ITPase Activity in Dry Blood Spots is Comparable with That in Fresh Erythrocytes

Tomková, Jana; Friedecký, David; Vyskočilová, Petra; Adam, Tomáš

doi:10.1080/15257770802143897

Nucleosides, Nucleotides and Nucleic Acids

2008

DOI: 10.1080/15257770802143897

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ITPase Activity in Dry Blood Spots is Comparable with That in Fresh Erythrocytes

Jana Tomková

David Friedecký

Petra Vyskočilová

et al.

Abstract: Inosine triphosphate pyrophosphohydrolase (ITPase) catalyzing the pyrophosphohydrolysis of inosine triphosphate, deoxyinosine triphosphate and xanthosine triphosphate is involved in the metabolism and tolerance of thiopurine drugs. ITPase activity plays an important role in the prediction of toxicity to thiopurine therapy. Activities in dry blood spots were compared with fresh erythrocytes. Samples were incubated with inosine triphosphate, then inosine monophosphate was determined by a capillary electrophoresi… Show more

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“…Most studies on ITPase activity and function used erythrocyte lysates (11)(12)(13). However, Sumi et al showed in a control population that ITPase activity in leukocytes is higher when compared to erythrocytes (14).…”

mentioning

confidence: 99%

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

Vroemen¹,

Munnix²,

Bakker³

et al. 2012

Cytometry Pt A

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The aim of this study was to assess inosine triphosphate (ITPase) expression in the different leukocyte populations present in peripheral blood samples of a nonimmune compromised control group. For this purpose, a multiparameter flow cytometric assay was developed and performed to study ITPase expression in peripheral leukocyte subpopulations of healthy volunteers (n 5 20). Qualitative ITPase expression was assessed by determining the percentage of ITPase-positive cells. Quantitative data were obtained by measuring the median fluorescent intensity (MFI). Subcellular localization of ITPase was analyzed using immunocytochemistry. Immunocytochemistry showed that ITPase is present in all leukocytes and localized intracellular. Based on this finding, a multiparameter flow cytometric assay was developed using a Fix & Perm strategy. Qualitative and quantitative ITPase expression remained stable (variation, \10%) for at least 48 h after blood sampling. MFI values showed that activated monocytes contained significantly more ITPase when compared to the total monocyte fraction (P \ 0.0001), which subsequently had a higher amount of expression than granulocytes (P \ 0.0001). In addition, the phagocyte subpopulations ([activated] monocytes and granulocytes) contained significantly higher levels of ITPase when compared to lymphocytes (P \ 0.0001). Within the lymphocyte fraction, it appeared that T-helper cells contained significantly higher ITPase levels when compared to cytotoxic T cells, B lymphocytes, and natural killer cells (P \ 0.0001). Our study is the first which describes a flow cytometry assay to analyze ITPase expression in leukocytes qualitatively as well as quantitatively and visualizes the intracellular localization of ITPase in leukocytes. ' International Society for Advancement of Cytometry Key termsITPase; flow cytometry; leukocyte subpopulations; thiopurines INOSINE triphosphatase (ITPase) is a ubiquitous enzyme, whose role in mammalian metabolism is still poorly understood (1). In humans, the gene encoding this enzyme is ITPA, a polymorphic gene located on chromosome 20p13. The primary role of ITPase is pyrophosphohydrolysis of (deoxy)inosine-5 0 -triphosphate ((d)ITP) to maintain the balance between (d)ITP and (deoxy)inosine-5 0 -monophosphate ((d)IMP) (2,3). Another, less highlighted function is the role of ITPA as a housekeeping gene. ITPase is involved in the maintenance of the degradation of noncanonical purine (deoxy) nucleotide triphosphates, thereby preventing the incorporation of these metabolites into RNA and DNA. Under abnormal physiological circumstances, such as metabolic stress or an inflammatory response, proper functioning of housekeeping genes is pivotal for cellular maintenance (4).The existence of nucleotide pyrophosphohydrolases was first described by Liakopoulou and Alivisatos who measured nucleotide pyrophosphohydrolase activity in erythrocytes (5). Later on, the presence of ITPase activity was also demonstrated in the different peripheral blood cells (granulocytes, lymphocyt...

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mentioning

confidence: 99%

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

Vroemen¹,

Munnix²,

Bakker³

et al. 2012

Cytometry Pt A

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show abstract

A comparative study of inosine triphosphatase activity in fresh erythrocytes and dried blood spots

Bakker

Lindhout

Dorland

et al. 2009

Clinica Chimica Acta

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ITPase Activity in Dry Blood Spots is Comparable with That in Fresh Erythrocytes

Cited by 2 publications

References 3 publications

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

A comparative study of inosine triphosphatase activity in fresh erythrocytes and dried blood spots

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