Validation of Blood-Based Assays Using Dried Blood Spots for Use in Large Population Studies

Assessment of health in large population studies has increasingly incorporated measures of blood-based biomarkers based on the use of dried blood spots (DBS). The validity of DBS assessments made by labs used by large studies is addressed by comparing assay values from DBS collected using conditions similar to those used in the field with values from whole blood samples. The DBS approach generates values that are strongly related to whole blood levels of HbA1c, cystatin C, and C-reactive protein. Assessing lipid levels reliably with DBS appears to be a greater challenge. However, even when DBS values and values from venous blood are highly correlated, they are often on a different scale, and using conventional cutoffs may be misleading.     

Enzyme-Linked Immunoassay-Based Quantitative Measurement of Apolipoprotein B (ApoB) in Dried Blood Spots,a Biomarker of Cardiovascular Disease Risk

Apolipoprotein B (ApoB) is a strong predictor of cardiovascular disease, which remains the leading cause of mortality in both higher and lower income countries. Here, we adapted an enzyme-linked immunosorbent assay (ELISA) development kit for quantitative determination of ApoB levels in serum and plasma for use with dried blood spots (DBS). After confirming the dilution linearity of the assay for DBS, we measured ApoB in 208 venous DBS samples. Then, using Passing-Bablok regression analysis and Spearman rank correlation analysis, we evaluated the correspondence in ApoB values between matched plasma and finger-prick DBS samples from 40 individuals who had ApoB values spanning the range of ApoB values observed in the 208 vDBS samples. We also evaluated assay precision and recovery, the effects of hematocrit, number of freeze-thaw cycles, and different storage temperatures on ApoB levels in DBS. There was a strong, significant correlation between plasma and DBS ApoB levels with little bias. Assay precision and recovery were within the range recommended by the U.S. government’s industry guidelines for bioanalytical assay validation. The assay was not affected by the DBS matrix or physiological hematocrit levels. This DBS-based ELISA assay will facilitate population-scale assessment of cardiovascular risk in previously unexplored populations.     

The effect of acetaldehyde concentrations on the relative rates of formation of acetaldehyde-modified hemoglobins

The effect of various concentrations of acetaldehyde (0, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 mM) on the relative rates of formation of hemoglobin acetaldehyde adducts detected in fractions eluted from cation exchange high-pressure liquid chromatography (HPLC) was investigated. When the hemoglobin and acetaldehyde mixtures were incubated at 37 degrees C for various time intervals up to 24 hr, increased amounts of HbA1c could be observed after 2 hr incubation with 1 mM or greater concentrations of acetaldehyde, or after 4 hr incubation with at least 0.5 mM acetaldehyde. An increase in the HbA1a + b fraction was not observed with 4 hr incubation time until the acetaldehyde level reached 1 mM. The HPLC method detected no difference in minor hemoglobins from alcoholic and normal subjects. Incubation of red blood cells at 37 degrees C for 1 hr with six consecutive pulses of 0.05 mM [14C]acetaldehyde showed no differences in the amounts of minor hemoglobins determined chromatographically at various pulse intervals. However, the measure of the 14C-label incorporation into hemoglobin showed that adducts eluting in the HbA1a+b fraction were formed at a faster rate than those eluting in the HbA1c or HbA0 fraction, respectively. The specific activities of the HbA1a+b fractions at 2, 4, and 6 pulses were 34, 128, and 949 cpm/mg hemoglobin; those of the HbA1c fraction were 15, 58, and 174 cpm/mg hemoglobin. This evidence of modification of hemoglobin by physiological levels of acetaldehyde from 14C-label incorporation suggests that an assay more sensitive than chromatographic separation of adducts might be clinically useful in detecting alcoholism or monitoring alcohol detoxification programs.     

Does polyamine oxidase activity influence the oxidative metabolism of children who suffer of diabetes mellitus?

Diabetes mellitus is a metabolic disease characterized by inadequate secretion of insulin. Polyamine oxidase (PAO), a FAD-containing enzyme is involved in the biodegradation of Sp and Spd, catalyzing the oxidative deamination of Sp and Spd, resulting in production of amonia (NH₃), corresponding amino aldehydes and H₂O₂. Malondialdehyde (MDA) and acrolein (CH2=CHCHO), potentially toxic agents, which induce oxidative stress in mammalian cells, are then spontaneously formed from aminoaldehydes. The main signs of oxidative stress in diabetic children were the values of HbA1c and MDA levels. Polyamines have an insulin-like action. Antiglycation property of spermine and spermidine has been recently confirmed. There are no data in the literature about plasma polyamine oxidase (PAO) activities in children with type 1 diabetes. The idea of this study was to evaluate the polyamine metabolism through the estimation of polyamine oxidase activity. We have study children with newly diagnosed type 1 diabetes mellitus (n = 35, age group of 5–16 years, as well as age-matched healthy control subjects (n = 25). The biochemical investigations were done on diabetic children who have the pathological values of glucose (9.11–17.33 mmol/l) and glycosylated Hb (7.57–14.49% HbA_1c). The children in the control group have referent values of glucose and glycated hemoglobin (4.11-5.84 mmol/L and HbA_1c 4.22-6.81% of the total Hb. Glucose levels in blood plasma and glycosylated hemoglobin in erythrocythes hemolysates (HbA1c) were measured by using standard laboratory methods. PAO activity in venous blood plasma and the amount of malondialdehyde (MDA) were measured by the spectrophotometric methods. PAO activity, glycemia, HbA1c and MDA were significantly increased in diabetic children compared to the control subjects. PAO activity in children with type 1 diabetes mellitus was very high. The findings of higher blood HbA_1C and MDA levels confirm the presence of oxidant stress in children with type 1 diabetes mellitus and demonstrate that PAO activity may participate in these circumstances.     

Studies on subfractions of hemoglobin A1b and hemoglobin A1c in diabetic subjects

Hemoglobin (Hb) obtained from the hemolysate of normal subjects and diabetic patients was separated into HbA1a1, HbA1a2, HbA1b, HbA1c and HbA0 (major Hb) by Bio-Rex 70 cation exchange column chromatography. The glycosylated Hbs were further separated reproductively by cation exchange high performance liquid chromatography (HPLC), using 50 mM sodium phosphate buffer pH 5.80 with 0-0.2 M NaCl linear gradient system. HbA1b and HbA1c were separated into two subfractions (HbA1b1 and HbA1b2) and three subfractions (HbA1c1, HbA1c2, HbA1c3), respectively. The percentages of each subfraction except HbA1c1 in diabetic patients were significantly higher than those in normal subjects. Furthermore, HbA1c1, HbA1c2 and HbA1c3 correlated well with fasting blood glucose levels in the prior 5 month period, while subfractions in HbA1b revealed no significant correlation with blood glucose levels. The percentages of each subfraction of HbA1c in patients either with diabetic cataracts or with diabetic neuropathy were almost the same as those in the patients without complications. However, the percentages of each of the three groups were markedly higher than those of the normal subjects. These results suggest that glycosylation of hemoglobin in diabetic patients may be increased in various sites of the molecule in parallel with the blood glucose levels during the preceding 4-5 months.     

HbA1c standardisation destination--global IFCC Standardisation. How, why, where and when--a tortuous pathway from kit manufacturers, via inter-laboratory lyophilized and whole blood comparisons to designated national comparison schemes

Glycohaemoglobins were first used in routine clinical laboratories for diabetes monitoring around 1977 and at the time all methods had either no calibrators, or used material with assayed values derived from individual manufacturers' assays. Over the next five to fifteen years, lyophilised and whole blood sample exchanges were shown to improve inter-laboratory variability markedly. The use of a precise HPLC method as the "standard method" in the Diabetes Control and Complications Trial (DCCT) led to significant further improvement. National standardisation schemes in the mid to late 1990s in the USA, Japan and Sweden further improved the quality and accuracy of HbA1c assays in clinical use. The work of the IFCC Working Group on Standardisation of HbA1c in establishing true International Reference Methods for HbA1c and the successful preparation of pure HbA1c calibration material should lead to further improvements in inter-method and inter-laboratory variability, essential to the long-term monitoring of patients with diabetes.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Assessment of hematologic indices and their correlation to hemoglobin A1c among Bosnian children with type 1 diabetes mellitus and their healthy peers

BackgroundAltered levels of many hematological parameters have been directly associated with diabetes in adults, while studies on children with type 1 diabetes mellitus are lacking. The aim of this study was to determine hematological indices in diabetic Bosnian children in comparison to healthy controls as well as to correlate their levels to blood glucose and hemoglobin A1c.Methods100 healthy and 100 children with type 1 diabetes mellitus (age 1-18) were included in this study. Complete blood count, hemoglobin A1c, and glucose were tested. Results were analysed by IBM SPSS Statistics version 23.ResultsSignificant differences (p<0.05) between healthy and diabetic children were found in relation to HbA1c, glucose, mean platelet volume, the number of white blood cells and erythrocytes, hematocrit, hemoglobin and MCH values. No gender differences or significant age differences were seen for hemoglobin, hematocrit, and MCV, while platelets, MPV, and MCH differed by age only in healthy children. When diabetic children were classified according to HbA1c levels, significant differences were seen for erythrocyte count and hematocrit value (p=0.013 and 0.019, respectively). The number of erythrocytes and white blood cells correlated significantly with HbA1c (p=0.037 and 0.027, respectively).ConclusionsLower levels of erythrocytes, hematocrit, and hemoglobin in diabetic compared to healthy children indicate possible development of anemia, while higher MCV, MCH, and MPV values indicate an alteration in erythrocyte morphology. Hematological indices could be a useful inexpensive tool in the diagnosis and follow up of type 1 diabetes in children.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

Association of the Common Catalase Gene Polymorphism rs1001179 With Glycated Hemoglobin and Plasma Lipids in Hyperlipidemic Patients

Catalase represents perhaps the most effective antioxidant defense in the body under conditions of increased oxidative stress, and rs1001179 (CAT-262C >T) is its most extensively studied gene polymorphism. Using an established PCR–RFLP method for genotyping, we examined the association of rs1001179 with glycated hemoglobin (HbA1c) and plasma lipids using univariate analyses with age, sex, body mass index (BMI), smoking, and alcohol abuse as covariates, in a group of dyslipidemic patients from northern Greece. Our results suggest that the TT genotype is a risk factor for increased HbA1c and plasma triglycerides, and that this association is modulated by the BMI and/or age of the patients.     

Bioelectrocatalytic detection of glycated hemoglobin (HbA1c) based on the competitive binding of target and signaling glycoproteins to a boronate-modified surface

We developed an electrochemical glycated hemoglobin (HbA(1c)) biosensor for diagnosing diabetes in whole human blood based on the competitive binding reaction of glycated proteins. Until now, no studies have reported a simple and accurate electrochemical biosensor for the quantification of HbA(1c) in whole blood. This is because it is very difficult to correctly distinguish HbA(1c) from large amounts of hemoglobin and other components in whole blood. To detect glycated hemoglobin, we used electrodes modified with boronic acid, which forms a covalent bond between its diol group and the cis-diol group of the carbohydrate moiety of glycated proteins. For accurate HbA(1c) biosensing, we first removed blood components (except for hemoglobin) such as glycated proteins and blood glucose as they interfere with the boronate-based HbA(1c) competition analysis by reacting with the boronate-modified surface via a cis-diol interaction. After hemoglobin separation, target HbA(1c) and GOx at a predetermined concentration were reacted through a competition onto the boronate-modified electrode, allowing HbA(1c) to be detected linearly within a range of 4.5-15% of the separated hemoglobin sample (HbA(1c)/total hemoglobin). This range covers the required clinical reference range of diabetes mellitus. Hence, the proposed method can be used for measuring %HbA(1c) in whole human blood, and can also be applied to measuring the concentration of various glycated proteins that contain peripheral sugar groups.     

Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

Alteration of T-state binding properties of naturally glycated hemoglobin, HbA1c

The thermodynamic and kinetic properties of the most abundant glycated hemoglobin in human blood, HbA1c, have been studied in detail. They display significant differences as compared to normal hemoglobin, HbA0, in that (1) the shape of the oxygen binding curve of HbA1c in the Hill plot is markedly asymmetrical, with a lower asymptote extending up to approximately 40% oxygen saturation, and the oxygen affinity of the T state being tenfold higher than in HbA0; (2) oxygen pulse experiments on HbA1c show a slower rate of ligand dissociation (k = 25 s-1) even at low levels of oxygen saturation, where the T state is largely predominant; (3) kinetics of CO combination to deoxy HbA1c followed by means of stopped-flow experiments reveal the presence of a quickly reacting component, whose fraction increases upon dilution of hemoglobin. These results show that in contrast to what has been stated by other authors, HbA1c displays functional properties markedly different from HbA0. Analysis indicates that glycation of human hemoglobin affects the T quaternary structure, bringing about a more "relaxed" T state and leading to preferential binding to one type of chain (which is unaffected by chloride ions).     

Structural analysis of hemolymph proteins from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda)

1. Five different molecular weight polypeptides from serum (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata, were examined by two-dimensional 125I-peptide mapping and high performance liquid chromatography (HPLC). 2. Peptide mapping indicated that all five radiolabeled polypeptides within and between the two snail strains had similar migration patterns when cleaved with pepsin or alpha-chymotrypsin, thus revealing a shared structural homology. All peptides chosen for analysis appeared to be structurally similar to the 160 kDa hemoglobin molecule. 3. Separations of the radiolabeled enzyme digests by HPLC confirmed results seen in the mapping experiments since all chromatograms had similar elution patterns. 4. Minor differences in the peptide maps and chromatograms within and between snail strains may be due to quantitative differences in the amount of protein present and/or variations in the primary amino acid sequences of the proteins chosen for analysis.     

Diabetes mellitus is associated with shortened activated partial thromboplastin time and increased fibrinogen values

Objective

This study was designed to examine the relationship between shortened activated partial thromboplastin time (APTT) and increased fibrinogen values with diabetes mellitus.

Methods

APTT, prothrombin time (PT), fibrinogen, fasting plasma glucose (FPG) and glycosylated hemoglobin A1c (HbA1c) levels were measured in 1,300 patients. Patients were divided into three groups according to their HbA1c and FPG levels.

Results

When participants were grouped according to their HbA1c levels, we found significantly shorter APTT values (26.9±5.6 s) and increased fibrinogen levels (3.1, 1.9–6.3 g/L) in the diabetes group when compared with the other two groups. When participants were grouped according to their FPG levels, we found significantly shorter APTT values (26.9±6.2 s) and increased fibrinogen levels (3.1, 1.8–6.2 g/L) in the diabetes group when compared with the euglycemic group.

Conclusions

Shorter APTT and increased fibrinogen levels might be useful hemostatic markers in patients with diabetes and in patients at high risk for diabetes.     

Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

We aimed to evaluate the association of exposure to lead with glycated hemoglobin (HbA1c), fasting glucose levels (FGLs), and the likelihood for dysglycemia. We accessed data from Canada Health and Measures Survey. General linear models were used to estimate the association between blood lead concentrations (BPb) and both HbA1c and FGLs, while controlling for confounders. Multivariate logistic regression was used for assessing the relation between BPb and the likelihood for dysglycemia. FGLs in participants with moderate BPb (2.5–5.0 μg/dL) were 1.03 (95 % CI 1.00–1.06) times higher compared with participants with BPb < 2.5 μg/dL. Equivalent figures for those with BPb ≥ 5.0 μg/dL were 1.10 (95 % CI 1.01–1.20) times, relative to the lowest stratum. This association was attenuated using HbA1c to define dysglycemia. Lead exposure was associated with the likelihood for neither FGLs ≥ 1.10 g/L nor HbA1c ≥ 5.7 %. The association between lead exposure and dysglycemia, if any, is likely to be very modest, at least at the population level.     

Effect of iron deficiency anaemia over glycated hemoglobin in non-diabetic women

Background: Glycated hemoglobin (HbA1c) is a form of hemoglobin bound to glucose and used as an index of glycaemic control reflecting glucose levels of the previous three months. Iron deficiency anemia (IDA) is the commonest form of anemia that affects HbA1c. Reports on the effects of IDA on HbA1c levels are inconsistent in India. Therefore, the study correlated the HbA1c and IDA in non-diabetic female patients. Methods: A correlative study between HbA1c and IDA was carried out at the Department of Biochemistry, A. J. Institute of Medical Sciences, Mangaluru, India. A total of 50 non-diabetic female patients, aged between 20-50 years, with decreased levels of Hb, MCV and MCHC were selected. Their ferritin levels were determined by ELISA method to confirm IDA. Forty confirmed iron-deficient samples whose serum ferritin levels were <90 pg/dL, were tested for HbA1c levels by nephelometry method. Results: HbA1c correlated positively with serum ferritin, Hb, MCV, MCH and MCHC (P<0.05). There was a significant decrease in mean value of HbA1c in those with severe anemia (4.50±0.34) compared to those with moderate anemia (5.18±0.35) (P<0.001). Conclusion: Results showed positive correlation of HbA1c with ferritin and hemoglobin. Therefore, iron status should be considered during the interpretation of the HbA1c concentrations in diabetes mellitus.     

Lipid metabolism in patients with insulin dependent diabetes. III. Effect of metabolic control of diabetes on the concentration of some blood serum lipid constituents in patients with insulin dependent diabetes]

The levels of the following blood serum lipid constituents: total cholesterol, triglycerides, phospholipids, HDL-cholesterol, lipoprotein fractions, as well as apolipoproteins AI, AII and B, have been determined in patients with insulin-dependent diabetes lasting from 3 months to 15 years in relation to the degree of metabolic control characterized by the levels of fructosamine and glycosylated hemoglobin HbA1c. The group of patients having the level of HbA1c exceeding 10% was characterized by significantly higher levels of cholesterol, triglycerides and Apo-B, and lower content of alpha-lipoprotein as compared to the group with HbA1c level beneath 10%. When fructosamine concentration was considered as an index of metabolic control of diabetes, it was found that the levels of cholesterol, phospholipids and apolipoproteins apo-A and apo-AI are highest in the group with the poor metabolic control and differ significantly from the respective values found in patients with mediocre and good metabolic control. Considering biological role of the individual lipids and lipoproteins, it should be stressed that the proper control of glycaemia is important for preventing the development of atherosclerosis in patients with insulin-dependent diabetes.     

Effect of nonenzymatic glycation on functional and structural properties of hemoglobin

HbA(1c), the major glycated hemoglobin increases proportionately with blood glucose concentration in diabetes mellitus. H(2)O(2) promotes more iron release from HbA(1c) than that from nonglycated hemoglobin, HbA(0). This free iron, acting as a Fenton reagent, might produce free radicals and degrade cell constituents. Here we demonstrate that in the presence of H(2)O(2), HbA(1c) degrades DNA and protein more efficiently than HbA(0). Formation of carbonyl content, an index of oxidative stress, is higher by HbA(1c). Compared to HbA(0), HbA(1c) is more rapidly autooxidized. Besides these functional changes, glycation also causes structural modifications of hemoglobin. This is demonstrated by reduced alpha-helix content, more surface accessible hydrophobic tryptophan residues, increased thermolability and weaker heme-globin linkage in HbA(1c) than in its nonglycated analog. The glycation-induced structural modification of hemoglobin may be associated with its functional modification leading to oxidative stress in diabetic patients.     

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe(3)O(4), which was synthesized with easy handling feature of Fe(3)O(4) by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe(3)O(4) magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 μg mL(-1), with the detection limit of 0.025 μg mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c.