Boronate Affinity Method Research Articles

HbA1c and Blood Pressure Correlation in Patients with Uncontrolled Type 2 Diabetes Mellitus

The primary metabolic symptom of diabetes mellitus is hyperglycemia. Analysing HbA1c levels can help determine blood glucose management. Elevations in blood pressure can be caused by abnormalities in the metabolism of glucose. Patients with uncontrolled diabetes mellitus may experience elevated blood pressure. To ascertain whether blood pressure and HbA1c levels are correlated in patients with uncontrolled DM. There were 90 subjects aged between 31-65 years consisting of 48 men and 42 women. HbA1c levels were checked using the boronate affinity method and blood pressure was measured using a sphygmomanometer. Data analysis used the independent difference t-test and Mann-Whitney U test as well as the Spearman correlation test with a significance level of α=0.05. Research subjects had an average HbA1c level of 10 ± 2.08% (7.1-15). Mean systolic blood pressure was 132.75 ± 18.04 mmHg (90-180). The average diastolic blood pressure was 83.28 ± 10.38 mmHg (60-120). Subjects who experienced hypertension were 36.66%. There were no differences in blood pressure between men and women (systolic p=0.70 and diastolic p=0.58). There was no correlation between HbA1c levels and blood pressure (systolic: r=0.022, p=0.81 and diastolic: r=0.022, p=0.81). There was no correlation between duration of DM and blood pressure (systolic: r=.093 and p=0.38 and diastolic: r= -.032 and p=0.76). There was no correlation between HbA1c levels and blood pressure

Interference of hemoglobin variants with HbA1c measurements by six commonly used HbA1c methods.

Glycated hemoglobin, or hemoglobin A1c (HbA1c), serves as a crucial marker for diagnosing diabetes and monitoring its progression. We aimed to assess the interference posed by common Hb variants on popular HbA1c measurement systems. A total of 63 variant and nonvariant samples with target values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-calibrated methods were included. We assessed 6 methods for measuring HbA1c in the presence of HbS, HbC, HbD, HbE, and fetal hemoglobin (HbF): 2 cation-exchange high-performance liquid chromatography (HPLC) methods (Bio-Rad D-100 and HLC-723 G8), a capillary electrophoresis (CE) method (Sebia Capillarys 3 TERA), an immunoassay (Roche c501), an enzyme assay system (Mindray BS-600M), and a boronate affinity method (Primus Premier Hb9210). The HbA1c results for nonvariant samples from the 6 methods were in good agreement with the IFCC-calibrated method results. The Bio-Rad D-100, Capillarys 3, Mindray BS-600M, Premier Hb9210, and Roche c501 showed no interference from HbS, HbC, HbD, and HbE. Clinically significant interference was observed for the HLC-723 G8 standard mode. Elevated HbF levels caused significant negative biases for all 6 methods, which increased with increasing HbF concentration. Elevated levels of HbF can severely affect HbA1c measurements by borate affinity, immunoassays, and enzyme assays.

PERBANDINGAN HASIL PEMERIKSAAN HBA1C METODE BORONATE AFFINITY DAN NEPHELOMETRY PHOTOMETRY

HbA1C is glycosylated hemoglobin at the N-terminal group, HbA1C is hemoglobin with a minor component closely related to blood glucose. HbA1C is referred to as the breakdown of sugar in the body's metabolism or glycosylated hemoglobin or glycohemoglobin. HbA1C has several examination methods such as the Boronate Affinity method and Nephelometry Photometry. The two main concepts of HbA1C measurement are based on the separation of hemoglobin fractions and chemical reactions. Both of these methods offer a fast, simple, precise and accurate alternative to the currently used methods and provide substantial freedom from many common hassles. This type of research is comparative analytic experimental. In this study, 25 samples were used during the November-January 2023 period. The purpose of this study was to evaluate the results of the HbA1C examination using two methods. In this study it was concluded that based on the Independent T Test it showed the results of Sig 0.913 > ? (0.05) so that it could be concluded that there was no significant difference in the results of the two examination methods Keywords : HbA1C, Boronate Affinity, Nephelometry Photometry

Porous boronate imprinted microsphere prepared based on new RAFT functioned cellulose nanocrystalline with multiple H-bonding at the emulsion droplet interface for highly specific separation of Naringin

For porous molecular imprinted polymers (MIPs), introduction of special functional groups and integration porous structure support will endow the MIPs with tunable surface properties and specific affinity performance. Herein, by combination of RAFT molecular imprinting, Pickering emulsion, and boronate affinity methods, a new kind of porous imprinted microspheres (BA-HPMIPs) using the low pKa value of APBA (1-allylpyridinium-3-boronic acid, pKa = 7.4) as functional monomer was prepared for specific molecular recognition and separation of NRG. Meanwhile, the novel RAFT functioned surfactants with dual amide multiple H-bonds were designed as stabilized particles was used to promote sustainability, which further confer them multifunctionality and/or by polymerizing the Pickering emulsion itself. Equilibrium adsorption was reached rapidly within 360 min, and the maximum equilibrium binding capacities of BA-HPMIPs were found to be 118.32 µmol g−1 for NRG. Furthermore, they had a stronger selectivity towards NRG than other competitive target (i.e rutin, catechol, ARS, o-nitrophenol). Moreover, BA-HPMIPs have shown outstanding reusability of 8.63 % loss in enrichment performance after five times repeatedly. As a proof of this concept, a commercially avaliable NRG with purity could be effectively extracted and concentrated to 96.75 % purity. These results demonstrated that, a new kind of porous imprinted microsphere with abundant porous structure and boronic acid recognizing sites has great potential for separation and purification of industrial samples under neutral conditions.

The First Case of Hemoglobin Görwihl [α2β25(A2) Pro→Ala] Identified in Türkiye

We present the first individual with hemoglobin (Hb) Görwihl in Türkiye and to our knowledge, this is the 3rd case reported worldwide. During premarital thalassemia screening an abnormal unidentified Hb peak [Retention Time (RT): 4.950 min., 42.4%] was determined by cation-exchange high-performance liquid chromatography (CE-HPLC) method at Premier Resolution (Trinity-Biotech) system. Variant Hb was eluted with HbA (RT: 2.47 min., 82%) at Variant II Turbo, Bio-Rad system. He was 22 years old and didn't show any clinical symptoms or haematological abnormality. HbA1c measured with Boronate affinity method at (Premier Hb9210, Trinity-Biotech) system was 5.2% and consistent with his blood glucose value (5.5 mmol/L) while it was 4.6% with CE-HPLC method at Variant II Turbo system. β-globulin gene sequencing revealed a heterozygote codon c16C>C variant which was identical to Hb Görwihl. Although clinically insignificant, Hb Görwihl may be important in hemoglobinopathy screening and HbA1c measurements.

Identification of Hemoglobin Variants Prevalent in China and Their Effects on Hemoglobin A1c Measurements.

We aimed to evaluate the effects of hemoglobin (Hb) variants prevalent in China on HbA1c measurements and to identify them during HbA1c measurements. We evaluated a cation-exchange high-performance liquid chromatography (HPLC) method (Bio-Rad D-100), a capillary electrophoresis (CE) method (Capillarys 3 TERA), an immunoassay (Cobas c501), and a boronate affinity method (Premier Hb9210, as a comparative method) for HbA1c measurements in the presence of Hb variants prevalent in China. The Bio-Rad D-100 and Capillarys 3 TERA gave specific retention times and numeric migration positions for each Hb variant, respectively, showing excellent interindividual reproducibility. All methods showed statistically significant differences (P < .01) for several variants. Clinically significant effects were observed for the Bio-Rad D-100 (Hb New York and Hb J-Bangkok), Capillarys 3 TERA (Hb New York and Hb J-Bangkok), and Cobas c501 (Hb New York). Among 297 samples with Hb variants, there were 75 (25.3%) unacceptable results for Bio-Rad D-100, 28 (9.4%) for Capillarys 3 TERA, and 19 (6.4%) for Cobas c501 compared with the results from Premier Hb9210. Some Hb variants prevalent in China affect HbA1c measurements. The HPLC retention time and CE migration position can aid in the presumptive identification of Hb variants.

Hb broomhill [α1 or α2 114(GH2) pro > ala;HBA1 or HBA2:c.343C > G]: a rare Hb variant found in a diabetic chinese individual

We report a clinically silent hemoglobin (Hb) variant, Hb Broomhill [α1 or α2 114(GH2) Pro > Ala;HBA1 or HBA2:c.343C > G] in a diabetic Chinese man. The Hb fractions of the subject were analyzed using various chromatographic and electrophoretic techniques. The glycated hemoglobin (HbA1c) levels measured using cation-exchange high-performance liquid chromatography (CE-HPLC) and the boronate affinity method showed nearly identical results. Analysis of the chromatogram of the CE-HPLC revealed an abnormal shoulder peak that appeared towards the end of the elution profile. Though the capillary electrophoresis method did not interpret the results, a manual examination revealed an abnormal shoulder on the HbA0 peak. Similarly, the electropherogram of the capillary zone electrophoresis also had an abnormal shoulder on the HbA peak. A missense mutation specific to the Hb Broomhill variant was found using Sanger sequencing.

Kadar HbA1c pada Pasien Wanita Penderita Diabetes Mellitus Tipe 2 di Rsud Dr. Doris Sylvanus Palangka Raya

Laboratory tests recommended by the ADA as a supportive examination for diagnosing DM are Hemoglobin Adult 1C (HbA1c), HbA1c examination can be used as a reference for monitoring diabetes mellitus because HbA1c can provide clearer information about the true condition in people with diabetes mellitus. HbA1c examination is an examination that reflects the average blood glucose level over a period of 2-3 months. The purpose of this study was to determine the description of HbA1c in female patients suffering from type 2 diabetes mellitus in RSUD dr. Sylvanus Palangka Raya. This research was conducted using a cross sectional study method with accidental sampling technique. The sample used in this study was patients suffering from type 2 diabetes mellitus in RSUD dr. Doris Sylvanus Palangka Raya taken by 15 people. Then examined using the boronate affinity method. The results obtained by the HbA1c category were 4 people (26.67%) good, 6 people (40%) medium and 5 people (33.33%) bad.

DIFFERENCE IN GLICATED HEMOGLOBIN LEVEL BETWEEN BORONATE AFFINITY METHOD AND ION EXCHANGE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD IN TYPE 2 DIABETES MELLITUS

Background: Glycated Hb (HbA1c) test is needed to control glycemic in high prevalence type 2 diabetes mellitus (DM) patients. Hemoglobin fraction separated and chemical reaction are two main concepts in HbA1c test. Ion exchange-high performance liquid chromatography (HPLC) and boronate affinity use first concept. Ion exchange-HPLC is reference method in most of clinical laboratorium. Point of care testing (POCT) with boronate affinity method that has been standardized by international institution is available. The aim of this study was to compare boronate affinity POCT method and ion exchange-HPLC method.Method: This cross sectional study was conducted to 22 type 2 DM patients those fullfill inclusion and exclusion criteria in January 2017 to February 2018. Level of HbA1c was assayed with boronate affinity POCT and ion exchange-HPLC method. T-test was used to analyse data and no significance difference if p&gt;0.005Results: Subjects of this study are women (59.1%) more than men (40.9%) with age mean 59.23 years old (8.1). Uncontroled type 2 DM (77.3%) more than controled type 2 DM (22.7%).Mean of HbA1 level was 8.0% (1.7) in boronate affinity POCT and 8.3% (1.8) in ion exchange-HPLC. T-test showed no significance difference between those two HbA1C assay methods (p&gt;0.005).Conclusion: There was no difference HbA1c level between boronate affinity POCT method and ion exchange-HPLC method.

Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing

BackgroundGlycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (βA) and HbS beta chain (βS) for the most common Hb variant trait (HbAS) are examined. MethodsWe analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. ResultsGlycation of βS chains was significantly higher (p<0.001) than βA chains; this difference was consistent across subjects. Total (α+β) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. ConclusionThis novel mass-spectrometric approach described allows for relative quantification of glycated forms of βS and βA. Although βS glycation was significantly higher than that of βA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Led Astray by Hemoglobin A1c

Hemoglobin A1c (A1c) is used frequently to diagnose and treat diabetes mellitus. Therefore, it is important be aware of factors that may interfere with the accuracy of A1c measurements. This is a case of a rare hemoglobin variant that falsely elevated a nondiabetic patient’s A1c level and led to a misdiagnosis of diabetes. A 67-year-old male presented to endocrine clinic for further management after he was diagnosed with diabetes based on an elevated A1c of 10.7%, which is approximately equivalent to an average blood glucose of 260 mg/dL. Multiple repeat A1c levels remained >10%, but his home fasting and random glucose monitoring ranged from 92 to 130 mg/dL. Hemoglobin electrophoresis and subsequent genetic analysis diagnosed the patient with hemoglobin Wayne, a rare hemoglobin variant. This variant falsely elevates A1c levels when A1c is measured using cation-exchange high-performance liquid chromatography. When the boronate affinity method was applied instead, the patient’s A1c level was actually 4.7%. Though hemoglobin Wayne is clinically silent, this patient was erroneously diagnosed with diabetes and started on an antiglycemic medication. Due to this misdiagnosis, the patient was at risk of escalation in his “diabetes management” and hypoglycemia. Therefore, it is important that providers are aware of factors that may result in hemoglobin A1c inaccuracy including hemoglobin variants.

Chemical and biological evaluation of nephrocizin in protecting nerve growth factor-differentiated PC12 cells by 6-hydroxydopamine-induced neurotoxicity

The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson's disease. This model is crucial in the search for compounds that diminish 6-OHDA-induced nerve growth factor (NGF)-differentiated PC12 cell death. Nephrocizin (luteolin-7-O-β-D-glucopyranoside), a flavone glycoside, was isolated from widely distributed plants. The protective effects of pre-treatment with nephrocizin on the induced neurotoxicity in PC12 cells by 6-OHDA and its oxidative products, H₂O₂-, and p-quinone, were evaluated herein. Nephrocizin promoted cell viability, scavenged ROS-related products, increased cellular glutathione (GSH) levels, and reduced caspase-3 and -8 activities in 6-OHDA-, H₂O₂-, or p-quinone-treated PC12 cells. Furthermore, nephrocizin-conjugated metabolites in PC12 cells were identified with the boronate-affinity method and LC-MS technology, and preferential regioselectivity at the C2' and C5' positions by the nephrocizin-GSH (or NAC) adduct method was observed. These lines of evidence established that nephrocizin could form a dimer to diminish the intracellular ROS. These results demonstrate the first neuroprotective mechanism of nephrocizin against 6-OHDA-, H₂O₂- or p-quinone-induced cytotoxicity in PC12 cells via chemical and biological studies. These dietary antioxidants are potential candidates for use in intervention in neurodegenerative diseases.

The Effect of Increased Fetal Hemoglobin on 7 Common Hb A1c Assay Methods

To the Editor: Hemoglobin variants and increases in circulating fetal hemoglobin (Hb F),1 (defined as an Hb F level >2%) have been reported to interfere with some assay methods for Hb A1c (1–3). In view of the relatively common occurrence of such Hb F increases (approximately 1.5% of the US population) and the fact that individuals with an increased Hb F level are usually asymptomatic, it is important to know whether Hb A1c methods show Hb F interference and, if so, at what level of Hb F. In this report, we evaluate the presence of Hb F interference with several HPLC methods. This study was approved by the Institutional Review Board of the University of Utah and Johns Hopkins Medical Institutional Review Board, where the samples originated. The following commercial HPLC methods were evaluated: the G7 and G8 Variant Mode (Tosoh Bioscience); the D-10™ Hemoglobin A1c Program (D-10), the VARIANT™ II Hemoglobin A1c Program (VII NU), the VARIANT™ II TURBO Hemoglobin A1c Program (VII Turbo), and the VARIANT™ II TURBO HbA1c Kit – 2.0 (VII Turbo 2.0) (all Bio-Rad Laboratories); and the ultra2™ A1c boronate affinity method (ultra2) (Trinity Biotech). The Hb F level was estimated from the G7 or G8 Hb F percentage. A small number of samples (n = 24) with and without an increased Hb F level were analyzed by both the IFCC capillary electrophoresis (CE) method (4) and the G7 method to verify …

Accuracy and Precision of the Axis-Shield Afinion Hemoglobin A1c Measurement Device

The Afinion HbA1c (Axis-Shield) is a newer point-of-care device for measurement of hemoglobin A1c (A1C) using a boronate affinity method unlike the more commonly used DCA immunoassay method (Siemens Medical Solutions Diagnostics). The Afinion's accuracy and precision, when compared with high-performance liquid chromatography (HPLC) and DCA methods, have not been established in pediatric practice settings. Capillary blood was collected from 700 subjects with diabetes mellitus at seven Pediatric Diabetes Consortium sites. Each subject's A1C was measured locally using Afinion and DCA devices, and by a central laboratory (University of Minnesota) using a Tosoh HPLC method. In addition, repeated measurements on six whole blood samples provided by the National Glycohemoglobin Standardization Program (NGSP) were taken at three clinical centers using the Afinion and DCA methods and centrally using the Tosoh HPLC method to assess the precision of each device. The coefficient of variation for measurements of whole blood samples for precision analysis was 2% for Afinion, 3% for DCA, and 1% for HPLC. In the patient samples measured at the seven clinic sites, the Afinion generated higher A1C results than the HPLC (mean difference = +0.15; p < 0.001), while the DCA produced lower values (mean difference = -0.19; p < 0.001). The absolute differences with HPLC were similar for the Afinion and DCA (median 0.2%) with a slight advantage for the Afinion when compared with DCA (p < 0.001 by rank test). The DCA tended to read lower than HPLC, particularly at high A1C levels (p < 0.001), while the Afinion's accuracy did not vary by A1C. When compared to the central laboratory HPLC method, the differences between the results of the Afinion and DCA devices are clinically insignificant, and the Afinion and DCA have similar accuracy and precision when used in pediatric practice settings.

Dosage délocalisé de l’hémoglobine A1c sur l’analyseur In2it™ dans le cadre d’une consultation de diabétologie pédiatrique

Summary The In2it™ is a POCT testing system for haemoglobin A1c (Hb A1c ) assay using a boronate affinity method. We evaluated its analytical qualities, practicability and the effect of this assay on patient follow-up, when using during a paediatric diabetic consultation. Imprecision was good (between-run CV less than 4%). Results are well-correlated with those obtained on D10™, high-pressure liquid chromatography analyser ( r = 0.978). Bland–Altman graph showed good agreement between the two methods. HbS did not interfere, but it was not identified. HbC induced 1% overestimation of result. Practicability was good. However the software could be improved. Knowledge of Hb A1c value during consultation permits a better adaptation of therapy and improves motivation and comfort of the patient. This activity has to be controlled according the Guide de bonne execution des analyses de biologie medicale (GBEA) and needs a good partnership between biologists and clinical staffs.

Effects of Hemoglobin Variants and Chemically Modified Derivatives on Assays for Glycohemoglobin

Glycohemoglobin (gHb), measured as hemoglobin (Hb) A(1c) or as total gHb, provides a common means for assessing long-term glycemic control in individuals with diabetes mellitus. Genetic variants and chemically modified derivatives of Hb can profoundly affect the accuracy of these measurements, although effects vary considerably among commercially available methods. The prevalence of genetic variants such as HbS, HbC, and HbE, and chemically modified derivatives such as carbamyl-Hb among patient populations undergoing testing is not insignificant. Clinical laboratories and sites responsible for point-of-care testing of gHb need to be aware of the interferences produced in assays by these Hbs. We conducted a review of the literature describing the effects of variant Hbs on gHb assay methods commonly used in clinical laboratories. This review summarizes the documented effects of both common and uncommon Hb variants and derivatives on the measurement of gHb. Where known, we discuss mechanisms of interference on specific assays and methodologies. We specifically address effects of commonly encountered Hbs, such as carbamyl-Hb, HbS, HbC, HbE, and HbF, on assays that use cation-exchange chromatography, immunoassays, or boronate affinity methods for measuring gHb. A variety of patient- and laboratory-related factors can adversely affect the measurement of gHb in patients harboring Hb variants or derivatives. Identification of the variant or derivative Hb before or during testing may allow accurate measurement of gHb by the selection of a method unaffected by the given variant or derivative. However, laboratories should make available alternative, non-Hb-based methods for assessing long-term glycemic control in individuals with HbCC, HbSS, or HbSC disease, or with other underlying disorders where the concentration of gHb does not accurately reflect long-term glycemic control.

Effects of Nine Hemoglobin Variants on Five Glycohemoglobin Methods

Most studies of the effects of variant hemoglobins (Hbs) on specific glycohemoglobin (gHb) methods have been case reports of a single variant Hb and one or two analytical methods (1)(2)(3)(4). Few studies have systematically examined multiple Hb variants with several widely used analytical methods. In this report, we describe the effects of nine heterozygous Hb variants on five gHb methods. A boronate affinity method was chosen as the comparative method because it has high specificity for glycated Hb and negligible interference by variant Hbs (5). Over 10 months, we studied 40 samples with nine variant Hbs; 38 were detected during routine gHb analysis by a cation-exchange method. Two samples (Hb E trait) were identified during routine Hb phenotype analysis. Samples were stored at 2–8 °C until analysis within 10 days of collection. Cation-exchange chromatography was performed on a Variant system with the Hb A1c program (Bio-Rad Laboratories) and on an A1c 2.2 Plus analyzer using a 3-min protocol (Tosoh Medics). Immunoassays were performed on a DCA 2000 (Bayer Corporation, Elkhart, IN) and on a Hitachi 717 with Tina-quant HbA1c II reagents (Roche Diagnostics, Indianapolis, IN). Boronate affinity analysis on a CLC 385 analyzer (Primus Corporation) served as the comparative method. All methods used the manufacturers’ reagents as recommended, had imprecision (CV) <5%, are certified traceable to the Diabetes Control and Complications Trial by the National Glycohemoglobin Standardization Program, and reported results as percentage of Hb A1c. Hb phenotype analysis was performed on all samples using a PVS99 system (Primus). This HPLC system uses a poly-aspartic acid cation-exchange column and a complex nonlinear salt and pH gradient at 40 °C. Rare phenotypes, including Grady, Hope, and Raleigh were identified using a poly-aspartic acid column with a nonlinear salt and pH gradient …

Glycohemoglobin Results in Samples with Hemoglobin C or S Trait: A Comparison of Four Test Systems

A wide variety of commercial methods are available to measure glycohemoglobin (gHb). These methods measure various species of gHb, e.g., total gHb, hemoglobin A1 (Hb A1), or Hb A1c. Some methods are based on charge differences between glycated and nonglycated hemoglobins (e.g., cation-exchange chromatography, electrophoresis, and isoelectric focusing), whereas boronate affinity methods depend upon the binding of the sugar groups on the hemoglobin molecule (1). Immunoassay of Hb A1c depends on the presence of an epitope that includes glucose and N-terminal amino acids of the β chain of hemoglobin. These methods usually measure Hb A1c as a percentage of total hemoglobin. Some immunoassay methods also measure Hb S1c or Hb C1c, whereas others do not (2). The National Glycohemoglobin Standardization Program (NGSP) was established to certify the various commercial methods so that they can be related to the candidate reference method used in the Diabetes Control and Complications Trial (DCCT) (3)(4). Recently, the effects of Hb AC and Hb AS on one gHb immunoassay method were assessed (5). Samples containing Hb C trait showed modestly higher results than the HPLC method used. Although hemoglobin variants are relatively rare in Caucasians of northern European descent, the African-American population has an 8% prevalence of Hb AS and a 3% prevalence of Hb AC (6)(7). In this study, we describe the influence of Hb AS and Hb AC on four DCCT-traceable gHb methods. A total of 129 samples were collected in evacuated tubes containing EDTA as an anticoagulant. Samples with hemoglobin variants were identified by comparison of retention times on the Diamat system (Bio-Rad Clinical Laboratories) to known retention times for Hb S and Hb C. Of the 129 samples collected, 47 were homozygous for Hb A, 39 were …

Determination of furosine in biomedical samples employing an improved hydrolysis and high-performance liquid chromatographic technique

Traditionally, the most sensitive and specific determination of non-enzymatic protein glycation has involved an 18–24-h acid hydrolysis in order to generate the compound furosine, which has been detected employing reversed-phase h.p.l.c. In this study, we have reported that significant quantities of furosine can be generated with much shorter hydrolysis times employing a 90-min vapor-phase acid hydrolysis procedure. The furosine generated by vapor-phase hydrolysis is then quantitated by pulsed amperometric detection using anion-exchange high-performance liquid chromatography. Employing this method, we were able to show that furosine generated from acid hydrolysis of purified hepatic membranes in a diabetic and non-diabetic animal model agreed with traditional methods assessing total glycated protein ( i.e., boronate affinity methods).

Liver and Kidney Tissue Membranes As Tissue Markers for Nonenzymatic Glycosylation

We investigated the relationship of serum protein glycosylation to peripheral tissue membrane glycosylation. We studied 27 Sprague-Dawley rats and induced diabetes in 20 of them. Blood glucose levels were treated in 10 of the diabetic animals with daily subcutaneous insulin. After 8 wk, liver and kidney tissue was removed, purified membranes were prepared, and the percentage of glycosylated membrane protein was determined for the liver and kidney membranes by boronate-affinity methods. The percentage of glycosylated membrane protein for both liver and kidney tissue was found to correlate significantly with the glycemic state of the animal as assessed with glycosylated serum albumin, total glycosylated serum proteins, and glycosylated hemoglobin determinations (P less than 0.001 for each glycosylated protein parameter). In addition, the percentage of glycosylated membrane protein in the liver tissue correlated significantly with the measured level in the corresponding kidney tissue (r = 0.78, P less than 0.001). To identify the nature of the glycosylated membrane proteins, boronate-affinity methods were used to separate the glycosylated and nonglycosylated membrane proteins. It was determined that two major glycosylated protein bands exist for the liver membrane (78,000 and 58,700 Mr) and four for the kidney membranes (ranging from 48,700 to 74,000 Mr). The ultrastructural location and identification of these glycosylated membrane proteins are not known. This study demonstrates that measurement of clinical glycemic state, as reflected in glycosylated blood protein parameters such as glycosylated serum albumin and glycosylated hemoglobin, correlates significantly with ongoing tissue membrane accumulation of glucose.