Pre-diagnostic biomarkers of type 2 diabetes identified in the UAE's obese national population using targeted metabolomics.

Currently, type 2 diabetes mellitus (T2DM) and obesity are major global public health issues, and their prevalence in the United Arab Emirates (UAE) are among the highest in the world. In 2019, The UAE diabetes national prevalence was 15.4%. In recent years there has been a considerable investigation of predictive biomarkers associated with these conditions. This study analysed fasting (8 h) blood samples from an obese, normoglycemic cohort and an obese, T2DM cohort of UAE nationals, employing clinical chemistry analysis, 1D 1H NMR and mass spectroscopy (FIA-MS/MS and LC-MS/MS) techniques. The novel findings reported for the first time in a UAE population revealed significant differences in a number of metabolites in the T2DM cohort. Metabolic fingerprints identified by NMR included BCAAs, trimethylamine N-oxide, ß-hydroxybutyrate, trimethyl uric acid, and alanine. A targeted MS approach showed significant differences in lysophosphatidylcholines, phosphatidylcholines, acylcarnitine, amino acids and sphingomyelins; Lyso.PC.a.C18.0, PC.ae.C34.2, C3.DC..C4.OH, glutamine and SM.C16.1, being the most significant metabolites. Pearson's correlation studies showed associations between these metabolites and the clinical chemistry parameters across both cohorts. This report identified differences in metabolites in response to T2DM in agreement with many published population studies. This contributes to the global search for a bank of metabolite biomarkers that can predict the advent of T2DM and give insight to its pathogenic mechanisms.

Early detection of metabolic changes in drug-induced steatosis using metabolomics approaches.

Steatosis is the accumulation of triglycerides in hepatic cells wherein fats exceed 5% of the entire liver weight. Although steatotic liver damage is reversible due to the liver's regenerative capability, protracted damage often and typically leads to irreversible conditions such as cirrhosis and hepatocellular carcinoma (HCC). Therefore, early steatotic detection is critical for preventing progression to advanced liver diseases. This also becomes particularly important given the higher prevalence of drug usage, as drugs are a frequent cause of liver damage. Currently, the recommendation to diagnose steatosis is using liver enzymes and performing a liver biopsy. Liver biopsy remains the gold standard method of detection, but the procedure is invasive and an unreliable diagnostic tool. Non-invasive, specific and sensitive diagnostic solutions such as biomarkers are therefore needed for the early detection of steatosis. Our aim is to identify changes in urinary metabolites in tetracycline-induced hepatic steatotic rats at different stages of the diseases using metabolomic-based techniques. Sprague Dawley male rats are treated by intraperitoneal injection (I.P.) with either 62.5 mg kg-1 or 125 mg kg-1 tetracycline, an antibiotic previously known to induce steatosis. We analyse the metabolic profile of the urinary tetracycline induced hepatic steatotic rats using 1H nuclear magnetic resonance (NMR), 2D 1H-1H TOCSY (total correlation spectroscopy) and electrospray liquid chromatography-mass spectrometry (ESI-LC-MS/MS) based metabolomics. The combined analysis of haematoxylin & eosin (H&E), oil red O (ORO) and direct measurement of triglyceride content in the liver tissues of the control samples against 125 mg kg-1 and 62.5 mg kg-1 treated samples, reveals that 125 mg kg-1 tetracycline exposure potentially induces steatosis. The combination of 1H NMR, 2D 1H-1H TOCSY and ESI-LC-MS/MS alongside multivariate statistical analysis, detected a total of 6 urinary metabolites changes, across 6 metabolic pathways. Furthermore, lysine concentration correlates with liver damage as tetracycline dose concentration increases, whilst both H&E and ORO fail to detect hepatocellular damage at the lowest dose concentration. We conclude that the combination of 1H NMR and ESI-LC-MS/MS suggests that these are suitable platforms for studying the pathogenesis of steatosis development, prior to morphological alterations observed in staining techniques and offer a more detailed description of the severity of the steatotic disease.

Correlation of histopathology, urinary biomarkers, and gene expression responses following hexachloro-1:3-butadiene-induced acute nephrotoxicity in male Hanover Wistar rats: a 28-day time course study.

Hexachloro-1:3-butadiene (HCBD) causes segment-specific injury to the proximal renal tubule. A time course study of traditional and more recently proposed urinary biomarkers was performed in male Hanover Wistar rats receiving a single intraperitoneal (ip) injection of 45 mg/kg HCBD. Animals were killed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 28 postdosing and the temporal response of renal biomarkers was characterized using kidney histopathology, urinary and serum biochemistry, and gene expression. Histopathologic evidence of tubular degeneration was seen from day 1 until day 3 postdosing and correlated with increased urinary levels of α-glutathione S-transferase (α-GST), albumin, glucose, and kidney injury molecule-1 (KIM-1), and increased gene expression of KIM-1, NAD(P)H dehydrogenase, quinone 1, and heme oxygenase (decycling) 1. Histopathologic evidence of tubular regeneration was seen from day 2 postdosing and correlated with raised levels of urinary KIM-1 and osteopontin and increased gene expression of KIM-1 and annexin A7. Traditional renal biomarkers generally demonstrated low sensitivity. It is concluded that in rat proximal tubular injury, measurement of a range of renal biomarkers, in conjunction with gene expression analysis, provides an understanding of the extent of degenerative changes induced in the kidney and the process of regeneration.

Nephrotoxicity of hexachloro-1:3-butadiene in the male Hanover Wistar rat; correlation of minimal histopathological changes with biomarkers of renal injury.

Hexachloro-1:3-butadiene (HCBD) causes damage specifically to the renal proximal tubule in rats. In the present study, injury to the nephron of male Hanover Wistar rats was characterized at 24 h following dosing with HCBD in the range 5-90 mg kg⁻¹ to determine the most sensitive biomarkers of damage, that is, the biomarkers demonstrating significant changes at the lowest dose of HCBD, using a range of measurements in serum and urine, renal histopathology, and renal and hepatic gene expression. Histologically, kidney degeneration was noted at doses as low as 10 mg kg⁻¹ HCBD. Significant changes in the hepatic and renal gene expression categories of xenobiotic metabolism and oxidative stress were observed at 5 mg kg⁻¹ HCBD, and in the kidney alone, evidence of inflammation at 90 mg kg⁻¹ HCBD. Increases in the urinary excretion of α-glutathione S-transferase (α-GST) and kidney injury molecule-1 (KIM-1) were seen at 10 mg kg⁻¹ HCBD, and increases in urinary excretion of albumin and total protein were evident at 15 mg kg⁻¹ HCBD. The most sensitive, noninvasive biomarkers of HCBD-induced renal toxicity in Hanover Wistar rats were urinary α-GST and KIM-1. Urinary albumin measurement is also recommended as, although it is not the most sensitive biomarker, together with α-GST, albumin showed the largest relative increase of all the biomarkers investigated, and the protein is easily measured.

Urinary biomarkers in hexachloro-1:3-butadiene-induced acute kidney injury in the female Hanover Wistar rat; correlation of α-glutathione S-transferase, albumin and kidney injury molecule-1 with histopathology and gene expression.

Hexachloro-1:3-butadiene (HCBD) causes kidney injury specific to the pars recta of the proximal tubule. In the present studies, injury to the nephron was characterized at 24 h following a single dose of HCBD, using a range of quantitative urinary measurements, renal histopathology and gene expression. Multiplexed renal biomarker measurements were performed using both the Meso Scale Discovery (MSD) and Rules Based Medicine platforms. In a second study, rats were treated with a single nephrotoxic dose of HCBD and the time course release of a range of traditional and newer urinary biomarkers was followed over a 25 day period. Urinary albumin (a marker of both proximal tubular function and glomerular integrity) and α-glutathione S-transferase (α-GST, a proximal tubular cell marker of cytoplasmic leakage) showed the largest fold change at 24 h (day 1) after dosing. Most other markers measured on either the MSD or RBM platforms peaked on day 1 or 2 post-dosing, whereas levels of kidney injury molecule-1 (KIM-1), a marker of tubular regeneration, peaked on day 3/4. Therefore, in rat proximal tubular nephrotoxicity, the measurement of urinary albumin, α-GST and KIM-1 is recommended as they potentially provide useful information about the function, degree of damage and repair of the proximal tubule. Gene expression data provided useful confirmatory information regarding exposure of the kidney and liver to HCBD, and the response of these tissues to HCBD in terms of metabolism, oxidative stress, inflammation, and regeneration and repair.

Dose response and time course studies on superoxide dismutase as a urinary biomarker of carbon tetrachloride-induced hepatic injury in the Hanover Wistar rat.

Previous studies have shown that the enzyme copper/zinc superoxide dismutase (SOD-1) is increased in the urine of rats with carbon tetrachloride (CCl(4))-induced hepatotoxicity. The present experiments aimed to investigate further the usefulness of urinary SOD-1 as a non-invasive biomarker of liver injury. Two investigations were carried out, a dose response study and a time course study. In the dose response study, rats were given a single dose of CCl(4) at 0 (control), 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.80 ml/kg and urine samples collected from 12 to 36 h postdosing. In the time course study, rats were dosed at 0.80 ml/kg CCl(4) and urine sampled at 4, 12, 24 and 36 h postdosing. In both studies, the presence of SOD-1 in the urine was confirmed by Western blotting with an SOD-1 antibody. In the dose response study, serum SOD activity was elevated in all CCl(4)-treated animals and urinary SOD-1 activity was increased 2.2 times at the lowest dose (0.10 ml/kg) and 60.4 times at the highest CCl(4) dose level (0.80 ml/kg). In the time course study, urinary SOD-1 was first detected in samples collected from 4 to 12 h postdosing. We conclude that urinary SOD-1 has potential as a sensitive non-invasive biomarker of CCl(4)-induced hepatocellular injury.

Proteomic investigation of urinary markers of carbon-tetrachloride-induced hepatic fibrosis in the Hanover Wistar rat.

Proteomic techniques such as two-dimensional gel electrophoresis (2-DGE) and mass spectrometry have become important tools for the identification of novel biomarkers of toxicity and disease. Ideally, such biomarkers need to be sensitive and organ specific, but, recently, it has become apparent that it would be an additional benefit to be able to measure biomarkers in samples obtained using non-invasive methods. The present study is concerned with the identification of novel urinary markers of hepatic fibrosis. In a carbon-tetrachloride-induced liver fibrosis rat model, analysis of urine by 2-DGE revealed an increase in the concentration of a number of proteins in animals with hepatic fibrosis. Using in-gel trypsin digest and nano-scale liquid chromatography combined with electrospray ionisation tandem mass spectrometry, protein spots were identified as copper/zinc superoxide dismutase, D: -dopachrome tautomerase, beta-2-microglobulin and neutrophil gelatinase associated lipocalin. These proteins are known to have important roles in the inflammatory response.

Comprehensive characterization of serum clinical chemistry parameters and the identification of urinary superoxide dismutase in a carbon tetrachloride-induced model of hepatic fibrosis in the female Hanover Wistar rat.

Carbon tetrachloride (CCl(4)) was used to induce liver fibrosis in the rat. Using this model, we have identified changes in serum and urinary clinical chemistry parameters, and characterized histopathological lesions in the liver. Two experiments were conducted. In Experiment 1, rats were dosed at six levels of CCl(4) (0.06-0.36 ml/kg) twice weekly for 6 weeks, followed by a 6-week non-dosing recovery period (week 12). Livers were removed for histology at 6 and 12 weeks and serum parameters analysed. In Experiment 2, rats were given seven dose levels of CCl(4) (0.4-1.0 ml/kg) twice weekly for 6 weeks, followed by a 6-week recovery period (week 12); urine samples were analysed at 3, 6, 9 and 12 weeks using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liver fibrosis was evident at 6 weeks in Experiments 1 and 2, and the activity of serum enzymes (including alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase) was increased. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis (Experiment 2) revealed a protein band at 18.4 kDa in urine from rats treated with CCl(4), not present in control urine, which was identified as copper/zinc superoxide dismutase (Cu/Zn SOD). Western blotting revealed that SOD was increased in urine from rats treated with CCl(4) at 3 and 6 weeks, but not at 9 and 12 weeks. We conclude that Cu/Zn SOD is a urinary marker of hepatic necrosis, but not hepatic fibrosis.