Advanced search
1 file | 2.95 MB Add to list

Development and evaluation of novel analytical methods for high-precision isotopic analysis of Ca and Mg via MC-ICP-MS for biomedical investigations

Rosa Grigoryan (UGent)
(2021)
Author
Promoter
(UGent) and (UGent)
Organization
Abstract
Natural variations in the isotopic composition have been observed for all elements with two or more isotopes as a result of mass-dependent isotope fractionation accompanying physical processes and/or (bio)chemical reactions. To detect and quantify such small variations in isotopic composition thus caused, high-precision isotope ratio measurements are required. Multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) is nowadays the preferred technique for this purpose due to the highly efficient ion source operated at atmospheric pressure, the excellent precision owing to the simultaneous monitoring of the isotopes of interest, an enhanced sample throughput and the straightforward sample introduction. Isotope ratio measurements of ‘heavy elements’ via MC-ICP-MS have been initially focused on geological, cosmological, archeological and environmental applications, but during the last years the isotopic analysis of essential mineral elements, such as Ca, Cu, Fe, S and Zn, have gained interest in the context of biomedical applications, and isotope ratios are explored as potential diagnostic and/or prognostic markers. The first part of this PhD research was focused on the development and validation of reliable analytical methods for isotopic analysis of Mg and Ca via MC-ICP-MS for biomedical applications. The second part explores the variation in the serum Mg isotopic composition of diabetic patients and after lipopolysaccharide-induced infection in mice. An exhaustive characterization of the new isotopic reference material ERM-AE143 was carried out to ensure traceability of the Mg isotope ratios determined in this work. Finally, a new analytical method for the ultra-sensitive Mg isotopic analysis of cerebrospinal fluid (CSF) micro-samples as small as a couple of microliters was developed using new high-gain 1013 Ω Faraday cup amplifiers and validated for further research on neurodegenerative diseases. A brief introduction on (i) Mg and (ii) Ca metabolism and related disorders is presented in Chapter 1. This chapter also contains (iii) a brief description of the basic operating principles of ICP-MS and MC-ICP-MS. Additionally, (iv) an overview of the existing methods for Mg and Ca isotopic analysis is provided. The last section of the chapter focuses on (v) the estimation of measurement uncertainty accompanying the isotope ratio results. Magnesium is considered a natural Ca antagonist, thus the isotopic analysis of both elements might be helpful to understand metal homeostasis, the interactions between these cations and/or alterations in their metabolism under disease conditions. Chapter 2 describes the development of a method for the sequential chromatographic isolation of Mg and Ca from biofluids and tissues from a single aliquot of sample using 1 mL of AG50W-X8 strong cation exchange resin and of the MC-ICP-MS measurement protocol. Additional purification of the Ca fraction from Sr was carried out with the Sr-specific resin Sr_SPEC. The elution profiles for biological fluids with different matrices, such as cerebrospinal fluid, serum and whole blood, were studied for optimization of the chromatographic isolation of both target elements. The procedures developed were validated using commercially available reference materials and isotopic reference standards. The Mg and Ca isotope ratio measurements by MC-ICP-MS were carried out at pseudo medium mass resolution and using a high-transmission jet interface. Also the potential effect of concomitant matrix elements (Ca, Cu, Fe, Zn), which may give rise to a pronounced matrix effect during the Mg isotope ratio measurement in the MC-ICP-MS was evaluated. Ca isotope ratio data were further validated via comparison with data obtained for the same samples using double-spike thermal ionization mass spectrometry (DS-TIMS). This chapter also provides the first dataset for the body distribution of Mg and Ca isotopes in mice. The use of isotopic reference materials certified for their isotope amount ratios and thus traceable to the International System of Units is crucial in high-precision isotopic analysis. The NIST SRM 980 isotopic reference material is available for Mg isotope ratio measurements, but due to its relatively large heterogeneity at the scale of single chips, DSM3 and Cambridge 1 were proposed as reference materials instead. However, neither of those are primary reference materials, thus they are not traceable to the SI. Recently, three candidate European Reference Materials (ERM)-AE143, -AE144 and -AE145 were characterized and the ERM-AE143 was proposed as primary isotopic reference material in the context of the determination of absolute Mg isotope ratios and delta values. The characterization of the new Mg isotopic reference material ERM-AE143 is presented in Chapter 3. The characterization of ERM-AE143 for the isotopic composition of Mg, was carried out by both a direct measurement approach and the intercept method. In addition, a set of 13 reference materials of geological and biological origin were measured against both the new (ERM-AE143) and the conventional (DSM3) isotopic reference material. Next to the Mg isotope ratios, also the corresponding expanded uncertainties are provided. Magnesium is an essential mineral element in the human body and its deficiency has been associated with diabetes mellitus. As the total serum Mg concentration does not necessarily reflect the individual Mg status, it was hypothesized that the serum Mg isotopic composition could provide valuable information on its status. Therefore, as reported in Chapter 4, Mg isotopic analysis of serum from diabetes type I patients and from healthy individuals was carried out and the resulting data was compared. The serum Mg isotopic composition in the diabetic patients was also determined one year later. On average, the serum Mg isotopic composition for diabetic patients is significantly lighter than that of the reference population. The same trend was observed for the samples taken one year later. However, for some diabetic patients, a substantial difference was observed between the serum Mg isotopic composition for the two sampling events; this observation still needs to be elucidated. Lipopolysaccharide (LPS)-induced infection is a well-established model for inducing endotoxemia, neuroinflammation and sepsis in mice, which leads to injuries in several organs, particularly the liver, kidneys, lungs and brain. Chapter 5 focuses on potential alterations in the Mg isotopic composition and in the concentrations of minor elements, such as Ca, K, Mg, Na, P and S, in a murine model of endotoxemia. The effect of the LPS-induced infection in the body by comparing the results obtained to those for age- and gender-matched controls. Two different age groups were selected for these experiments, in order to evaluate the potential effects of the LPS-induced infection and aging. Blood plasma of LPS-injected mice showed a significantly heavier Mg isotopic composition than that of matched controls. Altered electrolytes concentrations were detected in blood plasma, brain stem, colon content, intestine and urine of the LPS-injected mice. The Mg isotopic composition of the brain regions was heterogeneous, even for the healthy individuals. For further exploration of the potential use Mg isotopic variations in biomedicine, isotopic analysis of clinical micro-samples is required. The analysis of micro-samples, e.g. cerebrospinal fluid, bone marrow and biopsied material, can be challenging due to the invasiveness of the sampling and the often very limited amount of sample available. Chapter 6 describes the development of an analytical method for the ultra-sensitive Mg isotopic analysis of micro-samples, particularly of cerebrospinal fluid, for further research on neurodegenerative diseases. The concentration at which Mg isotope ratio measurements are conducted in MC-ICP-MS using the ‘conventional method’ previously developed (Chapters 2 and 4) is 150 µg L-1. Here, Mg isotope ratio measurements via MC-ICP-MS using high-gain 1013 Ω Faraday cup, amplifiers are presented at a concentration level of 7-10 µg L-1 for 5 µL volumes of human CSF samples only. For validation purpose, commercially available CSF and serum reference materials intended for quality control of elemental analysis were used. The method was finally applied to CSF samples from hydrocephalus patients, for whom larger volumes of CSF were available, as a proof-of-concept study. The Mg isotope ratios obtained at 7-10 µg L-1 level using the 1013 Ω amplifiers were in good agreement with those using the conventional method for standard volume samples at the concentration level of 150 µg L-1 using the 10^11 Ω amplifiers. Chapter 7 presents a general conclusion and describes future perspectives for Mg isotopic analysis in a biomedical context.
Keywords
Ca isotopes, Mg isotopes, Multi-collector ICP-Mass spectrometry, biomedical applications, chromatographic isolation, isotopic reference materials, type I diabetes, lipopolysaccharide, cerebrospinal fluid, 10^13 Ω amplifiers

Downloads

  • PhD Rosa Grigoryan.pdf
    • full text (Published version)
    • |
    • open access
    • |
    • PDF
    • |
    • 2.95 MB

Citation

Please use this url to cite or link to this publication:

MLA
Grigoryan, Rosa. Development and Evaluation of Novel Analytical Methods for High-Precision Isotopic Analysis of Ca and Mg via MC-ICP-MS for Biomedical Investigations. Ghent University. Faculty of Sciences, 2021.
APA
Grigoryan, R. (2021). Development and evaluation of novel analytical methods for high-precision isotopic analysis of Ca and Mg via MC-ICP-MS for biomedical investigations. Ghent University. Faculty of Sciences, Ghent, Belgium.
Chicago author-date
Grigoryan, Rosa. 2021. “Development and Evaluation of Novel Analytical Methods for High-Precision Isotopic Analysis of Ca and Mg via MC-ICP-MS for Biomedical Investigations.” Ghent, Belgium: Ghent University. Faculty of Sciences.
Chicago author-date (all authors)
Grigoryan, Rosa. 2021. “Development and Evaluation of Novel Analytical Methods for High-Precision Isotopic Analysis of Ca and Mg via MC-ICP-MS for Biomedical Investigations.” Ghent, Belgium: Ghent University. Faculty of Sciences.
Vancouver
1.
Grigoryan R. Development and evaluation of novel analytical methods for high-precision isotopic analysis of Ca and Mg via MC-ICP-MS for biomedical investigations. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2021.
IEEE
[1]
R. Grigoryan, “Development and evaluation of novel analytical methods for high-precision isotopic analysis of Ca and Mg via MC-ICP-MS for biomedical investigations,” Ghent University. Faculty of Sciences, Ghent, Belgium, 2021.
@phdthesis{8721310,
  abstract     = {{Natural variations in the isotopic composition have been observed for all elements with two or more isotopes as a result of mass-dependent isotope fractionation accompanying physical processes and/or (bio)chemical reactions. To detect and quantify such small variations in isotopic composition thus caused, high-precision isotope ratio measurements are required. Multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) is nowadays the preferred technique for this purpose due to the highly efficient ion source operated at atmospheric pressure, the excellent precision owing to the simultaneous monitoring of the isotopes of interest, an enhanced sample throughput and the straightforward sample introduction.

Isotope ratio measurements of ‘heavy elements’ via MC-ICP-MS have been initially focused on geological, cosmological, archeological and environmental applications, but during the last years the isotopic analysis of essential mineral elements, such as Ca, Cu, Fe, S and Zn, have gained interest in the context of biomedical applications, and isotope ratios are explored as potential diagnostic and/or prognostic markers. The first part of this PhD research was focused on the development and validation of reliable analytical methods for isotopic analysis of Mg and Ca via MC-ICP-MS for biomedical applications. The second part explores the variation in the serum Mg isotopic composition of diabetic patients and after lipopolysaccharide-induced infection in mice. An exhaustive characterization of the new isotopic reference material ERM-AE143 was carried out to ensure traceability of the Mg isotope ratios determined in this work. Finally, a new analytical method for the ultra-sensitive Mg isotopic analysis of cerebrospinal fluid (CSF) micro-samples as small as a couple of microliters was developed using new high-gain 1013 Ω Faraday cup amplifiers and validated for further research on neurodegenerative diseases.

A brief introduction on (i) Mg and (ii) Ca metabolism and related disorders is presented in Chapter 1. This chapter also contains (iii) a brief description of the basic operating principles of ICP-MS and MC-ICP-MS. Additionally, (iv) an overview of the existing methods for Mg and Ca isotopic analysis is provided.  The last section of the chapter focuses on (v) the estimation of measurement uncertainty accompanying the isotope ratio results. 

Magnesium is considered a natural Ca antagonist, thus the isotopic analysis of both elements might be helpful to understand metal homeostasis, the interactions between these cations and/or alterations in their metabolism under disease conditions. Chapter 2 describes the development of a method for the sequential chromatographic isolation of Mg and Ca from biofluids and tissues from a single aliquot of sample using 1 mL of AG50W-X8 strong cation exchange resin and of the MC-ICP-MS measurement protocol. Additional purification of the Ca fraction from Sr was carried out with the Sr-specific resin Sr_SPEC. The elution profiles for biological fluids with different matrices, such as cerebrospinal fluid, serum and whole blood, were studied for optimization of the chromatographic isolation of both target elements. The procedures developed were validated using commercially available reference materials and isotopic reference standards. The Mg and Ca isotope ratio measurements by MC-ICP-MS were carried out at pseudo medium mass resolution and using a high-transmission jet interface. Also the potential effect of concomitant matrix elements (Ca, Cu, Fe, Zn), which may give rise to a pronounced matrix effect during the Mg isotope ratio measurement in the MC-ICP-MS was evaluated. Ca isotope ratio data were further validated via comparison with data obtained for the same samples using double-spike thermal ionization mass spectrometry (DS-TIMS). This chapter also provides the first dataset for the body distribution of Mg and Ca isotopes in mice.

The use of isotopic reference materials certified for their isotope amount ratios and thus traceable to the International System of Units is crucial in high-precision isotopic analysis. The NIST SRM 980 isotopic reference material is available for Mg isotope ratio measurements, but due to its relatively large heterogeneity at the scale of single chips, DSM3 and Cambridge 1 were proposed as reference materials instead. However, neither of those are primary reference materials, thus they are not traceable to the SI. Recently, three candidate European Reference Materials (ERM)-AE143, -AE144 and -AE145 were characterized and the ERM-AE143 was proposed as primary isotopic reference material in the context of the determination of absolute Mg isotope ratios and delta values. The characterization of the new Mg isotopic reference material ERM-AE143 is presented in Chapter 3. The characterization of ERM-AE143 for the isotopic composition of Mg, was carried out by both a direct measurement approach and the intercept method. In addition, a set of 13 reference materials of geological and biological origin were measured against both the new (ERM-AE143) and the conventional (DSM3) isotopic reference material. Next to the Mg isotope ratios, also the corresponding expanded uncertainties are provided. 

Magnesium is an essential mineral element in the human body and its deficiency has been associated with diabetes mellitus. As the total serum Mg concentration does not necessarily reflect the individual Mg status, it was hypothesized that the serum Mg isotopic composition could provide valuable information on its status. Therefore, as reported in Chapter 4, Mg isotopic analysis of serum from diabetes type I patients and from healthy individuals was carried out and the resulting data was compared. The serum Mg isotopic composition in the diabetic patients was also determined one year later. On average, the serum Mg isotopic composition for diabetic patients is significantly lighter than that of the reference population. The same trend was observed for the samples taken one year later. However, for some diabetic patients, a substantial difference was observed between the serum Mg isotopic composition for the two sampling events; this observation still needs to be elucidated.

Lipopolysaccharide (LPS)-induced infection is a well-established model for inducing endotoxemia, neuroinflammation and sepsis in mice, which leads to injuries in several organs, particularly the liver, kidneys, lungs and brain. Chapter 5 focuses on potential alterations in the Mg isotopic composition and in the concentrations of minor elements, such as Ca, K, Mg, Na, P and S, in a murine model of endotoxemia. The effect of the LPS-induced infection in the body by comparing the results obtained to those for age- and gender-matched controls. Two different age groups were selected for these experiments, in order to evaluate the potential effects of the LPS-induced infection and aging. Blood plasma of LPS-injected mice showed a significantly heavier Mg isotopic composition than that of matched controls. Altered electrolytes concentrations were detected in blood plasma, brain stem, colon content, intestine and urine of the LPS-injected mice. The Mg isotopic composition of the brain regions was heterogeneous, even for the healthy individuals.

For further exploration of the potential use Mg isotopic variations in biomedicine, isotopic analysis of clinical micro-samples is required. The analysis of micro-samples, e.g. cerebrospinal fluid, bone marrow and biopsied material, can be challenging due to the invasiveness of the sampling and the often very limited amount of sample available. Chapter 6 describes the development of an analytical method for the ultra-sensitive Mg isotopic analysis of micro-samples, particularly of cerebrospinal fluid, for further research on neurodegenerative diseases. The concentration at which Mg isotope ratio measurements are conducted in MC-ICP-MS using the ‘conventional method’ previously developed (Chapters 2 and 4) is 150 µg L-1. Here, Mg isotope ratio measurements via MC-ICP-MS using high-gain 1013 Ω Faraday cup, amplifiers are presented at a concentration level of 7-10 µg L-1 for 5 µL volumes of human CSF samples only. For validation purpose, commercially available CSF and serum reference materials intended for quality control of elemental analysis were used. The method was finally applied to CSF samples from hydrocephalus patients, for whom larger volumes of CSF were available, as a proof-of-concept study. The Mg isotope ratios obtained at 7-10 µg L-1 level using the 1013 Ω amplifiers were in good agreement with those using the conventional method for standard volume samples at the concentration level of 150 µg L-1 using the 10^11 Ω amplifiers.

Chapter 7 presents a general conclusion and describes future perspectives for Mg isotopic analysis in a biomedical context.}},
  author       = {{Grigoryan, Rosa}},
  keywords     = {{Ca isotopes,Mg isotopes,Multi-collector ICP-Mass spectrometry,biomedical applications,chromatographic isolation,isotopic reference materials,type I diabetes,lipopolysaccharide,cerebrospinal fluid,10^13 Ω amplifiers}},
  language     = {{eng}},
  pages        = {{XXIV, 182}},
  publisher    = {{Ghent University. Faculty of Sciences}},
  school       = {{Ghent University}},
  title        = {{Development and evaluation of novel analytical methods for high-precision isotopic analysis of Ca and Mg via MC-ICP-MS for biomedical investigations}},
  year         = {{2021}},
}