Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating ...cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9-1.5 pmol mg(-1) protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3-5 pmol mg(-1) protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5-13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function.
Prognosis, diagnosis, and treatment of several diseases strongly rely on the sensitive, selective, and accurate determination of specific biomarkers in relevant biological samples. Free biliverdin ...and free bilirubin represent important new biomarkers of oxidative stress, however, the lack of suitable analytical methods for their determination has hindered progress in biomedical and clinical research.
Here, we introduce a first comprehensive approach for robust and simultaneous determination of these bilins in serum using liquid chromatography - mass spectrometry (LC-MS). The developed analytical method exhibits linearity for both analytes within the concentration range of 0.5-100 nM, with limits of detection and quantitation determined at 0.1 nM and 0.5 nM, respectively. Moreover, several analytical pitfalls related to the intrinsic molecular structures of free bilirubin and free biliverdin and their trace concentration levels in biological samples are discussed here in detail for the first time. We have shown that the solubility, chemical stability, and affinity of these bilins to various materials strongly depend on the solvent, pH, and addition of stabilizing and chelating agents. Finally, the validated LC-MS method was successfully applied to the analysis of both bilins in fetus bovine serums, yielding higher free bilirubin/biliverdin ratios compared with previously reported values for human serum.
Failure to recognize and address the challenges presented here often leads to substantial analytical errors and consequently biased interpretation of the obtained results. This pertains not only to LC-MS, but also to many other analytical platforms due to the compound-derived sources of error.
Diabetes mellitus (DM), a non-communicable endocrine disease that is marked by a differing degree of tolerance to insulin and dysfunction. The connection between diabetes and liver failure important ...to doctors in general practice diabetologists and hepatologists. DM is linked with an elevated risk of hepatic consequences and mortality of liver cirrhosis patients. DM may facilitate to insult the liver by inducing inflammation and fibrosis by elevating mitochondrial oxidative stress. The conventional liver function indices are bilirubin including Indirect Bilirubin (IBil), Direct Bilirubin (DBil), and Total Bilirubin (TBil). DBil, IBil, and TBil, have diverse clinical implications as the standard index of liver disorder. An elevated level of DBil may suggest damage to the hepatic cell whereas TBil is within the normal range. Thus, increased liver enzymes are correlated with hepatic insulin resistance in healthy subjects. Notably, a significant correlation between DBil levels and Insulin resistance risk could indicate a connection between liver dysfunction and diabetes mellitus risk. Thus, our primary goal via the current review to examine the impact of dietary vitamin D (VitD) in serum mediated risk reduction of insulin resistance and further incidence of DM through inflammatory liver associated high DBil. Therefore, modifying these inflammatory pathways may be a therapeutic alternative approach for diabetes treatment.
•Diabetes mellitus may facilitate to injury the liver by inducing inflammation and fibrosis.•DBil, IBil, and TBil, have diverse clinical implications as the standard index of liver disorder.•Increased liver enzymes are correlated with hepatic insulin resistance in healthy subjects.
•Novel approach to direct analysis of free bilirubin by HPLC-TLS.•Coupling of HPLC with TLS instead of DAD enabled 20-times lower LODs.•LOD of 90pM and LOQ of 250pM are the lowest published for ...bilirubin yet.•First reported direct determination of free bilirubin in animal and human serum.
Direct analysis of free bilirubin in human and animal blood serum samples is reported for the first time. A state-of-the-art system comprised of newly developed high-performance liquid chromatography (HPLC) on reverse-phase (RP) C18 support coupled with thermal lens spectrometric detection (TLS), based on excitation at λ=457.9nm by an argon laser was used for this purpose. This HPLC-TLS method enabled a baseline separation of all three structural isomers of bilirubin (XIII-α, IX-α and III-α) and the respective degradation products in isocratic mode in fewer than 7min. The method excels in ultra-high sensitivity with limit of detection (LOD) and limit of quantitation (LOQ) of 90pM and 250pM, respectively. Moreover, this method also affords high precision and accuracy, with correlation coefficients R2>0.997 over a broad linear range (0.250–150nM) and R2=0.9998 in a concentration range of clinical interest (0.500–25nM). The method's boosted sensitivity enabled to streamline sample preparation to just one serum ultrafiltration step, which made qualitative evaluation of sample preparation possible for the first time. The performance of the HPLC-TLS method was assessed to have 20-fold enhanced sensitivity when compared to a comparable method incorporating HPLC coupled with diode array detector (DAD), which is also a novel method by itself, and could be applied for free bilirubin determination in patients with elevated bilirubin levels.
We present the applicability of a new ultra-sensitive analytical method for the simultaneous determination of biliverdin and bilirubin in human serum. The method comprises isocratic reversed-phase ...(RP) C18 high-performance liquid chromatography (HPLC) and thermal lens spectrometric detection (TLS) based on excitation by a krypton laser emission line at 407nm. This method enables the separation of IX-α biliverdin and IX-α bilirubin in 11min with limit of detection (LOD) and limit of quantitation (LOQ) for biliverdin of 1.2nM and 3nM, and 1nM and 2.8nM for bilirubin, respectively. In addition, a step-gradient elution was set up, by changing the mobile phase composition, in order to further enhance the sensitivity for bilirubin determination with LOD and LOQ of 0.5nM and 1.5nM, respectively. In parallel, an isocratic HPLC-DAD method was developed for benchmarking against HPLC-TLS methods. The LOD and LOQ for biliverdin were 6nM and 18nM, and 2.5nM and 8nM for bilirubin, respectively. Additionally, both isocratic methods were applied for measuring biliverdin and free bilirubin in human serum samples (from 2 male and 2 female healthy donors). Combining isocratic HPLC method with TLS detector was crucial for first ever biliverdin determination in serum together with simultaneous free bilirubin determination. We showed for the first time the concentration ratio of free bilirubin versus unbound biliverdin in human serum samples.
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•The novel HPLC-TLS method is the most sensitive known method for biliverdin determination.•Coupling of HPLC with TLS instead of DAD enables 5-times lower LOD.•First simultaneous determination of unbound biliverdin and free bilirubin in human serum.•Ratio of free bilirubin versus unbound biliverdin in human serum is around 3:1.
Abstract Objective Low levels of bilirubin have recently been associated with obesity, diabetes mellitus, and metabolic syndrome. Here, we hypothesized that serum bilirubin levels might be already ...altered in overweight asymptomatic middle-aged individuals before full development of the metabolic syndrome. Methods Healthy nonsmoking adults aged 25–49 (64 women and 32 men) participated in this cross-sectional study. All participants who reported stable weight within the last three months underwent standard anthropomorphological measurements of body composition, blood pressure measurements, aerobic and anaerobic capabilities assessment, dietary intake evaluation, and fasting serological measurements of total and direct bilirubin, glucose, insulin, triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and C-reactive protein. Participants were divided into normal-weight and overweight groups. Linear correlation and multiple regression analyses were used to examine the association of serum bilirubin levels with all metabolic syndrome risk factor changes. Results Serum bilirubin levels were lower in overweight healthy individuals of both sexes, and were negatively associated with abdominal obesity, insulin resistance, fasting glucose, fasting insulin, fasting triglycerides, total cholesterol, low-density lipoprotein cholesterol, and C-reactive protein levels but positively associated with aerobic body capabilities. Conclusion Our findings suggest that serum bilirubin levels have the potential to be employed as an early biomarker for indicating asymptomatic individuals at increased risk of developing metabolic syndrome.
Growing analytical challenges have arisen for the detection of misuse of androgenic anabolic steroids (AAS) in athletes the last years. Therefore, consideration of additional indirect markers can ...substantially aid the efforts to detect AAS abuse in athletes. Moreover, this approach can also help physicians to suspect AAS abuse when treating athletes. Laboratory markers highly indicative of AAS abuse in athletes include the considerable downregulation of high density lipoprotein-cholesterol, elevation of haematocrit or serum γ-glutamyl transpeptidase levels and for males reduced serum levels of both luteinizing hormone and follicle-stimulating hormone. Moreover, physical signs suggestive of current AAS abuse are hypertension, apparent changes in behaviour making the athlete more irritable and aggressive and the sudden appearance of acne vulgaris in an adult athlete with no recent history of acne, while testicular atrophy and gynecomastia raise suspicion of current or past AAS abuse in male athletes.
Despite being reported to reduce the risk of cardiovascular diseases, little is known about acute direct effects of bilberry anthocyanins on whole mammalian heart under ischemia-reperfusion (I-R) ...conditions. Bilberry anthocyanins were prepared from the ripe bilberries and analyzed using HPLC-DAD. Their antioxidant activity was evaluated by measuring the intrinsic free radical-scavenging capacity and by cellular antioxidant assay (CAA) on endothelial cells, where we quantified the intracellular capacity to inhibit the formation of peroxyl radicals. Experiments on the isolated rat hearts under I-R were carried out according to the Langendorff method. Perfusion with low concentrations of bilberry anthocyanins (0.01-1 mg/L) significantly attenuated the extent of I-R injury as evidenced by decreasing the release rate of LDH, increasing the postischemic coronary flow, and by decreasing the incidence and duration of reperfusion arrhythmias. High concentrations (5-50 mg/L) diminished cardioprotection and show cardiotoxic activity despite having their radical scavenging and intracellular antioxidant capabilities increased in a concentration-dependent manner. This study reveals the biphasic concentration-dependent bioactivity of bilberry anthocyanins under I-R, which results in strong cardioprotective activity in low concentrations and cardiotoxic activity in high concentrations.
We report on our research in using literature-based discovery (LBD) to provide pharmacological and/or pharmacogenomic explanations for reported adverse drug effects. The goal of LBD is to generate ...novel and potentially useful hypotheses by analyzing the scientific literature and optionally some additional resources. Our assumption is that drugs have effects on some genes or proteins and that these genes or proteins are associated with the observed adverse effects. Therefore, by using LBD we try to find genes or proteins that link the drugs with the reported adverse effects. These genes or proteins can be used to provide insight into the processes causing the adverse effects. Initial results show that our method has the potential to assist in explaining reported adverse drug effects.
Nowadays, much attention has been paid to diet and dietary supplements as a cost-effective therapeutic strategy for prevention and treatment of a myriad of chronic and degenerative diseases. Rapidly ...accumulating scientific evidence achieved through high-throughput technologies has greatly expanded the understanding about the multifaceted nature of cancer. Increasingly, it is being realized that deregulation of spatio-temporally controlled intracellular signaling cascades plays a contributory role in the onset and progression of cancer. Therefore, targeting regulators of oncogenic signaling cascades is essential to prevent and treat cancer. A plethora of preclinical and epidemiological evidences showed promising role of phytochemicals against several types of cancer. Oleanolic acid, a common pentacyclic triterpenoid, is mainly found in olive oil, as well as several plant species. It is a potent inhibitor of cellular inflammatory process and a well-known inducer of phase 2 xenobiotic biotransformation enzymes. Main molecular mechanisms underlying anticancer effects of oleanolic acid are mediated by caspases, 5' adenosine monophosphate-activated protein kinase, extracellular signal-regulated kinase 1/2, matrix metalloproteinases, pro-apoptotic Bax and bid, phosphatidylinositide 3-kinase/Akt1/mechanistic target of rapamycin, reactive oxygen species/apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase, nuclear factor-κB, cluster of differentiation 1, CKD4, s6k, signal transducer and activator of transcription 3, as well as aforementioned signaling pathways . In this work, we critically review the scientific literature on the molecular targets of oleanolic acid implicated in the prevention and treatment of several types of cancer. We also discuss chemical aspects, natural sources, bioavailability, and safety of this bioactive phytochemical.
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