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1.  NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice 
Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins.
•Ablation of Nox4 results in lowered plasma homocysteine levels in mice.•This results from increased folate-independent remethylation of homocysteine.•As a consequence mice display reduced hepatic cysteine and glutathione.•Reduced glutathione levels render the mice susceptible to hepatotoxic agents.•Nox4 is a physiological regulator of partitioning of homocysteine metabolic flux.
PMCID: PMC4698376  PMID: 26472193
ALT, alanine-transaminase; AST, aspartate-transaminase; APAP, acetaminophen (paracetamol); BHMT, betaine-homocysteine methyltransferase; BIAM, N-(biotinoyl)-N'-(iodoacetyl)ethylenediamine; CBS, cysthathionine β-synthase; CGL, cystathionine γ-lyase; DMG, dimethylglycine; GCL, γ-glutamylcysteine ligase; GSH, Glutathione; GS, glutathione synthetase; GSSG, oxidised glutathione; GWAS, genome-wide association analysis; Hcy, Homocysteine; MS, methionine synthase; NAC, N-acetyl cysteine; NADQI, N-acetly-p-benzoquinone imine; Nox4, NADPH Oxidase 4; SNP, single nucleotide polymorphism; RNS, reactive nitrogen species; ROS, reactive oxygen species; Wt, wild-type.; Betaine-homocysteine-methyltransferase (BHMT); Glutathione; Hepatotoxicity; Nox4; Redox-signaling
2.  Elevated troponin I and its prognostic significance in acute liver failure 
Critical Care  2012;16(6):R228.
Acute liver failure (ALF) is a life-threatening multisystem illness complicated by multiple organ failure (MOF) and haemodynamic disturbances. Morbidity and mortality remains high and various prognostic and scoring models are in use to predict outcome. A recent observation in a large cohort of ALF patients suggested a prognostic value of troponin I (cTnI) and its role as a marker of subclinical myocardial injury and outcome.
Data from consecutive ALF patients over a four-year period from January 2007 to March 2011 were included. The aim of this study was to correlate any relationship that may exist between cTnI, mortality, severity of illness and non-hepatic organ failure.
A total of 218 subjects (age 36 (16 to 90) years, M:F 103:115) were studied, of which 136 had an elevated cTnI > 0.05 μg/L. Higher organ failure scores were found with positive cTnI: APACHE II (19.5 (3 to 51) vs 14 (2 to 51), P = 0.001), APACHE III (81 (15 to 148) vs 59 (8 to 172), P = < 0.001) SOFA (15 (4 to 20) vs 13 (2 to 21), P = 0.027) and SAPS (48 (12 to 96) vs 34 (12 to 97), P = 0.001). Patients with positive cTnI had higher serum creatinine (192 μmol/l (38 to 550) vs 117 μmol/l (46 to 929), P < 0.001), arterial lactate (0.25, P < 0.001) and a lower pH (-0.21, P = 0.002). Also a higher proportion required renal replacement therapy (78% vs 60%, P = 0.006). Patients with elevated cTnI more frequently required vasopressors-norepinephrine (73% vs 50%, P = 0.008). Elevated cTnI did not predict outcome as effectively as other models (AUROC 0.61 (95% CI 0.52 to 0.68)).
More than 60% of ALF patients in this study demonstrated elevated cTnI. Despite a close correlation with organ failure severity, cTnI was a poor independent predictor of outcome. cTnI may not represent true myocardial injury and may be better viewed as a marker of metabolic stress.
PMCID: PMC3672613  PMID: 23190744
3.  Total protein, albumin and low-molecular-weight protein excretion in HIV-positive patients 
BMC Nephrology  2012;13:85.
Chronic kidney disease is common in HIV positive patients and renal tubular dysfunction has been reported in those receiving combination antiretroviral therapy (cART). Tenofovir (TFV) in particular has been linked to severe renal tubular disease as well as proximal tubular dysfunction. Markedly elevated urinary concentrations of retinal-binding protein (RBP) have been reported in patients with severe renal tubular disease, and low-molecular-weight proteins (LMWP) such as RBP may be useful in clinical practice to assess renal tubular function in patients receiving TFV. We analysed 3 LMWP as well as protein and albumin in the urine of a sample of HIV positive patients.
In a cross-sectional fashion, total protein, albumin, RBP, cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL) were quantified in random urine samples of 317 HIV positive outpatients and expressed as the ratio-to-creatinine (RBPCR, CCR and NGALCR). Exposure to cART was categorised as none, cART without TFV, and cART containing TFV and a non-nucleoside reverse-transcriptase-inhibitor (TFV/NNRTI) or TFV and a protease-inhibitor (TFV/PI).
Proteinuria was present in 10.4 % and microalbuminuria in 16.7 % of patients. Albumin accounted for approximately 10 % of total urinary protein. RBPCR was within the reference range in 95 % of patients while NGALCR was elevated in 67 % of patients. No overall differences in urine protein, albumin, and LMWP levels were observed among patients stratified by cART exposure, although a greater proportion of patients exposed to TFV/PI had RBPCR >38.8 μg/mmol (343 μg/g) (p = 0.003). In multivariate analyses, black ethnicity (OR 0.43, 95 % CI 0.24, 0.77) and eGFR <75 mL/min/1.73 m2 (OR 3.54, 95 % CI 1.61, 7.80) were independently associated with upper quartile (UQ) RBPCR. RBPCR correlated well to CCR (r2 = 0.71), but not to NGALCR, PCR or ACR.
In HIV positive patients, proteinuria was predominantly of tubular origin and microalbuminuria was common. RBPCR in patients without overt renal tubular disease was generally within the reference range, including those receiving TFV. RBP therefore appears a promising biomarker for monitoring renal tubular function in patients receiving TFV and for distinguishing patients with normal tubular function or mild tubular dysfunction from those with severe renal tubular disease or Fanconi syndrome.
PMCID: PMC3444380  PMID: 22883485
Proteinuria; Albuminuria; Retinol-binding protein; RBP; Cystatin C; Neutrophil gelatinase-associated lipocalin; NGAL; Tenofovir; HIV
4.  Prediction of cognitive dysfunction after resuscitation from out‐of‐hospital cardiac arrest using serum neuron‐specific enolase and protein S‐100 
Heart  2007;93(10):1268-1273.
More than 50% of patients initially resuscitated from out‐of‐hospital cardiac arrest die in hospital.
To investigate the prognostic value of serum protein S‐100 and neuron‐specific enolase (NSE) concentrations for predicting (a) memory impairment at discharge; (b) in‐hospital death, after resuscitation from out‐of‐hospital cardiac arrest.
In a prospective study of 143 consecutive survivors of out‐of‐hospital cardiac arrest, serum samples were obtained within 12, 24–48 and 72–96 hours after the event. S‐100 and NSE concentrations were measured. Pre‐discharge cognitive assessment of patients (n = 49) was obtained by the Rivermead Behavioural Memory Test (RBMT). The relationship between biochemical brain marker concentrations and RBMT scores, and between marker concentrations and the risk of in‐hospital death was examined.
A moderate negative relationship was found between S‐100 concentration and memory test score, at all time points. The relationship between NSE and memory test scores was weaker. An S‐100 concentration >0.29 μg/l at time B predicted moderate to severe memory impairment with absolute specificity (42.8% sensitivity). S‐100 remained an independent predictor of memory function after adjustment for clinical variables and cardiac arrest timing indices. NSE and S‐100 concentrations were greater in patients who died than in those who survived, at all time points. Both NSE and S‐100 remained predictors of in‐hospital death after adjustment for clinical variables and cardiac arrest timing indices. The threshold concentrations yielding 100% specificity for in‐hospital death were S‐100: 1.20 μg/l (sensitivity 44.8%); NSE 71.0 μg/l (sensitivity 14.0%).
Estimation of serum S‐100 concentration after out‐of‐hospital cardiac arrest can be used to identify patients at risk of significant cognitive impairment at discharge. Serum S‐100 and NSE concentrations measured 24–48 hours after cardiac arrest provide useful additional information.
PMCID: PMC2000934  PMID: 17502328
cardiac arrest; hypoxia–ischaemia (brain); cognitive function
5.  Blood profile holds clues to role of infection in a premonitory state for idiopathic parkinsonism and of gastrointestinal infection in established disease 
Gut Pathogens  2009;1:20.
The two-stage neuroinflammatory process, containment and progression, proposed to underlie neurodegeneration may predicate on systemic inflammation arising from the gastrointestinal tract. Helicobacter infection has been described as one switch in the pathogenic-circuitry of idiopathic parkinsonism (IP): eradication modifies disease progression and marked deterioration accompanies eradication-failure. Moreover, serum Helicobacter-antibody-profile predicts presence, severity and progression of IP. Slow gastrointestinal-transit precedes IP-diagnosis and becomes increasingly-apparent after, predisposing to small-intestinal bacterial-overgrowth (SIBO). Although IP is well-described as a systemic illness with a long prodrome, there has been no comprehensive overview of the blood profile. Here, it is examined in relation to Helicobacter status and lactulose-hydrogen-breath-testing for SIBO.
A robust finding of reduced lymphocyte count in 126 IP-probands and 79 spouses (without clinically-definite IP), compared with that in 381 controls (p < 0.001 in each case), was not explained by Helicobacter-status or breath-hydrogen. This complements a previous report that spouses were 'down-the-pathway' to 'clinically-definite' disease. In 205 other controls without clinically-definite IP, there were strong associations between sporadic cardinal features and immunoglobulin class concentration, not explained by Helicobacter-status. Premonitory states for idiopathic parkinsonism associated with relative lymphopenia, higher serum immunoglobulin concentrations and evidence of enteric-nervous-system damage may prove viral in origin.
Although only 8% of the above 79 spouses were urea-breath-test-positive for Helicobacter, all 8 spouses with clinically-definite IP were (p < 0.0001). Transmission of a 'primer' to a Helicobacter-colonised recipient might result in progression to the diagnostic threshold.
Twenty-five percent of the 126 probands were seropositive for anti-nuclear autoantibody. In 20 probands, monitored before and serially after anti-Helicobacter therapy, seropositivity marked a severe hypokinetic response (p = 0.03). It may alert to continuing infection, even at low-density. Hyperhomocysteinemia is a risk factor for dementia and depression. Serum homocysteine exceeded the target in 43% of the 126 IP-probands. It was partially explained by serum B12 (12% variance, p < 0.001), but not by Helicobacter-status (gastric-atrophy uncommon in IP) or levodopa treatment. Immune-inflammatory activation increases homocysteine production. Since an estimated 60% of probands are hydrogen-breath-test positive, SIBO, with its increased bacterial utilisation of B12, is a likely cause. Thus, two prognostic indicators in established IP fit with involvement of Helicobacter and SIBO.
PMCID: PMC2795757  PMID: 19941660
6.  Folic Acid: A Marker of Endothelial Function in Type 2 Diabetes? 
Endothelial dysfunction is a common feature of type 2 diabetes. Recent studies suggest that the B-vitamin folic acid exerts direct beneficial effects on endothelial function, beyond the well known homocysteine lowering effects. Therefore, folic acid might represent a novel “biomarker” of endothelial function. We sought to determine whether plasma levels of folic acid determine endothelial-dependent vasodilation in patients with type 2 diabetes.
Forearm arterial blood flow (FABF) was measured at baseline and during intrabrachial infusion of the endothelial-dependent vasodilator acetylcholine (15 μg/min) and the endothelial-independent vasodilator sodium nitroprusside (2 μg/min) in 26 type 2 diabetic patients (age 56.5 ± 0.9 years, means ± SEM) with no history of cardiovascular disease.
FABF ratio (ie, the ratio between the infused and control forearm FABF) significantly increased during acetylcholine (1.10 ± 0.04 vs 1.52 ± 0.07, p < 0.001) and sodium nitroprusside (1.12 ± 0.11 vs 1.62 ± 0.06, p < 0.001) infusions. After correcting for age, gender, diabetes duration, smoking, hypertension, body mass index, microalbuminuria, glycated hemoglobin, low-density lipoprotein cholesterol, and homocysteine, multiple regression analysis showed that plasma folic acid concentration was the only independent determinant (p = 0.037, R2 = 0.22) of acetylcholine-mediated, but not sodium nitroprusside-mediated, vasodilatation.
Folic acid plasma concentrations determine endothelium-mediated vasodilatation in patients with type 2 diabetes. These results support the hypothesis of a direct effect of folic acid on endothelial function and the rationale for interventions aimed at increasing folic acid levels to reduce cardiovascular risk.
PMCID: PMC1993928  PMID: 17319100
folic acid; homocysteine; endothelium; type 2 diabetes

Results 1-7 (7)