Diabetes in pregnancy: Effect on glycation and acetylation of the different chains of fetal and maternal hemoglobin.

BACKGROUND: Measurement of glycated fetal hemoglobin to assess maternal glycemic control is unreliable. Electrospray ionization-mass spectrometry (ESI-MS) has been suggested as a definitive procedure. OBJECTIVE: This study aimed to evaluate glycation and acetylation by ESI-MS of the separate chains of neonatal fetal hemoglobin in comparison with glycation of maternal hemoglobin, to assess the impact of maternal diabetes. METHODS: Twenty-nine non-diabetic (31 neonates) and 15 diabetic women (15 neonates) were recruited. Whole blood was collected at delivery from the mothers and cord blood from their respective neonate. The blood samples were diluted in acetonitrile:water (50:50) and treated with a cation exchange resin to remove sodium and potassium adducts on the hemoglobin. The α- and ß-chain glycated maternal hemoglobin and α- and γ-chain glycated and acetylated hemoglobin of the neonate were measured by ESI-MS. HbA1c was measured on the Menarini 8160. RESULTS: Mean α- and γ-chain and overall glycated hemoglobin and acetylated fetal hemoglobin were 1.23±0.41%, 1.62±0.47%, 1.24±0.37%, and 8.56±0.84% in the infant of the non-diabetic mother (INDM) and 1.39±0.21%, 1.92±0.61%, 1.64±0.59%, and 8.44±0.63% in the infant of the diabetic mother (IDM). Mean maternal α- and ß-chain, overall glycated hemoglobin and HbA1c were 1.98±0.38%, 4.28±0.76%, 3.13±0.51%, 5.39±0.39% (non-diabetic) and 2.40±0.83%, 4.71±0.90%, 3.58±0.83%, 6.15±0.71% (diabetic). Overall glycated hemoglobin levels were significantly higher in the IDM (p=0.006) compared to the INDM. Maternal glycated hemoglobin was significantly higher than neonatal glycated hemoglobin in the control (p<0.0001) and diabetic group (p<0.0001). A significant correlation was observed between maternal glucose concentration and overall glycated fetal hemoglobin in the IDM (p=0.02). Glucose concentrations in the diabetic mother and the IDM were not significantly different (p=0.5) and were significantly correlated (p<0.0001). HbA1c was not significantly different in the two maternal groups. No significant difference was observed between AcHbF in IDM or INDM (p=0.61). CONCLUSIONS: Measurement of overall glycated fetal hemoglobin, incorporating α- and γ-chain glycations, seems best to assess abnormal glucose homeostasis during pregnancy. Accurate assay of this parameter is critical for stratification of risk of possible post birth developmental complications.

The potential of electrospray ionization mass spectrometry for the diagnosis of hemoglobin variants found in newborn screening.

Analytical procedures have been developed for the detection and diagnosis of sickle cell disease in newborn babies by analyzing the hemoglobin extracted from dried blood spots on Guthrie cards using electrospray ionization mass spectrometry (ESI-MS). An essential requirement is the ability to reliably differentiate two globin chains whose molecular weights differ by only 1 Da such as adult hemoglobin (Hb A) and Hb C. This has been achieved by improving the accuracy and precision of the molecular weight determination to a fraction of a dalton. We report the potential of mass spectrometry for screening neonates for these debilitating diseases by presenting results from 147 blood spots that had been characterized by phenotypic methods and which include samples from 20 sickle cell disease, 1 beta-thalassemia major, 57 sickle cell trait, and 39 normal babies. In all cases, the mass spectrometric results agreed with the results obtained using conventional analytical practice with high-performance liquid chromatography (HPLC) and isoelectric focusing (IEF). We show that mass spectrometry is a viable technique for the diagnosis of newborns with sickle cell disease or homozygous beta0-thalassemia.

The number of unexpected HbA variants may be a greater problem in routine practice than is generally realized.

BACKGROUND: The majority of variant and abnormal haemoglobins are clinically silent but may not be biochemically silent when it comes to HbA1c estimation. CASE REPORT: We describe several cases in which there were problems in the determination of HbA1c for monitoring diabetes in patients that would not normally be classified as being at risk. Four of these cases were sufficiently unusual to warrant individual publication but all came from a very restricted geographical area with a population of only approximately 500,000. The significance of this is not that variant haemoglobins affect HbA1c analysis but that the occurrence of unexpected/unusual variant haemoglobins may be more frequent than most clinicians would expect, considering that only a small proportion of the basal population are diabetic and have their HbA1c monitored. Differences will exist between different areas due to differences in both the prevalence of variants and the analytical methods employed. CONCLUSIONS: Consequently, we propose greater interaction between diabetologists and the laboratory in an attempt to identify these clinically but not biochemically silent variants to achieve a true estimation of the glycaemic control in affected patients.

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

Mass spectral analysis of asymmetric hemoglobin hybrids: demonstration of Hb FS (alpha2gammabetaS) in sickle cell disease.

Formation of the asymmetric hemoglobin hybrid FS (alpha2gammabetaS) inhibits hemoglobin S (Hb S) polymerization in vitro and underlies the protective effect of fetal hemoglobin (Hb F) in homozygous sickle cell disease. Conventional methods for separating Hb reveal only symmetric Hb tetramers because of the rapid dissociation of tetramers to dimers relative to the separation time for electrophoresis and chromatography. To gain insight into the quantitative distribution of asymmetric Hb FS and other tetrameric species in sickle cell disease, the noncovalent association of Hb subunits in hemolysates was studied by a novel application of electrospray ionization mass spectrometry (ESI-MS). Mass spectra of both patient and fetal blood revealed predominance of tetrameric species with dimer and monomer subunits in lower abundance. ESI-MS analysis revealed the hybrid Hb AF (alpha2gammabetaA) in hemolysates shown by conventional high-performance liquid chromatography to contain only the symmetric species Hb A (alpha2betaA2) and Hb F (alpha2gamma2). A unique tetramer of average mass 64,558 Da was identified in hemolysates from patients with sickle cell disease in accordance with the calculated mass of the asymmetric Hb hybrid FS. Hybrid Hb species were stable under the ESI-MS conditions employed allowing concurrent determination of the proportions of Hb FS and the symmetrical Hb S (alpha2betaS2). The ratios of Hb FS to Hb S correlated closely (r2 = 0.96) with those predicted under physiological conditions.

Rapid identification of hemoglobin variants by electrospray ionization mass spectrometry.

The precise identification of human hemoglobin variants, over 700 human hemoglobin variants are known, is essential for prediction of their clinical and genetic significance. A systematic approach to their rapid identification is described. Traditionally this requires protein or DNA characterization which entails lengthy analytical procedures. To overcome these obstacles a rapid approach to variant hemoglobin identification has been developed using conventional phenotypic methods combined with electrospray ionization-mass spectrometry (ESI-MS). The latter requires only a small amount of whole blood (10 microl) but in most cases 2 microl would have been sufficient and no preanalytical steps, such as separation of red cells or globin chains, are necessary. Aged, hemolyzed blood samples can also be analyzed. This approach has been used to positively identify 95% of the variants in over 250 samples. The remaining 5% in which a variant was detected by phenotypic techniques were not resolved by mass spectrometry. Ninety-nine different abnormalities comprising 36 alpha-chain variants, 59 beta-chain variants (including 2 extensions), and 4 hybrid hemoglobins were identified. These include 15 novel variants. The application of ESI-MS described requires approximately 1 h to prepare and analyze each sample and has minimal reagent costs. The turnaround time on a single sample can be as little as 2 h. This technique can now be considered a useful additional tool for reference laboratories.

Observation of large, non-covalent globin subassemblies in the approximately 3600 kDa hexagonal bilayer hemoglobins by electrospray ionization time-of-flight mass spectrometry.

A non-covalent globin subassembly comprising 12 globin chains (204 to 214 kDa) was observed directly by electrospray ionization time-of-flight mass spectrometry in the native hexagonal bilayer hemoglobins from the oligochaetes Lumbricus terrestris and Tubifex tubifex, the polychaetes Tylorrhynchus heterochaetus, Arenicola marina, Amphitrite ornata and Alvinella pompejana, the leeches Macrobdella decora, Haemopis grandis and Nephelopsis oscura and the chlorocruorin from the polychaete Myxicola infundibulum, over the pH range 3.5-7.0. The Hb from the deep-sea polychaete Alvinella exhibited in addition, peaks at approximately 107 kDa and at approximately 285 kDa, which were assigned to subassemblies of six globin chains and of 12 globin chains with three non-globin linker chains, respectively. The experimental masses decreased slightly with increased de-clustering potential (60 to 160 V) and were generally 0.1 to 0.2 % higher than the calculated masses, due probably to complexation with cations and water molecules.

Chiropractic hospitals in America: A case study of the Bakkum Hospital (1936-1950).

OBJECTIVE: The purpose of this article is to relate the story of the Bakkum Chiropractic Clinic and Hospital and offer evidence regarding the lost days of chiropractic hospitalization and inpatient care. DISCUSSION: The number of chiropractic facilities offering inpatient care peaked in the period between World Wars I and II. Little information is available about the vast majority of these facilities. One of them was the Bakkum Chiropractic Clinic and Hospital in Waukon, Iowa. The proprietor was Roy C. Bakkum, DC; he and his wife, Jessie H. Bakkum, DC, opened their 30-bed facility in 1936. Patients received hole-in-one chiropractic adjustments, rest, and nursing care at this facility. Dr Roy Bakkum not only envisioned chiropractors as true primary caregivers but also implemented that vision to the best of his ability. He saw chiropractic as the principal type of health care that the vast majority of people should receive, even as inpatients; instead of being the usual and customary type of hospital care, pharmaceutical and surgical intervention would be called on only as needed. Mainly because of economic pressures, the Bakkums' facility closed in 1950. CONCLUSION: The Bakkum Chiropractic Clinic and Hospital may have been fairly typical of small chiropractic facilities offering inpatient care. Like many private hospitals of various kinds, the Bakkums' facility closed in the post-World War II period, primarily because of economic and political losses. This article traces the history of one chiropractic facility to add historical context to chiropractors' long struggle to own and operate chiropractic hospitals.

Isometric back extension endurance tests: a review of the literature.

OBJECTIVE: To review the literature that describes and evaluates the use of isometric back extension endurance tests. DATA COLLECTION: Relevant articles in English were retrieved through a search of MEDLINE and the Index to Chiropractic Literature. Key search terms were back muscle endurance, isometric back endurance, trunk extensors, back muscle performance, and Sorensen test. DATA SYNTHESIS: The principal criterion for inclusion was as follows: any study that discussed or tested an isometric type of back endurance extension test. Studies that were excluded did not use an isometric testing protocol. Thirty-seven of the initial studies are included in this review. RESULTS: Six different types of isometric back extension endurance testing methods were found. Three of these procedures require special testing devices. Much of the research on this topic has centered on a procedure known as the Sorensen test. Normative databases have been established for the Sorensen test and 2 other test types. Validity and reliability have been assessed for some of the procedures. CONCLUSIONS: The influence of motivation and effort exerted by the subject are limiting factors in all of the tests reviewed. These psychologic factors warrant further research. On the basis of the literature reviewed, we determined that the Sorensen is probably the most clinically useful of these tests; it is easy to perform, requires no special equipment, and enjoys the most support from the literature.

Long-term evaluation of electrospray ionization mass spectrometric analysis of glycated hemoglobin.

BACKGROUND: Electrospray ionization mass spectrometry (ESIMS) has been successfully applied to the identification of hemoglobin (Hb) variants and the presence of glucose adducts (mass difference of 162 Da) on the separate Hb alpha and beta chains. To establish the potential of ESIMS as a routine and/or a reference method for the quantification of glycohemoglobin (HbA1c), we carried out a detailed evaluation over a 4-month period in a routine laboratory environment. METHODS: We optimized a procedure using ESIMS suitable for the routine quantitative analysis of HbA1c. We determined reliability and reproducibility over 4 months and assessed the potential for automated sample injection. We then compared values of 1022 blood samples from diabetic patients with a routine HPLC-based ion-exchange procedure (HA-8140; Menarini). RESULTS: Results of HbA1c measurement by ESIMS were available within 3 min. The analytical imprecision (CV) was 1.6-5.0% for both manual and automated injections. Data collection over the m/z 980-1400 range confirmed lower glycation of the alpha chain relative to the beta chain (0.66:1). Only one glycation was observed per globin chain. The overall glycohemoglobin (i.e., the average of alpha- and beta-chain glycations) measured by ESIMS (x) on 1022 blood samples was lower than by HPLC (y): y = 1.0432x + 0.4815. However, the ss-chain glycation measured by ESIMS was up to 20% higher than the value measured by ion-exchange HPLC and showed a close conformity, particularly at 5-10% HbA1c, with the ion-exchange Diabetes Control and Complications Trial (DCCT)-corrected and the United Kingdom National External Quality Assessment Scheme DCCT mean return values. CONCLUSIONS: ESIMS provides a precise measurement of HbA1c and, in particular, glycation of the beta chain. The method is robust and could be proposed as a procedure to substantiate HbA1c measurement and/or calibration.

Polypeptide chain composition diversity of hexagonal-bilayer haemoglobins within a single family of annelids, the alvinellidae.

Following previous analysis of the structure of Alvinella pompejana heaxagonal-bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep-sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi-angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 +/- 14 kDa (A. caudata), 3822 +/- 28 kDa (P. grasslei) and 3750 +/- 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI-MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull. 179, 366-373.

Gas transfer system in Alvinella pompejana (Annelida polychaeta, Terebellida): functional properties of intracellular and extracellular hemoglobins.

Alvinella pompejana is a tubicolous polychaete that dwells in the hottest part of the hydrothermal vent ecosystem in a highly variable mixture of vent (350 degrees C, anoxic, CO(2)- and sulfide-rich) and deep-sea (2 degrees C, mildly hypoxic) waters. This species has developed distinct-and specifically respiratory-adaptations to this challenging environment. An internal gas exchange system has recently been described, along with the report of an intracellular coelomic hemoglobin, in addition to the previously known extracellular vascular hemoglobin. This article reports the structure of coelomic hemoglobin and the functional properties of both hemoglobins in order to assess possible oxygen transfer. Coelomocytes contain a unique monomeric hemoglobin with a molecular weight of 14,810+/-1.5 Da, as determined by mass spectrometry. The functional properties of both hemoglobins are unexpectedly very similar under the same conditions of pH (6.1-8.2) and temperature (10 degrees -40 degrees C). The oxygen affinity of both proteins is relatively high (P50=0.66 Torr at 20 degrees C and pH 7), which facilitates oxygen uptake from the hypoxic environment. A strong Bohr effect (Phi ranging from -0.8 to -1.0) allows the release of oxygen to acidic tissues. Such similar properties imply a possible bidirectional transfer of oxygen between the two hemoglobins in the perioesophagal pouch, a mechanism that could moderate environmental variations of oxygen concentration and maintain brain oxygenation.

Electrospray ionization mass spectrometric determination of the molecular mass of the approximately 200-kDa globin dodecamer subassemblies in hexagonal bilayer hemoglobins.

Hexagonal bilayer hemoglobins (Hbs) are approximately 3.6-MDa complexes of approximately 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a approximately 2:1 mass ratio found in annelids and related species. Studies of the dissociation and reassembly of Lumbricus terrestris Hb have provided ample evidence for the presence of a approximately 200-kDa linker-free subassembly consisting of monomer (M) and disulfide-bonded trimer (T) subunits. Electrospray ionization mass spectrometry (ESI-MS) of the subassemblies obtained by gel filtration of partially dissociated L. terrestris and Arenicola marina Hbs showed the presence of noncovalent complexes of M and T subunits with masses in the 213. 3-215.4 and 204.6-205.6 kDa ranges, respectively. The observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from approximately 215,400 Da at 60 V to approximately 213,300 Da at 200 V. In contrast, the mass of the A. marina complex decreased linearly from 60 to 120 V and reached an asymptote at approximately 204,600 Da (180-200 V). The decrease in mass was probably due to the progressive removal of complexed water and alkali metal cations. ESI-MS at an acidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M(3)T(3). Because there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M(3)T(3) subassemblies are not unique. A random assembly model was used to calculate the mass distributions of the subassemblies, using the known ESI-MS masses and relative intensities of the M and T subunit isforms. The expected mass of randomly assembled subassemblies was 213,436 Da for Lumbricus Hb and 204,342 Da for Arenicola Hb, in good agreement with the experimental values.

Comparison between collision induced dissociation of electrosprayed protonated peptides in the up-front source region and in a low-energy collision cell.

A systematic comparison between the up-front Collision induced dissociation (CID) mass spectra and low-energy CID tandem mass spectra from twenty-one singly and/or doubly charged peptides has been made. CID spectra of the peptides were recorded at different electrode voltages in the up-front source region of a single quadrupole instrument and different collision energies in the collision cell of a tandem quadrupole instrument. It was observed that up-front CID and low-energy CID yielded comparable product ion spectra from protonated peptides, and that the instrumental settings necessary for obtaining comparable CID mass spectra from the two methods are correlated.

Effects of altering cycling technique on gluteus medius syndrome.

OBJECTIVE: We discuss how altering the cycling technique of a cyclist receiving periodic chiropractic care helped in the management of gluteus medius syndrome. CLINICAL FEATURES: A 24-year-old male amateur cyclist had numbness and tingling localized to a small region on the superior portion of the right buttock. The area involved demonstrated paresthesia to light touch sensory evaluation. The cyclist had received chiropractic adjustments 2 days before the onset of the symptoms. One week earlier, the patient began riding a new bicycle with different gearing than his previous one. Manual-resisted muscle testing created soreness in the lumbosacral area and buttocks. Trigger points were identified in the right gluteus medius. Standing lumbar spine flexion was 70 degrees, limited by tight hamstrings. INTERVENTION AND OUTCOME: Because the patient was already receiving periodic chiropractic care, no passive therapy was used. Patient education regarding the difference in gear selection in bicycles of a higher quality was provided. He was instructed to train in lower gears than he had previously used and to maintain a cadence of 70 to 90 revolutions of the pedals per minute. After 2 days, the paresthesia on the right buttock resolved. The trigger points were only mildly tender with minimal residual soreness of the involved muscles. CONCLUSION: Management of gluteus medius syndrome by altering the cadence and gear development for a bicyclist is discussed. Either frank or cumulative injury to the gluteus medius muscle is the typical etiologic factor for this syndrome. Repetitive strain of the patient's gluteus medius muscle as a result of poor cycling technique appeared to be the cause here. Knowledge of bicycle fitting, training techniques, and bicycle mechanics appeared necessary to resolve the problem.