydroxylysine-O-glycosides were evident between diabetic and control GBMC. The results suggest that non-enzymatic glucosylation, but not glycosylation catalyzed by collagen glucosyl and galactosyl transferases, is increased in diabetes. The increased carbohydrate content of collagen may lead to decreased turnover and/or excessive accumulations of basement membrane collagen thus contributing to the vascular complications of diabetes.

Characterization and Quantitation of Glycosylated Hemoglobins in Canine Blood

Canine hemolysates exhibited two minor and one major hemoglobins when chromatographed on Bio Rex-70 column with 0.05 M phosphate-0.01 M KCN (pH 6.5) containing 0.015 M NaCl. The first minor fraction was electrophoretically heterogeneous, constituting at least two hemoglobins. The second was completely homogeneous with somewhat faster anodal mobility than the major one and the difference was shown to be localized solely in the beta subunits. Thiobarbituric acid reaction was strongly positive for this hemoglobin, indicating the presence of glycosyl ketoamine linkage. We describe a simple and reliable procedure for quantitation of the canine glycosylated hemoglobins with a home-made Bio Rex-70 mini-column.

Improvement of the thiobarbituric acid assay for serum glycosylprotein determination

This study shows that the thiobarbituric acid (TBA) reaction as applied to the measurement of non-enzymatically glycosylated serum protein(s) [1], yields erroneous results unless strictly standardized conditions are followed. Critical points are in particular (a) the concentrations of NaBH 4 required for the preparation of the blanks by reduction of the ketoamine linkages; (b) the amounts of protein which should be identical in all serum samples to be compared; (c) dialysis for removal of glucose and NaBH 4 prior to hydrolysis; (d) increase in the yield of 5-hydroxymethylfurfural (HMF) by appropriate conditions of hydrolysis. From our data obtained with an improved TBA-assay it appears that about 90% of total glycosylated serum protein is accounted for by glycosylated albumin. Thus changes in the latter used for diagnostic purposes in diabetes, may well be reflected by total serum glycosylprotein measurement.

Hemoglobin Carbamylation in Uremia

CHROMATOGRAPHY of hemolysates of red cells resolves four minor hemoglobin components from the main hemoglobin A (HbA) fraction. These minor components — HbA1a1, HbA1a2, HbAlb, and HbA1c — collectively referred to as the HbA1 fraction, make up approximately 7 per cent of total hemoglobin in normal subjects and result mainly from the post-translational, nonenzymatic glycosylation of HbA.1 In HbAlc, the most abundant of the glycosylated hemoglobins, glucose is attached to the N-terminal amino group of the β chains by a stable ketoamine linkage; HbA1c is present in increased amounts . . .

1103 SCLERODERMA-LIKE SYNDROME AND THE NON-ENZYMATIC GLUCOSYLATION OF COLLAGEN IN CHILDREN WITH POORLY CONTROLLED INSULIN DEPENDENT DIABETES (IDDM)

104 children with IDDM were examined for induration and thickening of the skin and for joint contractures. Five patients had both multiple joint involvement and skin changes; 3 were studied in detail. Their diabetes ranged from 7-16 years in duration and was characterized by short stature, hepatomegaly, 4 to 6 hospitalizations each year, and glucosylated Hgb levels of 12-16%. All 3 had restrictive pulmonary lung disease. Histopathology of skin biopsies (bxs) demonstrated increased accumulation of collagen in the lower dermis. Three skin bxs were homogenized and compared to 6 bxs from age matched controls. The extractability of collagen in 0.5 N acetic acid was decreased to about ½ normal in 2 of the patients, suggesting increased cross-linkage of collagen. The amount of hexose bound to protein in a ketoamine linkage was assayed as the 5-hydroxymethylfurfural derivative. In patients, the mean non-enzymatic glucosylation was 4.0 (range 0.9-8.5) ng hexose/ug hydroxyproline which was 13 times that of controls (mean=0.3, range 0.2-0.4, ng hexose/ug hydroxyproline). The results suggest that non-enzymatic glucosylation may alter the packing, cross-linking and turnover of collagen, thus contributing to the development of a scleroderma-like cutaneous syndrome in IDDM.