Protein-bound uremic toxins, inflammation and oxidative stress: a cross-sectional study in stage 3-4 chronic kidney disease.

BACKGROUND AND AIMS: Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) are nephro- and cardiovascular toxins, produced solely by the gut microbiota, which have pro-inflammatory and pro-oxidative properties in vitro. We undertook this study to investigate the associations between IS and PCS and both inflammation and oxidative stress in the chronic kidney disease (CKD) population. METHODS: In this cross-sectional observational cohort study, participants with stage 3-4 CKD who enrolled in a randomized controlled trial of cardiovascular risk modification underwent baseline measurements of serum total and free IS and PCS (measured by ultraperformance liquid chromotography), inflammatory markers (interferon gamma [IFN-γ], interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-α]), antioxidant and oxidative stress markers (plasma glutathione peroxidase [GPx] activity, total antioxidant capacity [TAC] and F2-isoprostanes) and pulse wave velocity (PWV), a marker of arterial stiffness. RESULTS: There were 149 CKD patients (59% male; age 60 ± 10 years; 44% diabetic) with a mean eGFR of 40 ± 9 mL/min/1.73 m(2) (range 25-59). Serum free and total IS were independently associated with serum IL-6, TNF-α and IFN-γ, whereas serum free and total PCS were independently associated with serum IL-6 and PWV. Free IS and PCS were additionally independently associated with serum GPx but not with TAC or F2-isoprostanes. CONCLUSIONS: IS and PCS were associated with elevated levels of selected inflammatory markers and an antioxidant in CKD patients. PCS was also associated with increased arterial stiffness. Inflammation and oxidative stress may contribute to the nephro- and cardiovascular toxicities of IS and PCS. Intervention studies targeting production of IS and PCS by dietary manipulation and the subsequent effect on cardiovascular-related outcomes are warranted in the CKD population.

In chemico evaluation of prohapten skin sensitizers: behavior of 2-methoxy-4-(¹³C)methylphenol in the peroxidase peptide reactivity assay (PPRA) as an alternative to animal testing.

In chemico methods, based on the assessment of a hapten's reactivity toward peptides, have been proposed as alternative methods for the assessment of the skin sensitizing potential of chemicals. However, even with these approaches showing promise, a major drawback is the activation of prohaptens, i.e. molecules needing a metabolic activation to become reactive and therefore sensitizing. Recently, it has been proposed to couple an enzymatic activation step based on horseradish peroxidase (HRP)/hydrogen peroxide to such peptide reactivity assays. To evaluate this approach, the behavior of 2-methoxy-4-methylphenol (2M4MP), reported as a moderate sensitizer according to the Local Lymph Node Assay (LLNA), has been investigated in this assay. To follow the reaction with the peptides and characterize more easily intermediates and adducts, the molecule was first (13)C isotopically substituted at the most probable reactive position. When 2M4MP was incubated with HRP/H2O2 in a mixture PBS (pH 7.4, 0.1M)/acetonitrile 2:1, two main products were formed deriving from the formation of a quinone methide 2M4MQ subsequently trapped by either H2O2 or H2O to form a benzylic hydroperoxide or alcohol, respectively. When nucleophiles such as GSH or a peptide containing a cysteine residue (Pep-Cys) were present in the reaction medium, the quinone methide 2M4MQ was trapped by the more nucleophilic thiol function to form thio-adducts. No modifications of 2M4MP were observed when the same reactions were carried out without HRP confirming that the activation of the molecule was enzyme related. Amino nucleophiles were shown to be far less reactive towards the quinone methide 2M4MQ with only tiny formation of adducts with lysine or arginine side chains. In addition we demonstrated that the same enzymatic activation could also take place in a microemulsion based on sodium dodecyl sulfate/tert-butanol/chloroform/buffer.

The requirement of ammonium or other cations linked with p-cresol sulfate for cross-reactivity with a peptide of myelin basic protein.

Urinary myelin basic protein-like material (MBPLM), so designated because of its immunoreactivity with a polyclonal antibody directed against a cryptic epitope located in residues 83-89 of myelin basic protein (MBP), exists in humans normally but increases in concentration in patients with multiple sclerosis who have progressive disease. Given its possible role in reflecting events of neural tissue destruction occurring in multiple sclerosis, urinary MBPLM is a candidate surrogate marker for this phase of the disease. Previously, it has been demonstrated that p-cresol sulfate (PCS) is the dominant component of MBPLM; however, another component(s) was essential in enabling p-cresol sulfate to have molecular mimicry with MBP peptide 83-89 detected by immunoreactivity. In the present investigation, this remaining component(s) was characterized by a combination of high performance size exclusion chromatography followed by nuclear magnetic resonance spectroscopy and shown to be ammonium. The monovalent cation ammonium could be substituted in vitro by several different monovalent and divalent cations, most notably zinc, in restoring to deprotonated p-cresol sulfate its immunoreactivity as MBPLM. These findings indicate the basis for the unexpected molecular mimicry between an epitope of an encephalitogenic protein and a complex containing a small organic molecule, p-cresol sulfate. Furthermore, the reaction of either ammonium or other cations with p-cresol sulfate may represent an in vivo process directly related to damage of axonal membranes.