Oxidative stress: Determination of 4-hydroxy-2-nonenal by gas chromatography/mass spectrometry in human and rat plasma

Abstract

The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography–mass spectrometry (GC–MS) method based on derivatization of HNE with pentafluorobenzylhydroxylamine–HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d₁₁) as internal standard. This excellent stable and precise GC–MS method was carefully validated for HNE, and showed good linearity (r² = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4–6.1% and between-day precisions were 5.2–10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5–250 nmol/L) of HNE.

To examine the versatility of this modified GC–MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats.

Taken together, the GC–MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.     

Comparison of 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract

Background. Measurement of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p <?0.001). According to ROC analysis, the AUC value of HbA1c was higher than the value of the AUC of 8-OHdG (0.882 and 0.771, respectively). Conclusions. This study shows that the urine 8-OHdG levels increase in diabetic patients. However, urinary 8-OHdG is not a useful clinical marker, compared with UACR, to predict the development of diabetic nephropathy in diabetic patients.     

Influence of plant growth regulators on micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br

An efficient protocol for micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br. was developed using shoot tip explants. The physiological role of cytokinin and its combination with auxins on micropropagation and in vitro flowering was investigated. The highest number of shoots (9.94 ± 0.10) and the maximum average shoot length (5.56 ± 0.35 cm) were recorded on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) (4.44 μM) and naphthaleneacetic acid (NAA) (2.69 μM). The effect of sucrose concentration on in vitro floral development was studied in plantlets cultured on MS medium supplemented with gibberellic acid (GA₃) and BAP. The highest percentage of flowering (93.2%) was obtained on MS medium supplemented with GA₃ (1.44 μM), BAP (1.33 μM) and sucrose (30 g l^?1). Root formation from the adventitious shoots was easily achieved on MS medium containing indole-3-butyric acid (IBA) (2.46 μM). The regenerated plantlets showed 86% survival rate and were phenotypically normal. The described method can be successfully employed for large-scale multiplication and in vitro flowering of T. indicum.     

Mass Spectroscopy and Chromatography of the Trichloromethyl Radical Adduct of Phenyl Ter T-Butyl Nitrone

Positive structural identification of the PBN-trichloromethyl spin adduct in vim was accomplished with the use of high pressure liquid chromatography and/or gas chromatography coupled with mass spectrome-try. Both thin layer and liquid chromatography were used to separate a complex mixture of compounds from rat liver extracts treated with CCI, in vitro and in vivo. Deuterated PBN's (PBN-d, text-butyl deuteration, or PBN-d₁₄; both phenyl and tert-butyl deuteration) were also used to aid in the mass spectral analysis of spin adducts from liver extracts of CCI, exposed rat livers, since the rerr-butyl group fragment ion, C₄D₉+ (m/z = 66) is always present for PBN and PBN spin adducts. In addition, the masses of the ion peaks increase by the amount of deuteration, i.e. an increase of 9 for PBN-d, or PBN-d₁₄ in comparison to normally synthesized PBN.     

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.     

The Plasma Proteome: High Abundance versus Low Abundance

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Emerging liquid chromatography–mass spectrometry technologies improving dried blood spot analysis

Dried blood spots (DBS), a micro blood sampling technique, has recently gained interest in drug discovery and development due to its inherent advantages over the conventional whole blood, plasma or serum sample collection. Since the regulatory authorities have agreed to the use of blood as an acceptable biological matrix for drug exposure measurements, its applications have been extended not only to therapeutic drug monitoring but also to toxicokinetic and pharmacokinetic studies. The pharmaceutical industry is keen to promote DBS as a prominent tool in bioanalytical applications due to the financial, ethical and organizational issues involved in clinical trials. This could be accomplished due to the latest advances in modern analytical technology, particularly liquid chromatography–mass spectrometry. The present review discusses some of the emerging liquid chromatography–mass spectrometry technologies in improving DBS analysis for its innovative applications in the development of new drugs.     

Detection of Prothrombin and Osteopontin in a Renal Stone Found in a Hyperuricemic Patient Using 2D‐PAGE and LC‐MS Analysis

The liquid chromatography‐mass spectrometry (LC‐MS) following on from the two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) technique was applied for the analysis of proteins in a renal stone found in a hyperuricemic patient. This technique was sensitive enough to detect small quantities of proteins even in a renal stone.     

HPLC/Tandem Ion Trap Mass Detector Methods for Determination of Inosine Monophosphate Dehydrogenase (IMPDH) and Thiopurine Methyltransferase (TPMT)

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 ± 0.2 nmol/hour/10⁶ PBMC and TPMT activity of 19.9 ± 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.