Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

A Cross-Reactive Monoclonal Antibody to Nematode Haemoglobin Enhances Protective Immune Responses to Nippostrongylus brasiliensis

Background

Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection.

Methodology/Principal Findings

Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.

Conclusion

The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.     

Genetic parameters for haemoglobin levels in pigs and iron content in pork

Genetic parameters were obtained for iron content in m. longissimus dorsi (2255 records) and haemoglobin levels recorded at 5 (4974 records) and 21 (2405 records) weeks of age in two sire lines from September 2009 until January 2011. The measure of iron in pork was the mean of two replicates. Genetic associations of haematological traits with meat quality traits (2255 records), as well as growth rate and backfat (close to 60 000 records), were estimated. Analyses were based on an animal model using residual maximum likelihood procedures. Iron content in pork was moderately heritable (0.34 ± 0.07) and genetic correlations with haemoglobin measures ranged from 0.39 ± 0.24 to 0.58 ± 0.13, indicating their potential use as selection criteria for increasing iron levels in pork. However, heritabilities for haemoglobin levels were low, ranging from 0.04 ± 0.2 to 0.18 ± 0.04. Procedures to measure haemoglobin on farm may require refinement. Redness of pork, quantified by a* value, had high genetic correlations with iron content (0.90 ± 0.04 to 0.94 ± 0.03) and moderate genetic correlations with haemoglobin levels (0.31 ± 0.22 to 0.55 ± 0.15). Iron content had significant genetic associations with L* measures (−0.61 ± 0.14 to −0.54 ± 0.23), b* value (0.60 ± 0.14 for dorsal b* measure, 0.50 ± 0.15 for average of dorsal and ventral b* measures) and pH at 45 min post mortem (−0.42 ± 0.14). These high genetic correlations between colour measurements and iron content in pork provide further avenues for selection strategies to improve iron content in pork. Current selection practices are not expected to affect iron content in pork, as no significant genetic correlations between performance and haematological traits were found.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

High-performance liquid chromatographic separation of human haemoglobins: Simultaneous quantitation of foetal and glycated haemoglobins

A high-performance liquid chromatographic system, which uses a weak cation exchanger (PolyCATA) together with Bis-Tris buffer (pH 6.47–7.0) and sodium acetate gradients, is described. Samples from adults and newborns were analysed and a clean separation of many minor and major normal and abnormal haemoglobin (Hb) variants was greatly improved. The method allows the separation of minor foetal haemoglobin (HbF) variants and the simultaneous quantitation of HbF and glycated HbA. HbF values correlated well with those obtained by the alkali denaturation method (r=0.997). The glycated haemoglobin (HbAr_Ic) levels measured in patients with high HbF concentrations correlated with the total glycated haemoglobin determined by bioaffinity chromatography (r=0.973). The procedure is useful for diagnostic applications and affords an effective and sensitive way of examining blood samples for haemoglobin abnormalities.     

Evaluation of genetic risks of alkylating agents IV. Quantitative determination of alkylated amino acids in haemoglobin as a measure of the dose after-treatment of mice with methyl methanesulfonate.

The present study explores the possibilities of using specific amino acids in haemoglobin for tissue dosimetry of alkylating agents. The well-known directly alkylating compound methyl methanesulfonate has been used as a model compound.In one experiment ³H-labelled methyl methanesulfonate was given to mice intraperitoneally at three dose levels. The degree of alkylation of haemoglobin exhibited a linear dependence on the quantity of methyl methanesulfonate injected. The degree of alkylation of guanine-N-7 in DNA indicated a slight positive deviation from linearity at high doses.After a single injection the degree of alkylation of cysteine-S and histidine-N-3 in haemoglobin decreased linearly with time reaching the value zero after about 40 days (the life-time of the erythrocytes in the mouse). This demonstrates a stability of these alkylated products, which is fundamental to their use as integral dose monitors.In a second experiment mice were treated with methyl methanesulfonate once a week over a period of 8 weeks. The experiment demonstrated an accumulation of alkylated groups in haemoglobin in agreement with expectation.A method for the quantitative determination of S-methylcysteine in a protein hydrolysate by gas chromatography was developed.     

Biochemische und cytogenetische Untersuchungen zur Natur des Hämoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidivittatus

The haemoglobin of chironomids is dissolved in the body fluid of the larvae and can be separated electrophoretically in Chironomus tentans into 10, and in C. pallidivittatus into 8, different bands. The molecular weight determined under the electrophoretic conditions was 15,000±1,000 for each haemoglobin band. This means that each haemoglobin band represented a single protein chain. In each species 7 haemoglobins could be characterised as species specific according to their different electrophoretic mobilities, developmental characteristics and the fact that one haemoglobin could be correlated genetically with a specific chromosome inversion. The inheritance of all these species specific haemoglobins was found to be co-dominant. With cytogenetic methods it was possible to define the loci of the species specific haemoglobin genes as being restricted to certain parts of chromosome 3. This finding suggests gene duplication as the most likely mecanism of the evolution of haemoglobins in Chironomus.     

Molecular size and shape of the native and reassociated forms of the extracellular haemoglobin of Tubifex tubifex

The extracellular haemoglobin of Tubifex tubifex and the product of its reassociation at neutral pH subsequent to dissociation at alkaline pH, were examined by small-angle X-ray scattering. The following molecular parameters were determined for the native and reassociated molecules, respectively: maximum diameter 30.0±1.0 and 32.0±1.0nm; radius of gyration 10.66±0.15 and 11.07±0.15 nm; molecular weight (3.09±0.15) × 10⁶ and (2.99±0.15) × 10⁶ dalton. Although the scattering curves of the native and reassociated haemoglobin possess similar shapes the distance distribution functions exhibit slight differences in their shape as well as in the position of their maximum. The best fit with the experimental distribution functions was obtained with models consisting of 12 spheres arranged in two hexagonal layers. In the case of the native haemoglobin each of the 12 spheres has a diameter of 9.3 nm while for the reassociated haemoglobin each of the 12 spheres has a diameter of 11.5 nm. The results suggest that although their molecular weights are the same, the reassociated molecule is slightly larger than the native molecule     

Mechanism of charge compensation and impairment of co-operative functions in haemoglobin Tacoma (Arg B12(30)beta leads to Ser).

A difference Fourier synthesis of deoxyhaemoglobin Tacoma minus deoxyhaemoglobin A at 3.5 Å resolution has been calculated. The map shows a large negative peak due to the removal of the guanidinium group of Arg B12(30)β, surrounded by positive and negative peaks indicative of some atoms moving towards, and others away from, the vacated site. Among the latter, the most important is the carboxylate of Glu B8(26)β which is hydrogen-bonded to the guanidinium group of the arginine in haemoglobin A, but swings round its α-β carbon bond towards the imidazoles of histidines G18(116) and 19(117)β in haemoglobin Tacoma. This movement would raise the pK values of the histidines, so that their positive charges compensated for the loss of the arginine. This may explain why haemoglobin Tacoma has the same electrophoretic mobility as haemoglobin A. It is shown that haemoglobin Tacoma has a lower oxygen equilibrium constant K_T and a larger allosteric constant L than haemoglobin A. The lowering of K_T may be due to the loosening of the T structure in haemoglobin Tacoma consequent upon the removal of the hydrogen bonds made by the guanidinium group of Arg B12(30)β at the α₁β₁ contact. Their removal also accounts for the decreased stability of haemoglobin Tacoma. We cannot yet explain its diminished Bohr effect, nor the increase in L.     

Circular dichroism and absorption spectra of haemoglobin Bart's

Circular dichroism spectra of haemoglobin Bart's (γ₄) in the region from 240 to 600 nm were different from those of human adult haemoglobin, but closely similar to those of the β-subunit of human adult haemoglobin. The amplitude of the positive circular dichroism maximum of deoxygenated haemoglobin Bart's in the Soret region was much less than that of human adult haemoglobin. The peak molar extinction coefficient of deoxygenated haemoglobin Bart's in the Soret region was found to be lower than that of deoxygenated human adult haemoglobin. These data indicate that haemoglobin Bart's, which is composed of four identical chains and lacks co-operativity, is structurally similar to haemoglobin H (β₄).     

Expression,Characterization, and Cellular Localization of Knowpains,Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.     

Ligand binding kinetics of des arginine haemoglobin

Des arginine 141 a haemoglobin (the haemoglobin in which the C-terminal arginine of the a chain has been removed) has a high affinity for oxygen and a reduced co-operativity in its oxygen equilibrium binding. The kinetic consequences of this modification are investigated in this paper. Deoxy des Arg haemoglobin binds carbon monoxide faster than does haemoglobin A, whilst oxy des Arg haemoglobin loses oxygen more slowly. These results are correlated with the oxygen equilibrium binding properties of des Arg haemoglobin. The carbon monoxide binding kinetics have been interpreted as implying a change in the parameter c (of the allosteric model), as well as L, when this arginine is removed from haemoglobin.     

Three-dimensional structure of abnormal human haemoglobins Chesapeake and J Capetown

The replacement of the invariant residue, arginine FG4(92)α, by a leucine in the mutant human haemoglobin Chesapeake causes drastically abnormal functional properties. When the arginine is replaced by a glutamine in haemoglobin J Capetown the mutant protein is almost normal. Crystallographic studies at 5.5 Å resolution show that the deoxy form of these two mutants have no significant structural distortions. In contrast, the structure of oxyhaemoglobin Chesapeake is considerably distorted. It appears that in the oxy form, the leucine side chain introduces impermissibly close van der Waal's contacts which disrupt the structure. This disturbance of the oxy structure is probably responsible for the abnormal properties displayed by haemoglobin Chesapeake. The structural basis for the milder abnormalities of haemoglobin J Capetown is as yet unknown.     

Effects of 2,4-dichlorophenoxyacetic acid on haematological indices in West African dwarf goats

We investigated the possible toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in West African dwarf goats. The goats (20) were randomly divided into four equal groups; three of which were exposed to graded levels (low, medium and high doses) of 2,4-D for a period of 6 weeks. Blood samples were collected from the treatment group goats as well as the control group goats on weeks 0, 2, 4 and 6. The blood samples were used for analysis of haematologic indices such as packed cell volume (PCV), erythrocyte count (EC), total leucocyte count (TLC), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Total leucocyte counts were significantly reduced (p < 0.05) by the test herbicide (2,4-D) in all the treatment groups. Significant reduction in the levels of PCV, Hb concentration, MCV, MCH and MCHC were also recorded in all treatment groups. There was no significant change (p > 0.05) in the erythrocyte count value of all the four groups. The significant reduction in the haematologic indices of West African dwarf goats (WADG) as evidenced from the result of this study, suggest the possibility of 2,4-D toxicity in these goats.     

Uptake of homologous haemolymph protein by salivary glands of Chironomus thummi

Radioisotopically labelled homologous protein fractions were injected into the haemolymph of Chironomus larvae. Among four different protein fractions, fraction II appears to enter the salivary gland and accumulates in the glandular lumen. Lack of blood pigmentation in the gland and secretion lumen indicates that the haemoglobin is probably not secreted, but a portion of the haemoglobin molecule or a fraction co-precipitating with haemoglobin in fraction II is being transported by the gland. Since only a portion of fraction II is taken up and secreted, the specificity of the process suggests to us a molecular basis for the activity which must ultimately be reflected in the genes of the transporting tissue.     

Histochemical and ultrastructural observations on digestion in Tetrameres fissispina Diesing, 1861 (Nematoda: Spiruridea) with special reference to intracellular digestion

A large proportion of the blood ingested by Tetrameres fissispina is digested extracellularly to haematin. The probable site of extracellular haemoglobin degradation is the glycocalyx of the microvilli which may carry adsorbed enzymes functional in contact digestion. A smaller proportion of the haemoglobin released from haemolysed erythrocytes is endocytosed in an unchanged state by isolated groups of absorptive cells. In the latter, haemoglobin-containing phagosomes apparently fuse with primary lysosomes ultimately to produce large, heterogeneous, multiple phagolysosomes (digestive complexes). Lipid droplets produced during digestion are extruded from these at intervals. Haemosiderin is the end-product of intracellular haemoglobin breakdown—the differences in residues of the extracellular and intracellular processes (haematin and haemosiderin) reflecting differences in the two enzyme systems employed. Haemosiderin is accumulated as sphaerocrystals in dilated cisternae of the ER. It is suggested that the purpose of intracellular digestion is to provide a source of ferric ions (in the form of haemosiderin) for the biosynthesis of endogenous haemoglobin which the extracellular degradation of haemoglobin cannot supply.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Plasmodium falciparum Malaria in Children Aged 0-2 Years: The Role of Foetal Haemoglobin and Maternal Antibodies to Two Asexual Malaria Vaccine Candidates (MSP3 and GLURP)

Background

Children below six months are reported to be less susceptible to clinical malaria. Maternally derived antibodies and foetal haemoglobin are important putative protective factors. We examined antibodies to Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate-rich protein (GLURP), in children in their first two years of life in Burkina Faso and their risk of malaria.

Methods

A cohort of 140 infants aged between four and six weeks was recruited in a stable transmission area of south-western Burkina Faso and monitored for 24 months by active and passive surveillance. Malaria infections were detected by examining blood smears using light microscopy. Enzyme-linked immunosorbent assay was used to quantify total Immunoglobulin G to Plasmodium falciparum antigens MSP3 and two regions of GLURP (R0 and R2) on blood samples collected at baseline, three, six, nine, 12, 18 and 24 months. Foetal haemoglobin and variant haemoglobin fractions were measured at the baseline visit using high pressure liquid chromatography.

Results

A total of 79.6% of children experienced one or more episodes of febrile malaria during monitoring. Antibody titres to MSP3 were prospectively associated with an increased risk of malaria while antibody responses to GLURP (R0 and R2) did not alter the risk. Antibody titres to MSP3 were higher among children in areas of high malaria risk. Foetal haemoglobin was associated with delayed first episode of febrile malaria and haemoglobin CC type was associated with reduced incidence of febrile malaria.

Conclusions

We did not find any evidence of association between titres of antibodies to MSP3, GLURP-R0 or GLURP-R2 as measured by enzyme-linked immunosorbent assay and early protection against malaria, although anti-MSP3 antibody titres may reflect increased exposure to malaria and therefore greater risk. Foetal haemoglobin was associated with protection against febrile malaria despite the study limitations and its role is therefore worthy further investigation.     

Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge, Chironomus thummi

The fat body of developing mid- and late fourth instar larvae of a midge, Chironomus thummi, has been investigated by means of the benzidine reaction for the localization of haemoglobin within cells. In the subepidermal fat body the reaction deposits of the haemoglobin pseudo-peroxidase activity appear predominantly in the intracisternal cavities of ER and the Golgi, and later, in the pharate pupal stage, in small dense granules (0.5–1 μm in. diameter).All the major protein bands of fat body extracts, which are resolved in electrophoresis, give the benzidine reaction and show incorporation of ¹⁴C-amino levulinic acids, in this case a specific marker for haemoglobin synthesis. In addition, labelled proteins show identical electrophoretic mobility as the haemoglobins of the haemolymph, suggesting that haemoglobins are synthesized in the fat body. Two types of fat body cells seem to differ with respect to their rôle in haemoglobin metabolism.     

Simultaneous quantification of haemoglobin adducts of ethylene oxide,propylene oxide,acrylonitrile, acrylamide and glycidamide in human blood by isotope-dilution GC/NCI-MS/MS

Haemoglobin adducts are highly valuable biomarkers of cumulative exposure to carcinogenic substances. We have developed and applied an analytical method for the simultaneous quantification of five haemoglobin adducts of important occupational and environmental carcinogens. The N-terminal adducts were determined with gas chromatography as pentafluorophenylthiohydantoine derivatives according to the modified Edman-procedure and subsequent acetonization of the glycidamide adduct N-(R,S)-2-hydroxy-2-carbamoylethylvaline (GAVal). The use of self-synthesized labelled internal standards in combination with tandem mass spectrometry using negative chemical ionisation guarantees both high accuracy and sensitivity of our determination. The limit of detection for N-2-hydroxyethylvaline (HEVal), N-(R,S)-2-hydroxypropylvaline (HPVal), N-2-carbamoylethylvaline (AAVal) and N-(R,S)-2-hydroxy-2-carbamoylethylvaline (GAVal) was 2 pmol/g globin, for N-2-cyanoethylvaline (CEVal) it was determined as 0.5 pmol/g globin, which was sufficient to determine the background levels of these adducts in the non-smoking general population. The between-day-precision for all analytes using a human blood sample as quality control material ranged from 4.7 to 12.3%. We investigated blood samples of a small group (n = 104) of non-smoking persons of the general population for the background levels of these haemoglobin adducts. The median values for HEVal, HPVal, CEVal, AAVal and GAVal in a group of 92 non-smoking persons were 18.1, 4.1, <0.5, 29.9 and 35.2 pmol/g globin, respectively. The adduct levels in 12 persons reporting exposure to passive smoke at home were similar for most adducts with median values of 17.2, 4.1, 1.0, 24.9 and 29.7 pmol/g globin for HEVal, HPVal, CEVal, AAVal and GAVal, respectively. Our results point to an elevated uptake of acrylonitrile caused by passive smoking as indicated by higher levels of the corresponding haemoglobin adduct CEVal.