Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment.

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.

Detection of the mitochondrial and catalytically active alanine aminotransferase in human tissues and plasma.

Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Three forms of human ALT have been identified, ALT1 and 2 and an alternative splice variant of ALT2 (herein called ALT2_2). The standard ALT activity assay does not discriminate between ALT from different organs, or the isoforms measured in the plasma. Here, we show that ALT1 and 2 possess similar enzymatic activity for alanine and pyruvate but with different Km and kcat values, while recombinant ALT2_2 protein does not possess any enzymatic activity. Isolation of organelles from cultured human skeletal muscle cells, showed localisation of ALT2 to the mitochondrial fraction and endoplasmatic reticulum (ER), but not to the cytosol. In human hepatocytes, on the other hand, ALT1 was only localised to the cytosol and ER, with no detection in mitochondria. ALT2 was not detected in cultured human hepatocytes, liver extract or tissue using Western blotting or immunohistochemistry. The islet of Langerhans and cardiomyocytes were other examples of cells with high expression of catalytic ALT2. A clinical method for selective measurement of ALT1 and 2 in human plasma is described, and both ALT1 and 2 were immunoprecipitated from human plasma and structurally detected using Western blotting techniques.

The mammalian alcohol dehydrogenases interact in several metabolic pathways.

Mammalian alcohol dehydrogenases (ADHs), including ADH1-ADH5/6, interact extensively in the oxidation and reduction of alcohols and aldehydes. ADH1 and ADH2 are involved in several metabolic pathways besides the oxidation of ethanol and have also been shown to be involved in drug transformations. The ADH2 enzymes show further complexity among the species, e.g. in enzymatic characteristics where the rodent forms essentially lack ethanol-oxidizing capacity. ADH3 (glutathione-dependent formaldehyde dehydrogenase) has been shown to catalyze the reductive breakdown of S-nitrosoglutathione, indicating involvement in nitric oxide metabolism. Mass spectrometry identified the major enzymatic product as glutathione sulfinamide. This reductive breakdown directly interferes with the formaldehyde scavenging that has been proposed to be the physiological action of ADH3. The human ADH5 and rodent ADH6 seem to be the corresponding enzymes due to their similar behavior. None of these latter ADHs have so far been assigned to any function. They can be expressed as recombinant proteins but no enzymatic activity has been detected.

Analysis of mammalian alcohol dehydrogenase 5 (ADH5): characterisation of rat ADH5 with comparisons to the corresponding human variant.

Alcohol dehydrogenase 5 (ADH5) is a member of the mammalian alcohol dehydrogenase family of yet undefined functions. ADH5 was first identified at the DNA level in human and deer mouse. A rat alcohol dehydrogenase structure of similar type has been isolated at the cDNA level using human ADH5 as a screening probe, where the rat cDNA structure displayed several atypical properties. mRNA for rat ADH5 was found in multiple tissues, especially in the kidney. In vitro translation experiments indicated that rat ADH5 is expressed as efficiently as ADH1 and furthermore, rat ADH5 was readily expressed in COS cells fused to Green Fluorescent Protein. However, no soluble ADH5 protein could be heterologously expressed in Escherichia coli cells with expression systems successfully used for other mammalian ADHs, including fused to glutathione-S-transferase. Molecular modelling of the enzyme indicated that the protein does not fold in a productive way, which can be the explanation why no stable and active ADH5 has been isolated. These results indicate that ADH5, while readily expressed at the mRNA level, does not behave similarly to other mammalian ADHs investigated. The results, in vitro and in silico, suggest an unstable ADH5 structure, which can explain for why no active and stable protein can be isolated. Further possibilities are conceivable: the ADH5 protein may have to interact with a stabiliser, or the gene is actually a pseudogene.

Isoforms of alanine aminotransferases in human tissues and serum--differential tissue expression using novel antibodies.

Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Two forms of ALT have been identified, ALT1 and ALT2, encoded by separate genes. The cellular and tissue distribution of the different ALT proteins has not been characterized in humans, and their relative contribution to serum is unknown. Here, we describe the development of novel isoenzyme specific ALT1 and ALT2 antibodies and the expression of the enzymes in human cells and organs. In normal human tissue, high expression of ALT1 was found in liver, skeletal muscle and kidney and low levels in heart muscle and not detectable in pancreas. High ALT2 reactivity was detected in heart and skeletal muscle, while no ALT2 expression was found in liver or kidney. Using immunohistochemistry, strong ALT1 reactivity was found in hepatocytes, renaltubular epithelial cells and in salivary gland epithelial cells, while ALT2 was expressed in adrenal gland cortex, neuronal cell bodies, cardiac myocytes, skeletal muscle fibers and endocrine pancreas. Immunoprecipitation using ALT antibodies on normal human serums showed ALT1 to be mainly responsible for basal ALT activity. Together, the results points to a differential expression of ALT1 and ALT2 in human organs and substantiate a need for investigations regarding the possible impacts on ALT measurements.

A simplified 2-D electrophoresis protocol with the aid of an organic disulfide.

2-DE is still a relatively cumbersome and labor intensive method. Given the successful cysteinyl protection concept with hydroxyethyl disulfide (specific oxidation) during the first dimension separation, the possibility for a simplified equilibration procedure was investigated. This was achieved by maintaining the S-mercaptoethanol modified cysteinyls throughout the 2-D workflow including second dimension separation, spot handling, protein digestion, and protein identification. The traditional equilibration protocol encompassing thiol reduction and alkylation was compared with a one-step protocol employing continuous exposure to hydroxyethyl disulfide. Both equilibration protocols gave equally well-resolved spot maps with analytical protein loads regardless of IPG strip pH range. Using preparative protein loads, narrow range IPG strips gave comparable results for the two protocols while preparative load on wide range IPG strips was the only condition where classical reduction/alkylation outperformed hydroxyethyl disulfide equilibration. Moreover, with analytical protein loads, the hydroxyethyl disulfide equilibration time could be significantly reduced without apparent loss of spot map quality or quantitative protein transfer from the first- to the second dimension gel. MALDI-TOF mass spectrometric protein identification was successfully performed with either iodoacetamide or hydroxyethyl disulfide as the cysteine modifier, yielding comparable identification results with high confidence in protein assignment, sequence coverage, and detection of cysteine-containing peptides. The results provide a novel and simplified protocol for 2-DE where the concept of hydroxyethyl disulfide as the cysteinyl protecting agent is extended to cover the entire 2-D work flow.

Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry.

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a kcat/Km approximately twice the kcat/Km for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxymethylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD+ ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.

Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen-immortalized oral keratinocytes.

The metabolic detoxification capacity may critically regulate the susceptibility of human tissues to cancer development. We used standardized and quantitative, reverse transcription-polymerase chain reaction (StaRT-PCR) and microarray chip techniques to analyze transcript levels of multiple detoxification enzymes in cultured normal human oral keratinocytes (NOK) and the Siman virus 40 T antigen-immortalized oral keratinocyte line SVpgC2a, viewing the latter as a model of a benign tumor state. With good agreement between the 2 methodologies, NOK and SVpgC2a were found to express transcripts for cytochrome P450 enzymes (CYPs), factors related to CYP induction and enzymes involved in conjugation reactions or detoxification of reactive oxygen. The cell types expressed similar levels of CYP 2B6/7, CYP 2E1, P450 oxidoreductase, the aryl hydrocarbon receptor nuclear translocator, sulfotransferase 1A1, sulfotransferase 1A3, epoxide hydrolase, glutathione S-transferase M3, glutathione S-transferase pi and catalase, superoxide dismutase 1, glutathione peroxidase 1 and glutathione peroxidase 3. In contrast, SVpgC2a exhibited comparatively higher levels of CYP1A1, 1B1, aryl hydrocarbon receptor, glutathione S-transferase M1, 2, 4, 5, glutathione S-transferase theta 1 and superoxide dismutase 2 and comparatively lower levels of UDP glycosyltransferase 2 and microsomal glutathione S-transferase 1. Some transcripts, e.g., CYP 2A6/7, were not detected by either standard, non quantitative RT-PCR or the above methods, whereas others were barely quantifiable by StaRT-PCR, i.e., were present at 1-10 molecules/10(6) molecules of actin. Overall, the expression analysis demonstrated presence of mRNA for multiple enzymes involved in foreign compound metabolism and detoxification pathways, including several enzymes not previously reported for oral epithelium. Generally, the comparison of NOK from 2 individuals indicated relatively similar transcript levels of these enzymes. In contrast, differences between NOK and SVpgC2a, e.g., for CYP1B1, may reflect alteration caused by immortalization and aid identification of early stage tumor markers in oral epithelium.