scholarly journals Markedly Increased Vitamin B12 Concentrations Attributable to IgG–IgM–Vitamin B12 Immune Complexes

2006 ◽  
Vol 52 (11) ◽  
pp. 2107-2114 ◽  
Author(s):  
Raffick AR Bowen ◽  
Steven K Drake ◽  
Rachna Vanjani ◽  
Edward D Huey ◽  
Jordan Grafman ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Size Exclusion Chromatography ◽  
Immune Complexes ◽  
Size Exclusion ◽  
Protein G ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Tof Ms

Abstract Background: High serum vitamin B12 concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B12 concentration in a patient without these underlying conditions. Methods: Affinity and size-exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B12 concentrations in this patient’s serum. Results: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B12 treatment for anemia had vitamin B12 concentrations of <74 pmol/L, whereas the vitamin B12 concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins in the patient’s serum revealed an abnormal vitamin-B12-binding protein. SDS–PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient’s eluates was associated with the IgG to form IgG-IgM immune complexes. Conclusions: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B12 concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B12 and illustrates techniques that can be used to identify this occurrence.

Significantly Elevated Vitamin B12 Levels in Autoimmune Lymphoproliferative Syndrome (ALPS), a Rare Lymphoproliferative Disorder with Apoptosis Defect

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4898-4898 ◽  
Author(s):  
Raffick AR Bowen ◽  
Janet K Dale ◽  
Margaret Brown ◽  
Steven K Drake ◽  
Tarsilla Moura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Vitamin B12 ◽  
Size Exclusion Chromatography ◽  
Serum Protein ◽  
Binding Proteins ◽  
Reference Interval ◽  
Size Exclusion ◽  
Sds Page ◽  
Exclusion Chromatography

Abstract Vitamin B12 is an essential micronutrient that plays a fundamental role in cell division and one-carbon metabolism. Vitamin B12 also plays an important role in hematopoiesis and maintaining the integrity of the nervous system. Vitamin B12 in serum is bound to two proteins, haptocorrin (HC) and transcobalamin (TC). HC is a glycosylated serum protein of ~68kDa that is produced mainly by myeloid cells and binds the majority of circulating vitamin B12 (70–90%). The exact function of this HC-bound vitamin B12 is unknown (holo-HC), but it is believed to be biologically unavailable to most cells. The remainder of vitamin B12 is bound to TC (holo-TC). TC is a 43 kDa non-glycosylated serum protein that is synthesized primarily in the enterocytes and carries vitamin B12 to the tissues via a specific receptor with a high affinity for holo-TC. Only vitamin B12 bound to TC is available for cellular uptake and use as coenzymes for L-methylmalonyl-CoA mutase and methionine synthase that synthesize succinyl-CoA and methionine, respectively. ALPS is a rare autoimmune disease in children resulting from defective lymphocyte apoptosis and is characterized by lymphoproliferation, peripheral accumulation of DNT cells (double-negative T cells; TCR alpha/beta +, CD3+ CD4− CD8−), often leading to autoimmune multilineage cytopenias. Type Ia ALPS with Fas mutations accounting for ~75% of all reported cases is the most prevalent, High serum vitamin B12 concentrations have been observed in patients with ALPS studied at NIH Clinical Center. Levels of Vitamin B12 detected in these patients range from 742 to 35365 pmol/L (median: 2489 pmol/L; mean: 4123 pmol/L; reference interval: 162–708 pmol/L) among 137 patients compiled for this study (Vitamin B12: 1pmol/L=1.355 pg/mL). To date, the mechanism of the elevated vitamin B12 concentrations in ALPS patients has not been determined and we sought to explore the source of elevated vitamin B12. Radio- and enzyme immunoassays, size-exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and flow cytometry techniques were used to identify and quantitate the source of the elevated vitamin B12 concentrations in peripheral blood specimens from ALPS patients and controls. Total and holo-HC levels were 36- and 32-fold higher than the upper limit of the reference interval (Total HC: 250–840 pmol/L; Holo-HC: 240–680 pmol/L), respectively, in ALPS patients. However, total and holo-TC levels (1210 pmol/L; reference interval: 500–1500 pmol/L and 143 pmol/L; reference interval: >40 pmol/L, respectively) were not elevated in ALPS patients. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins did not reveal any abnormal vitamin B12-binding proteins in ALPS. SDS-PAGE and western blot analysis revealed the presence of HC in lymphocyte lysates of ALPS but not apparently healthy controls. Flow cytometry studies also showed elevated levels of HC in all white blood cell types from ALPS patients compared to controls. The results of this study show for the first time that elevated concentrations of vitamin B12 found in ALPS patients were due to increased leukocyte (including lymphocytes) expression of HC. Further studies are needed to elucidate the mechanism for the increased synthesis and secretion of HC from lymphocytes of ALPS patients, but altered cytokine profiles, including increased interleukin-10 levels in ALPS may contribute.

SEC/MALDI-TOF Mass spectrometry study of hydroxy-, carboxy- and methyl ester-terminated aliphatic polyesters

e-Polymers ◽  
2005 ◽  
Vol 5 (1) ◽  
Author(s):  
Slim Salhi ◽  
Martine Tessier ◽  
Jean-Claude Blais ◽  
Rachid El Gharbi ◽  
Alain Fradet
Keyword(s):  
Dimethyl Ester ◽  
Calibration Method ◽  
Size Exclusion ◽  
Aliphatic Polyesters ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Calibration Curves ◽  
End Groups ◽  
Poly Ethylene ◽  
Tof Ms

AbstractPoly(ethylene adipate) (PEA) and poly(ethylene dodecanedioate) (PED) with dicarboxy-, dihydroxy-, dimethyl ester- and hydroxy-carboxy end-groups were synthesized and characterized by 13C NMR and MALDI-TOF MS. Size-exclusion chromatography (SEC) fractions of these polyesters were analyzed by MALDI-TOF MS and used as standards to obtain SEC calibration curves leading to the absolute molar masses of the unfractionated polyesters. PEA and PED calibration curves were different from each other, but the same calibration curve was obtained for each type of polyester, irrespective of the nature of end-groups, showing the predominant role of the nature of repeating units on the hydrodynamic volume of oligoesters. The study also shows that the conventional SEC calibration method using polystyrene standards leads to a significant overestimation of polyester average molar masses.

Electroelution of Intact Proteins from SDS-PAGE Gels and Their Subsequent MALDI-TOF MS Analysis

2006 ◽  
pp. 353-364 ◽  
Author(s):  
Zhentian Lei ◽  
Ajith Anand ◽  
Kirankumar S. Mysore ◽  
Lloyd W. Sumner
Keyword(s):  
Maldi Tof Ms ◽  
Intact Proteins ◽  
Maldi Tof ◽  
Ms Analysis ◽  
Sds Page ◽  
Tof Ms

scholarly journals 2155. Accelerated Confirmation of Porin Loss in Carbapenem-Resistant Enterobacterales: A MALDI-TOF Mass Spectrometry-Based Approach

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S731-S731
Author(s):  
Carlos Correa-Martinez ◽  
Evgeny A Idelevich ◽  
Karsten Becker
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Gold Standard ◽  
Sodium Dodecylsulfate ◽  
Resistant Bacteria ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Carbapenem Resistant ◽  
Sds Page ◽  
Tof Ms

Abstract Background The accurate identification of carbapenem resistance mechanisms is decisive for the appropriate selection of antibiotic regimens. Numerous methods can detect carbapenemase-producing carbapenem-resistant bacteria (CPCR). However, non-CPCR (NCPCR) are routinely assumed to display porin loss as a diagnosis of exclusion. No further confirmatory tests are performed since the gold standard (sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS–PAGE) is laborious and time consuming. We propose a test for rapid and easy detection of porin loss by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Clinical meropenem-resistant Enterobacterales strains (10 CPCR, 10 NCPCR) and control strains recommended by EUCAST (5 carbapenemase-producing, one with porin loss, one-negative control) were analyzed. Membrane proteins were extracted by successive centrifugation of bacterial suspensions (McFarland 0.5) and addition of ethanol, formic acid and acetonitrile. MALDI-TOF MS of the protein extracts was performed on a 96-spot target (Bruker Daltonics, Germany). Peaks between 35 and 40 kDa were analyzed for the presence of porins and compared with the bands observed in the SDS–PAGE of the protein extracts. Results Within the molecular weight range of 35–40 kDa, the MALDI-TOF MS-based method revealed peaks in all CPCR isolates corresponding to those observed in the carbapenemase-producing control strains. In contrast, the control strain with porin loss as well as all CNCR isolates showed a lower quantity of peaks in this range. All peaks observed correlated with the bands observed in the SDS–PAGE of the protein extracts at the corresponding molecular weight (Figure 1). Conclusion Yielding results that reliably correspond to the current gold standard, we propose a method for accelerated detection of porin loss as an alternative to the diagnosis of exclusion usually made in routine settings. With a processing time of approximately 20 minutes, the method can be easily implemented in the clinical setting. Applying this MALDI-TOF MS-based approach, valuable information will be provided about a resistance mechanism that otherwise remains unexplained. Disclosures All authors: No reported disclosures.

scholarly journals A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

2016 ◽  
Author(s):  
Rahul Mahadev Shelake ◽  
Yuki Ito ◽  
Junya Masumoto ◽  
Eugene Hayato Morita ◽  
Hidenori Hayashi
Keyword(s):  
Helicobacter Pylori ◽  
Metal Binding ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Metal Species ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Gel Shift ◽  
Tof Ms

AbstractSodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed “gel shifting” appears to be common for histidine-rich proteins but not yet studied in detail. We investigated “gel shifting” in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining “gel shifting” of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed “metal gel-shift” demonstrating an intimate link between Ni2+ binding and “gel shifting”. To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and “metal-gel shift” models are discussed.

scholarly journals Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

2011 ◽  
Vol 46 (11) ◽  
pp. 2144-2151 ◽  
Author(s):  
Francisco Adriano O. Carvalho ◽  
José Wilson P. Carvalho ◽  
Patrícia S. Santiago ◽  
Marcel Tabak
Keyword(s):  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Tof Ms ◽  
Page Electrophoresis

Synthesis, characterization and ring-closing depolymerization of furanic polyesters

e-Polymers ◽  
2005 ◽  
Vol 5 (1) ◽  
Author(s):  
Wided Kamoun ◽  
Slim Salhi ◽  
Souhir Abid ◽  
Martine Tessier ◽  
Rachid El Gharbi ◽  
...  
Keyword(s):  
High Temperature ◽  
Size Exclusion Chromatography ◽  
Side Reaction ◽  
Size Exclusion ◽  
Chain Flexibility ◽  
Furoic Acid ◽  
End Groups ◽  
Exclusion Chromatography ◽  
High Yields ◽  
Tof Ms

AbstractFuranic polyesters were prepared by the bulk polycondensation of 5,5'- isopropylidene-bis(2-furoic acid) with 1,2-ethanediol, 1,4-propanediol and 1,6- hexanediol, and were characterized by size exclusion chromatography, NMR and MALDI-TOF MS. The ring-closing depolymerization of these polyesters was then investigated at 10 g/L concentration in 1-methylnaphthalene at 130 - 240°C. Chain flexibility, the use of zinc diacetate catalyst and, particularly, the presence of hydroxy end-groups were found to be favourable factors for the formation of high yields of cyclic oligoesters. A side reaction leading to the formation of non-reactive furyl-terminated species took place when the reactions were carried out at high temperature (240°C), limiting the yield of cyclic oligoesters.

Identification of LAB and Fungi in Laru, a Fermentation Starter, by PCR-DGGE, SDS-PAGE, and MALDI-TOF MS

2018 ◽  
Vol 28 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Lenny S. F. Ahmadsah ◽  
Eiseul Kim ◽  
Youn-Sik Jung ◽  
Hae-Yeong Kim
Keyword(s):  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Fermentation Starter ◽  
Tof Ms
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