Β-chain Of Hemoglobin Research Articles

Measuring postmortem glycated hemoglobin – A comparison of three methods

Glycated hemoglobin (HbA1c) is a glycoprotein formed as a result of the non-enzymatic addition of d-glucose to the hemoglobin β-chain. The amount of HbA1c in the blood is dependent on mean glucose levels present during the 1-2 months preceding measurement, and it is an indicator of long-term glycemic levels. HbA1c is a useful marker in postmortem biochemistry in determining cause of death in acetonemic cases by allowing to distinguish diabetic ketoacidosis (DKA) from starvation or alcoholic ketoacidosis (AKA) and intoxication by acetone or isopropanol. We evaluated three methods for postmortem HbA1c measurement: Mono S cation exchange HPLC, affinity chromatography using point-of-care analyzer and a direct enzymatic method by. Additionally, optimization of sample storage and preservatives was performed and interpretation of the results in autopsy cases is discussed. Our results indicate that the HbA1c levels of postmortem samples can be reliably measured with Mono S HPLC. EDTA is the preferable preservative, as samples can be measured after as long as 4 weeks storage at +4°C. Our study shows that HbA1c analysis is a valuable tool alongside glucose and ketone body analysis in determining the metabolic state of deceased persons in medicolegal autopsies.

Hematopoietic Stem Cell Transplantation for Thalassemia

Thalassemia is an autosomal recessive disorder associated with defective synthesis of the α- or β-chain of hemoglobin. For β-thalassemia major patients, therapeutic options are either monthly red cell transfusions and chelation therapy or allogeneic stem cell transplant. Patients undergoing transfusion therapy remain at risk for transmitted infections and iron overload with associated tissue damage. Stem cell transplant is the only curative approach and success is inversely correlated with the degree of iron overload and hepatic damage. Overall outcomes following stem cell transplant with a matched sibling donor are excellent with over 90% of low-risk children becoming transfusion free. Hypertransfusion therapy and aggressive chelation in addition to hydroxyurea, azathioprine and fludarabine is a new approach for high-risk patients to decrease graft rejection by suppressing endogenous erythropoiesis pretransplant. The use of unrelated donors and novel approaches such as gene therapy are under current investigation.

Potamotrygon cf. henlei stingray mucus: Biochemical features of a novel antimicrobial protein

Antimicrobial molecules are important components of the innate immune system in vertebrates. They have been studied widely in several fishes, but little is known about these defence factors in stingrays, which are thought to have less sophisticated adaptive immune systems when compared to other teleosts. Stingrays from the specie Potamotrygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the cause of numerous envenomations occurring in the dry seasons.In a previous study, we reported that the mucus of the stingray P. cf. henlei shows antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf. henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-MS, and Edman degradation. A protein similar to the β-chain of hemoglobin was identified, isolated and partially sequenced by Edman degradation. This protein has a molecular weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new protein in the microcirculation environment were also evaluated. The results obtained provide fundamental information for future basic research, clinical diagnosis and development of new therapies to accident treatment. To the best of our knowledge, this is the first description of a bioactive polypeptide from the mucus of a stingray.

Characterization of the Hepatic Cellular Uptake of α1-Acid Glycoprotein (AGP), Part 1: A Peptide Moiety of Human AGP Is Recognized by the Hemoglobin β-Chain on Mouse Liver Parenchymal Cells

Human α(1) -acid glycoprotein (AGP), a serum glycoprotein, is known to have anti-inflammatory activity. We recently reported that AGP was mainly incorporated into the liver in mice via a receptor-mediated pathway, although the mechanism for this was largely unknown. The objective of this study was to identify the specific cellular surface protein that recognizes the peptide moiety of AGP. Pharmacokinetic studies of (111) In-AGP and (111) In -recombinant glycan-deficient AGP (rAGP) in mice demonstrated that both AGPs are mainly distributed to the liver and kidney, but hepatic and renal uptake clearance of rAGP was higher than that for AGP. Hepatic uptake of rAGP was inhibited in the presence of 100-fold excess of unlabeled AGP, indicating that the hepatic uptake of rAGP shared a common route with that of AGP and that it recognized the peptide moiety of AGPs. In ligand blotting analyses using crude cellular membrane fraction of mice liver, a band corresponding to a 16 kDa protein was observed to bind to both AGPs. Interestingly, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MALDI-TOF-MS and western blotting analyses indicated that this 16 kDa protein is the hemoglobin β-chain (HBB). It, therefore, appears that HBB is associated with the hepatic uptake of AGP via a direct interaction with its peptide moiety.

Characterization of Hepatic Cellular Uptake of α1-Acid Glycoprotein (AGP), Part 2: Involvement of Hemoglobin β-Chain on Plasma Membranes in the Uptake of Human AGP by Liver Parenchymal Cells

Human α(1) -acid glycoprotein (AGP), a lipocalin family member, serves as a carrier for basic drugs and endogenous hormones. It is mainly distributed in the liver and also has anti-inflammatory effects. We previously discovered a protein in liver parenchymal cells that interacts with AGP and it was identified as hemoglobin β-chain (HBB). The purpose of this study was to clarify the role of HBB in the hepatic cellular uptake of AGP. Ligand blotting experiments showed that the interaction of (125) I-AGP with hemoglobin was saturable and was significantly suppressed in the presence of excess unlabeled AGP. In addition, the cellular uptake of fluorescein isothiocianate-AGP by HepG2 cells was saturable and temperature dependent. This uptake was inhibited by fillipin and methyl-β-cyclodextrin, but not chlorpromazine, suggesting that AGP is taken up via caveolae/lipid rafts endocytic pathway. Immunostaining showed that HBB and caveolin-1, exclusively expressed in caveolae, were partially colocalized on the plasma membranes of HepG2 cells. HBB knockdown with siRNA decreased the uptake of AGP by HepG2 cells by 40%, and exogenous hemoglobin inhibited the uptake by 40%-50%. These findings indicate that HBB is located on the liver plasma membrane and that it contributes to the intracellular uptake of AGP.

LVV-hemorphin 7 and angiotensin IV in correlation with antinociception and anti-thermal hyperalgesia in rats

Hemorphins, a family of atypical endogenous opioid peptides, are produced by the cleavage of hemoglobin β-chain. Hemorphins were proved to bind to the μ-opioid receptors (agonist) and angiotensin IV receptors (insulin-regulated aminopeptidase; IRAP) (inhibitor). Among the hemorphins, LVV-hemorphin-7 (LVV-H7) was found to be abundant and with a longer half life in the CNS. Using intrathecal and intracerebroventricular injections, LVV-H7 and angiotensin IV were given to the rats, which were then subjected to the plantar test and the tail-flick test. Our results showed that LVV-H7 attenuated carrageenan-induced hyperalgesia at the spinal level, which could not be reversed by the co-administration of naloxone. At the supraspinal level, LVV-H7 also produced a significant anti-hyperalgesia effect but with a lower extent. Angiotensin IV showed a similar anti-hyperalgesia effect at the spinal level, but had no effect at the supraspinal level. In the tail-flick test and paw edema test, both peptides showed no effect. These results suggest that LVV-H7 mainly exert the anti-hyperalgesia effect at the spinal level, possibly through IRAP but not μ-opioid receptors. In addition, we observed the expression of IRAP in the CNS of animals with/without carrageenan-induced hyperalgesia. Our results showed a significant expression of IRAP in the spinal cord of rats. However, there was no significant quantitative change of IRAP after the development of hyperalgesia. The serum level of LVV-H7 was also found to be with no change caused by hyperalgesia. These results indicated that the endogenous LVV-H7 and IRAP may not regulate the severity of hyperalgesia through a quantitative change.

Carotid atherosclerotic plaques: Proteomics study after a low‐abundance protein enrichment step

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin β-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.

Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer

Abstractβ-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

Analysis of the mitochondrial proteome in multiple sclerosis cortex

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin β-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.

Adduct formation of Thimerosal with human and rat hemoglobin: a study using liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOF-MS)

Thimerosal (THI) is used as a preservative in many vaccines throughout the world. Ethylmercury (EtHg(+)), released from THI in aqueous media, has a high affinity to thiol functions of proteins. In blood, hemoglobin is a likely target protein because of its high abundance and its several free thiol functions. In comparison to hemoglobin of human origin, hemoglobin of rats exhibits almost twice as many free thiol groups, which might lead to different binding behavior and therefore a limited comparability between the situation in man and in rats, which are frequently used as models for mercury species toxicity investigations. Thus, the adduct formation of EtHg(+) with hemoglobin of humans and rats was compared under simulated physiological conditions by using gradient reversed-phase liquid chromatography (LC) with electrospray time-of-flight mass spectrometry (ESI-TOF-MS) detection. The binding stoichiometry correlated with the number of free thiols in the α- and β-chain of hemoglobin. The use of rats to verify the safety of additives in vaccines like Thimerosal is therefore doubtful and should be reevaluated.

Probing the oxyferrous and catalytically active ferryl states of Amphitrite ornata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I.

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O(2)] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O(2)(2-)] heme species, reflecting the presence of conformational substates in the oxyferrous precursor. (1)H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His N(ε)H and heme-bound O(2) in these conformers are similar to those in the β-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g(⊥) = 3.75 and g(∥) = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)═O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H(2)O(2). However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)═O)(porphyrin)/Tyr(•)]. Addition of the substrate analogue 2,4,6-trifluorophenol (F(3)PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)═O]. These assumptions are also supported by the observation that F(3)PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F(3)PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes*

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.

Quantitative enzyme-linked immunosorbent assay determination of an abundant hemoglobin-derived anti-infective peptide in human placenta

A fragment of the human β-chain of hemoglobin (HEM), hHEMβ111–146, was shown to have broad antimicrobial properties. The 3.9-kDa peptide was postulated to occur in high concentrations in placenta tissue. We established a reliable method to quantify hHEMβ111–146 in placenta tissue. Our methodology consists of a tissue extraction step (step 1), a chromatographic enrichment step (step 2), and a final quantification step (step 3) by enzyme-linked immunosorbent assay (ELISA). The specificity of the ELISA reaction was confirmed by parallel analysis of the samples via Western blot (step 4). The ELISA measured the absorbance of a tetramethylbenzidine substrate at 450 nm. It showed no cross-reactivity with the corresponding γ- and α-HEM regions and low cross-reactivity with the β-HEM region and full-length HEM. The sample preparation procedure enabled a prepurification of hHEMβ111–146, completely eliminating cross-reactive proteins and HEM peptides. The linear range of detection in step 3 was 20–200 ng/well (200–2000 μg/L) with a limit of quantification of 23 ng/well (230 μg/L) and a limit of detection of 7 ng/well (70 μg/L). The assay was characterized by good linearity ( r 2 > 0.99), intraday precision (coefficient of variation [CV] = 2.2–8.3%), interday precision (CV = 1.8–9.1%), and accuracy (76–109%). The mean recovery of the ELISA was determined to be 97%, and the overall recovery during steps 1–3 was found to be 40.3 ± 2.5%. We measured concentrations from 0.28 to 0.74 mg/g placenta tissue of the hHEMβ111–146 in different placenta samples with an average concentration of 0.57 mg/g. This abundant concentration supports an important physiological role of hHEMβ111–146 in the placenta infective barrier.

Unexpected expression of α- and β-globin in mesencephalic dopaminergic neurons and glial cells

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for alpha- and beta-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing alpha- and beta-globin chains, changes in genes involved in O(2) homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.

A novel mutation of −50 (G→A) in the direct repeat element of the β-globin gene identified in a patient with severe β-thalassemia

Dear Editor, β-Thalassemia is a heterogeneous genetic disease associated with defective expression of the β-chain of human hemoglobin. So far, more than 200 mutations have been defined and affect almost every known stage of β-globin gene expression resulting in a reduction (β) or complete absence (β) of β-chain synthesis from the affected allele [1, 2]. In southern China, the carrier rate of β-thalassemia in Guangdong province was 2.54% [3], with five common mutations accounting for 90% of all β-thalassemia individuals. Identification of new mutations and update of the mutation spectrum of thalassemia in one specific ethnic population is always needed for improving genetic counseling and prenatal diagnosis. We here describe a novel promoter mutation of −50 (G→A) within the conserved direct repeat element (DRE) leading to β-thalassemia major when associated with a β-thalassemia mutation. A 3-year-old Chinese boy with his parents was seen at our genetics clinic. The boy suffered from β-thalassemia major and had been transfused every 2 months since the age of 6 months. Molecular analyses performed by reverse dot blots (RDB) at other clinics revealed that he was a heterozygote for the common mutation of codons (CDs) 41–42 (−TTCT) of the β-globin gene, but the other mutational allele was not determined. At this referral, the family was planning for another pregnancy and counseled about prenatal diagnosis. Blood samples were obtained from all three family members. Hematological investigations showed that both of the parents had a classical β-thalassemia trait and the son manifested with severe β-thalassemia (Table 1). Determination of the β-thalassemia mutations was performed using the polymerase chain reaction (PCR)–RDB method which can simultaneously detect 17 types of β-globin gene mutations found in Chinese population. This confirmed the heterozygous presence of the CDs 41–42 (−TTCT) mutation in the son and the father, but the mutation was not found in the mother. Gap PCR was also undertaken in the three family members to exclude the coinheritance of αthalassemia, and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−α, −α, –). To further investigate the underlying mutation in this family, the β-globin gene was amplified by PCR from DNA of the mother, and the product was analyzed by direct nucleotide sequencing using the BigDyeTM Terminator Cycle Sequencing Kit and the ABI PRISMTM 310 genetic analyzer (Applied BioSystems). Direct sequencing of the β-globin gene showed the normal sequence except for the heterozygous G→A mutation at position −50 relative to the Cap site (Fig. 1). This so far undescribed mutation was also revealed in the son by DNA sequencing. Thus the patient was confirmed to be a compound heterozygote with a genotype of −50 (G→A)/CDs 41–42 (−TTCT). In order to evaluate the functional effect of this novel mutation, two other family members were investigated. A semiquantitative reverse transcription PCR assay was used to measure β globin mRNA in the reticulocytes from three heterozygous subjects (the mother, grandmother, and an aunt) and eight normal individuals. These analyses showed a mildly reduced β-globin mRNA level (23.4 %) in heterozygotes compared with that in normal subjects. Ann Hematol (2009) 88:1149–1150 DOI 10.1007/s00277-009-0732-8

Gene Therapy for Homozygous β -thalassemia. Is it a Reality?

The β-thalassemias are genetic disorders that are caused by the absent or insufficient production of the β-chain of hemoglobin. This deficiency causes ineffective erythropoiesis and hemolytic anemia. Without treatment, the severe form of the disease is lethal within the first decade of life. The only curative therapeutic option to date is allogeneic bone marrow transplantation from a matched, related donor, which carries a low risk of morbidity and mortality. Most patients, however, lack a matched donor and are thus managed with palliative therapy, consisting of lifelong transfusion therapy combined with pharmacological chelation to curb iron accumulation. Despite a major improvement in the chelation therapy and supportive care, the major cause of death in these patients is cardiac failure due to secondary hemochromatosis. The goal of globin gene therapy is to offer a potentially curative treatment to patients lacking a matched, related donor, based on the transfer of a regulated β-globin gene in autologous CD34+ hematopoietic cells collected following G-CSF mobilization. Our clinical trial at Memorial Sloan-Kettering Cancer Center builds on a 20-year long investigation to develop an erythroid-specific vector to regulate β-globin transgene expression in the progeny of transduced hematopoietic stem cells. To minimize the risks to the patient, the genetically modified cells will be infused after extensive biosafety testing of the transduced cells and following the administration of a reduced intensity (non-myeloablative) conditioning regimen. The protocol will be offered to patients with transfusion-dependent ß-thalassemia who are 15 years or older and lack a matched, related donor.

Biological markers of diabetes mellitus: recent developments and their clinical relevance

The availability of robust biological markers of diabetes mellitus is crucial for preventing severe long-term complications. Among them are markers of non-enzymatic glycation, due to the spontaneous binding of sugars to amino groups of proteins. Glycation generates a variety of products that eventually form complex and deleterious compounds named advanced glycation end products (AGEs). HbA 1c , characterised by the binding of glucose to the N-terminal valine of hemoglobin β-chains, is the key parameter for retrospectively assessing glycaemic balance. HbA 1c level is closely linked to the development of degenerative complications. The International Federation of Clinical Chemistry (IFCC) reference method is high performance liquid chromatography (HPLC) coupled with mass spectrometry or capillary electrophoresis, which provides HbA 1c percentages lower than obtained by the currently used national glycohemoglobin standardization program (NGSP). Results should be expressed as mmol HbA 1c /mol Hb. International societies of Biology and of Diabetology have agreed to report HbA 1c results using both units, in order to ensure the best evolution of medical practices. When HbA 1c determination is not informative (alterations of haemoglobin structure or metabolism), fructosamine determination (i.e., plasma glycated proteins) is a valuable alternative. New methods for evaluating AGEs (e.g., pentosidine, carboxymethyllysine) in vitro (proteomics, LC-tandem MS) or in vivo (transdermic measurement of AGEs) represent promising fields of investigation.

Genetic complexity in sickle cell disease

Sickle cell disease (SCD) was the first human monogenic disorder to be characterized at the molecular level (1). It results from the substitution of glutamic acid by valine at position 6 of the β-chain of hemoglobin. The clinical manifestations of SCD arise from the tendency of sickle hemoglobin (known as HbS or α2βS2) to polymerize at reduced oxygen tensions and deform red cells into the characteristic rigid sickle cell shape. Such inflexible red cells cannot pass through the microcirculation efficiently, and this results in anemia (due to destruction of the red cells) and intermittent vasoocclusion causing tissue damage and pain (2). Although all patients with homozygous SCD have exactly the same molecular defect, there is considerable clinical variation, ranging from death in early childhood (3) to a normal life span with few complications (4). Genetic modifiers of SCD include α-thalassemia (5), and it has been known for many years that patients with increased levels of fetal Hb (HbF or α2γ2) often tend to have a relatively mild clinical course (2) because HbF reduces the tendency of HbS to polymerise within the red cell. Increased HbF may result from rare deletions within the β-globin gene cluster or from point mutations in the promoters of the fetal γ-globin genes (hereditary persistence of fetal hemoglobin, HPFH), but additional loci are known to increase HbF levels in adult life. Identifying such loci has been a painstaking task, but a combination of genome-wide analysis within a large kindred (6) and within twin pairs (7) has identified two quantitative trait loci (QTL) with major influences on fetal hemoglobin levels in adults. In this issue of PNAS, Lettre et al. (8) now show that a significant proportion of the variation in HbF levels and the frequency of painful crises in patients with SCD is accounted for by five common single-nucleotide polymorphisms (SNPs) at these loci (Fig. 1).

Modified HPLC-Electrospray Ionization/Mass Spectrometry Method for HbA1c Based on IFCC Reference Measurement Procedure

Monitoring of hemoglobin A(1c) (HbA(1c)) is important in the management of diabetes. The IFCC reference measurement procedure for HbA(1c) is based on the ratio of glycated to nonglycated N-terminal hexapeptides of the beta-chains of hemoglobin after digestion with Glu-C endoproteinase. We developed a modification of the original reference measurement procedure with HPLC-electrospray ionization/mass spectrometry (ESI/MS). We performed chromatographic separation of the hexapeptides using a C12 reversed-phase column and a binary gradient system consisting of a mixture of H(2)O/acetonitrile/formic acid. Using this method, we obtained higher signal intensities and improved system stability compared with the reference measurement procedure. In the range of 3% to 14% HbA(1c), intralaboratory CVs were 0.71% to 1.86%. Deviations from IFCC target values were -0.87 to 1.00 relative %. These values fulfill acceptability criteria for HbA(1c) determination set by the IFCC Working Group on HbA(1c) Standardization. This procedure for the determination of HbA(1c) improves the existing reference measurement procedure.

Phenotypic analysis of Atlantic cod hemoglobin chains using a combination of top-down and bottom-up mass spectrometric approaches

Amino acid sequence information of both α- and β-chains of hemoglobin collected from Atlantic cod was obtained using a combination of mass spectrometry-based top-down and bottom-up approaches. Although complete sequencing is a very challenging undertaking due to the high degree of phenotypic variation, we were able to obtain between 38%, 28%, 46%, and 18% sequence coverage for the α-1-globin, α-2-globin, β-1-globin, and β-2-globin, respectively. The results of this work provide some evidence that the hemoglobin of Atlantic cod caught off the New England shoreline in the United States show genotypic/phenotypic variation from the hemoglobin of the same species caught off the coast of Norway. Though full coverage of the sequences was not accomplished, this work demonstrates the importance of further study to catalog the phenotypic variation of the American populations of Atlantic cod.