Inflammatory phenotype, clinicopathologic variables, and effects of long-term methylene blue in dogs with hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency.

OBJECTIVE: To determine whether dogs with cytochrome b5 reductase (CYB5R) deficiency have a constitutive proinflammatory phenotype, characterize hematologic and serum chemistry results, and describe changes in methemoglobin (MetHb) levels and serum C-reactive protein (CRP) concentrations after long-term per os (PO) methylene blue (MB) therapy. ANIMALS: 21 client-owned dogs (CYB5R deficient, n = 10; healthy controls, 11). PROCEDURES: In this prospective, case-control study, methemoglobin levels were measured using a blood gas analyzer with co-oximetry. Plasma tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations were measured using a canine-specific multiplex bead-based assay. Serum CRP concentrations were measured with a canine-specific commercial ELISA kit. Serum CRP concentration and MetHb levels were measured in 6 dogs with CYB5R deficiency after ≥ 60 days of PO MB therapy. RESULTS: As expected, MetHb levels were higher in dogs with CYB5R deficiency compared to controls (P < .001). Plasma TNF-α, IL-6, IL-10, and serum CRP concentrations were no different between CYB5R-deficient and control dogs. Dogs with CYB5R deficiency had lower absolute lymphocyte (P = .005) and eosinophil counts (P = .04) and higher alanine transaminase (P = .04) and alkaline phosphatase activity (P = .02) than controls, but these changes were not clinically relevant. Methemoglobin levels decreased after PO MB therapy (P = .03). CLINICAL RELEVANCE: These results suggest that otherwise healthy dogs with CYB5R deficiency do not have a constitutive proinflammatory phenotype and clinically relevant abnormalities in hematologic and serum chemistry panels are not expected. Dogs with decreased quality of life attributed to methemoglobinemia from CYB5R deficiency might benefit from PO MB therapy.

The First Korean Family with Hemoglobin-M Milwaukee-2 Leading to Hereditary Methemoglobinemia.

Hemoglobin M (HbM) is a group of abnormal hemoglobin variants that form methemoglobin, which leads to cyanosis and hemolytic anemia. HbM-Milwaukee-2 is a rare variant caused by the point mutation CAC>TAC on codon 93 of the hemoglobin subunit beta (HBB) gene, resulting in the replacement of histidine by tyrosine. We here report the first Korean family with HbM-Milwaukee-2, whose diagnosis was confirmed by gene sequencing. A high index of suspicion for this rare Hb variant is necessary in a patient presenting with cyanosis since childhood, along with methemoglobinemia and a family history of cyanosis.

Novel Redox Active Tyrosine Mutations Enhance the Regeneration of Functional Oxyhemoglobin from Methemoglobin: Implications for Design of Blood Substitutes.

Heme mediated oxidative toxicity has been linked to adverse side effects in Hemoglobin Based Oxygen Carriers (HBOC), initiated by reactive ferryl (FeIV) iron and globin based free radical species. We recently showed that the addition of a redox active tyrosine residue in the beta subunit (ßF41Y) of recombinant hemoglobin had the capability to decrease lipid peroxidation by facilitating the reduction of FeIV iron by plasma antioxidants such as ascorbate. In order to explore this functionality further we created a suite of tyrosine mutants designed to be accessible for both reductant access at the protein surface, yet close enough to the heme cofactor to enable efficient electron transfer to the FeIV. The residues chosen were: ßF41Y; ßK66Y; ßF71Y; ßT84Y; ßF85Y; and ßL96Y. As with ßF41Y, all mutants significantly enhanced the rate of ferryl (FeIV) to ferric (FeIII) reduction by ascorbate. However, surprisingly a subset of these mutations (ßT84Y, and ßF85Y) also enhanced the further reduction of ferric (FeIII) to ferrous (FeII) heme, regenerating functional oxyhemoglobin. The largest increase was seen in ßT84Y with the percentage of oxyhemoglobin formed from ferric hemoglobin in the presence of 100 µM ascorbate over a time period of 60 min increasing from 10% in ßF41Y to over 50% in ßT84Y. This increase was accompanied by an increased rate of ascorbate consumption. We conclude that the insertion of novel redox active tyrosine residues may be a useful component of any recombinant HBOC designed for longer functional activity without oxidative side effects.

Diagnosis of a rare fetal haemoglobinopathy in the age of next-generation sequencing.

Neonatal cyanosis resulting from a fetal methaemoglobin variant is rare. Most such variants are only described in a few published case reports. We present the case of a newborn with unexplained persistent cyanosis, ultimately determined to have a γ-chain mutation causing Hb FM-Fort Ripley. This neonatal haemoglobinopathy can be challenging to diagnose, as significant oxygen desaturation may result from barely detectable levels of the mutant haemoglobin and co-oximetry studies may show a falsely normal methaemoglobin level. Our analysis of the infant's haemoglobin included high-performance liquid chromatography, cellulose acetate electrophoresis and citrate agar electrophoresis, which showed trace amounts of a suspected variant. Ultimately, the diagnosis was made through a novel application of next-generation sequencing (NGS). NGS-based diagnostic approaches are becoming increasingly available to clinicians, and our case provides a framework and evidence for the utilisation of such testing paradigms in the diagnosis of a rare cause of neonatal cyanosis.

Congenital methemoglobinaemia due to Hb F-M-Fort Ripley in a preterm newborn.

Methemoglobinaemia is a rare cause of cyanosis in newborns. Congenital methemoglobinaemias due to M haemoglobin or deficiency of cytochrome b5 reductase are even rarer. We present a case of congenital methemoglobinaemia presenting at birth in a preterm infant. A baby boy born at 29 weeks and 3 days of gestation had persistent central cyanosis immediately after delivery, not attributable to a respiratory or cardiac pathology. Laboratory methemoglobin levels were not diagnostic. Cytochrome b5 reductase levels were normal and a newborn screen was unable to pick up any abnormal variants of fetal haemoglobin. Genetic testing showed a γ globin gene mutation resulting in the M haemoglobin, called Hb F-M-Fort Ripley. The baby had no apparent cyanosis at a corrected gestational age of 42 weeks. Although rare, congenital methaemoglobin aemia should be considered in the differential in a preterm with central cyanosis and investigated with genetic testing for γ globin chain mutations if other laboratory tests are non-conclusive.

Comparative analysis of oxy-hemoglobin and aquomet-hemoglobin by hydrogen/deuterium exchange mass spectrometry.

The function of hemoglobin (Hb) as oxygen transporter is mediated by reversible O2 binding to Fe(2+) heme in each of the α and ß subunits. X-ray crystallography revealed different subunit arrangements in oxy-Hb and deoxy-Hb. The deoxy state is stabilized by additional contacts, causing a rigidification that results in strong protection against hydrogen/deuterium exchange (HDX). Aquomet-Hb is a dysfunctional degradation product with four water-bound Fe(3+) centers. Heme release from aquomet-Hb is relatively facile, triggering oxidative damage of membrane lipids. Aquomet-Hb crystallizes in virtually the same conformation as oxy-Hb. Hence, it is commonly implied that the solution-phase properties of aquomet-Hb should resemble those of the oxy state. This work compares the structural dynamics of oxy-Hb and aquomet-Hb by HDX mass spectrometry (MS). It is found that the aquomet state exhibits a solution-phase structure that is significantly more dynamic, as manifested by elevated HDX levels. These enhanced dynamics affect the aquomet α and ß subunits in a different fashion. The latter undergoes global destabilization, whereas the former shows elevated HDX levels only in the heme binding region. It is proposed that these enhanced dynamics play a role in facilitating heme release from aquomet-Hb. Our findings should be of particular interest to the MS community because oxy-Hb and aquomet-Hb serve as widely used test analytes for probing the relationship between biomolecular structure in solution and in the gas phase. We are not aware of any prior comparative HDX/MS experiments on oxy-Hb and aquomet-Hb.

Hemoglobin Shady Grove: a novel fetal methemoglobin variant.

Recommendations by the US Department of Health and Human Services Secretary's Advisory Committee on Heritable Disorders in Newborns and Children aim to increase congenital heart disease screening by pulse oximetry in the nursery. Here, we describe a novel fetal methemoglobin variant discovered in a newborn found to have oxygen saturations significantly below normal upon pulse oximetry screening for congenital heart disease. As universal newborn screening with pulse oximetry is implemented, hereditary variant hemoglobins should be considered in the diagnostic work-up in otherwise well newborns with low SpO2 .

Hb M Dothan [beta 25/26 (B7/B8)/(GGT/GAG-->GAG//Gly/Glu-->Glu]; a new mechanism of unstable methemoglobin variant and molecular characteristics.

A new unstable beta globin chain variant associated with methemoglobin (Met-Hb) phenotype was found in a Caucasian infant. Molecular analysis of the beta globin gene using polymerase chain reaction (PCR) amplification and sequencing led to the detection of a new in frame deletion in exon-1. Direct sequencing of the PCR product revealed a 3 bp deletion (-GTG) between codons 25/26, which resulted in the loss of a single amino acid (-Gly). We propose that this newly discovered unstable M-hemoglobin (M-Hb) variant, named Hb Dothan [GGT/GAG-->GAG//Gly/Glu-->Glu], is caused by a shift in the amino acid sequence and altered packing of the B and E helices during beta globin synthesis, and also changes the orientation of the critical proximal and distal histidine in the F and E helices respectively. Phenotype/Genotype features and molecular characteristics of this new beta chain are presented in this communication.

Red cell enzymes.

Mutations leading to red cell enzyme deficiencies can be associated with diverse phenotypes that range from hemolytic anemia, methemoglobinemia, polycythemia, and neurological and developmental abnormalities. While most of these mutations occur sporadically, some such as common glucose-6-phosphate dehydrogenase (G6PD) mutants are endemic and rarely cause disease. Common G6PD mutants likely reached their prevalence because they provide some protection against severe malarial complications. In this review G6PD, pyruvate kinase, 5' nucleotidase, and cytochrome b5 reductase deficiencies will be discussed in greater detail. Limitations of commonly used screening tests for detection of these disorders will also be emphasized, as well as emerging knowledge about non-enzymatic function of the glycolytic enzymes.

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine.

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.

Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity.

Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure.

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study.

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.

Solution 1H-NMR structure of the heme cavity in the low-affinity state for the allosteric monomeric cyano-met hemoglobins from Chironomus thummi thummi. Comparison to the crystal structure.

Solution 1H-NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S = 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82-94]. The crystal structure for the low-pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientation, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338]. All heme pocket residues for the low-pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic-induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.

Serine-proline replacement at residue 127 of NADH-cytochrome b5 reductase causes hereditary methemoglobinemia, generalized type.

Hereditary methemoglobinemia is an autosomal recessive disorder characterized by NADH-cytochrome b5 reductase (b5R) deficiency. In an attempt to clarify the molecular mechanisms involved in the enzyme deficiency, we isolated the b5R gene from a patient homozygous for hereditary methemoglobinemia, generalized type, and compared its nucleotide sequence with that of the normal NADH-cytochrome b5R gene. Only one difference was observed; a thymidine at the first position of codon 127 (TCT) was altered to a cytidine in the b5R gene of the patient, resulting in replacement of serine with proline. Dot blot hybridization of the amplified DNA samples with allele-specific oligonucleotide probes showed that the proband and her brothers were homozygous for this mutation and that their father was heterozygous. Although the activity of b5R in lymphoblastoid cells from homozygotes was reduced to 10% of the normal level, RNA blot and protein blot analyses of the lymphoblastoid cells showed that synthesis of b5R messenger RNA and the b5R polypeptide were normal. Serine at residue 127 is presumed to be in an alpha-helix structure that is part of a nucleotide-binding domain. These observations suggest that replacement of Pro-127 causes a significant conformation change in the nucleotide-binding domain that affects electron transport from NADH to cytochrome b5. Functional enzyme deficiency results in a generalized type of hereditary methemoglobinemia.

Mutant fetal hemoglobin causing cyanosis in a newborn.

A well but cyanotic newborn was found to have a mutant gamma-globin chain, leading to a functionally abnormal fetal hemoglobin. A single amino acid substitution was found in a site consistent with known adult M hemoglobins. This patient showed no clinical evidence of cyanosis at 5 weeks of age as gamma-chain synthesis was replaced by beta-chain synthesis. A sibling born 20 months later was also cyanotic and the same mutant hemoglobin was found.