A liquid chromatography-tandem mass spectrometry based method for the quantification of adenosine nucleotides and NAD precursors and products in various biological samples

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions

Krüppel-like zinc finger proteins in end-stage COPD lungs with and without severe alpha1-antitrypsin deficiency

ABSTRACT: BACKGROUND: Chronic obstructive pulmonary disease (COPD) is influenced by environmental and genetic factors. An important fraction of COPD cases harbor a major genetic determinant, inherited ZZ (Glu342Lys) alpha1-antitrypsin deficiency (AATD). A study was undertaken to investigate gene expression patterns in end-stage COPD lungs from patients with and without AATD. METHODS: Explanted lungs of end-stage ZZ AATD-related (treated and non-treated with AAT augmentation therapy) and "normal" MM COPD, and liver biopsies from patients suffering from liver cirrhosis with and without ZZ AATD were used for gene expression analysis by Affymetrix microarrays or RT-PCR. RESULTS: A total of 162 genes were found to be differentially expressed (p-value [less than or equal to] 0.05 and |FC| [greater than or equal to] 2) between MM and ZZ COPD patients. Of those, 134 gene sets were up-regulated and 28 were down-regulated in ZZ relative to MM lung tissue. A subgroup of genes, zinc finger protein 165, snail homolog 1 (Drosophila) (SNAI1), and Kruppel-like transcription factors (KLFs) 4 (gut), 9 and 10, perfectly segregated ZZ and MM COPD patients. The higher expression of KLF 9 and KLF10 has been verified in the replication cohort with AATD-related end-stage lung emphysema and liver cirrhosis. Furthermore, higher expression of KLF9, SNAI1 and DEFA1 was found in ZZ COPD lungs without augmentation therapy relative to MM COPD or ZZ COPD with augmentation therapy. CONCLUSIONS: These results reveal the involvement of transcriptional regulators of the zinc-finger family in COPD pathogenesis and provide deeper insight into the pathophysiological mechanisms of COPD with and without AATD

DataSheet_1_A liquid chromatography-tandem mass spectrometry based method for the quantification of adenosine nucleotides and NAD precursors and products in various biological samples.docx

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions.</p

Krüppel-like zinc finger proteins in end-stage COPD lungs with and without severe alpha1-antitrypsin deficiency

Abstract Background Chronic obstructive pulmonary disease (COPD) is influenced by environmental and genetic factors. An important fraction of COPD cases harbor a major genetic determinant, inherited ZZ (Glu342Lys) α1-antitrypsin deficiency (AATD). A study was undertaken to investigate gene expression patterns in end-stage COPD lungs from patients with and without AATD. Methods Explanted lungs of end-stage ZZ AATD-related (treated and non-treated with AAT augmentation therapy) and “normal” MM COPD, and liver biopsies from patients suffering from liver cirrhosis with and without ZZ AATD were used for gene expression analysis by Affymetrix microarrays or RT-PCR. Results A total of 162 genes were found to be differentially expressed (p-value ≤ 0.05 and |FC| ≥ 2) between MM and ZZ COPD patients. Of those, 134 gene sets were up-regulated and 28 were down-regulated in ZZ relative to MM lung tissue. A subgroup of genes, zinc finger protein 165, snail homolog 1 (Drosophila) (SNAI1), and Krüppel-like transcription factors (KLFs) 4 (gut), 9 and 10, perfectly segregated ZZ and MM COPD patients. The higher expression of KLF 9 and KLF10 has been verified in the replication cohort with AATD-related end-stage lung emphysema and liver cirrhosis. Furthermore, higher expression of KLF9, SNAI1 and DEFA1 was found in ZZ COPD lungs without augmentation therapy relative to MM COPD or ZZ COPD with augmentation therapy. Conclusions These results reveal the involvement of transcriptional regulators of the zinc-finger family in COPD pathogenesis and provide deeper insight into the pathophysiological mechanisms of COPD with and without AATD.</p