Metabolic and Genetic Risk Factors Are the Strongest Predictors of Severity of Alcohol-Related Liver Fibrosis.

BACKGROUND & AIMS: Individual risk for developing alcohol-related liver disease (ALD) varies greatly. We hypothesized that metabolic risk factors and genetic polymorphisms predict severity of ALD. METHODS: Biopsy-controlled, cross-sectional study in patients with a history of excessive drinking. We measured the homeostatic model assessment of insulin resistance (HOMA-IR), plasma triglycerides, high- and low-density lipoproteins (HDL, LDL), and total cholesterol. Moreover, we genotyped four single nucleotide polymorphisms in PNPLA3 (rs738409C>G), TM6SF2 (rs58542926C>T), MBOAT7 (rs641738C>T), and HSD17B13 (rs72613567T>TA). We assessed predictors of higher fibrosis stage using multivariable ordered logistic regression. RESULTS: Of 325 included patients, 25% had severe fibrosis or cirrhosis and 59% had HOMA-IR ≥2.5. HOMA-IR increased for each fibrosis stage, while there was a similar decrease in LDL and total cholesterol. Individuals with risk variant PNPLA3 rs738409-G or TM6SF2 rs58542926-T had higher fibrosis stage. In multivariable regression, HOMA-IR ≥2.5 (OR = 3.04, 95% CI 1.90-4.87), LDL <2.60 mmol/L (OR = 2.05, 95% CI 1.33-3.16), TM6SF2 rs58542926-T (OR = 1.99, 95% CI 1.17-3.37), age above 50 years (OR = 1.66, 95% CI 1.03-2.70), and PNPLA3 rs738409-G (OR = 1.54, 95% CI 1.11-2.12) independently predicted higher fibrosis stage. Independent predictors of hepatic inflammatory activity were HOMA-IR, active drinking, age, and PNPLA3 risk variant. Active drinking, elevated triglycerides, and PNPLA3 risk variant predicted steatosis. CONCLUSIONS: Insulin resistance is the strongest predictor of liver fibrosis stage and hepatic inflammation in patients with alcohol-related liver disease. Genetic susceptibility further aggravates this risk. These data highlight the clinical value of detailed metabolic and genetic profiling of patients with excessive alcohol use.

Lack of association between cystatin C and different coronary atherosclerotic manifestations.

Cystatin C (CysC) is known to be related to cardiovascular disease (CVD), including the presence and severity of coronary artery disease (CAD) and future clinical events. In this study, the association between CysC levels and (1) coronary artery calcification (CAC) in asymptomatic individuals from the general population as well as (2) different subgroups of patients with suspected or definite acute myocardial infarction (MI) was investigated. CysC levels were measured in serum from asymptomatic individuals as part of a screening study for CAC using non-contrast cardiac CT scan (N = 1039) as well as in subgroups of hospitalized patients with a suspected MI (N = 769). CysC was not associated with CAC in asymptomatic individuals after adjusting for relevant risk factors. No difference in CysC levels was observed between patients with type 1 MI (1.07 mg/L) and patients with normal troponin (with or without prior CAD: 1.14 and 1.01 mg/L, respectively). However, patients with type 2 MI and patient subgroups with elevated troponin but without MI had significantly higher CysC levels (1.24, 1.23 and 1.31 mg/L), even after adjusting for other risk factors. CysC was not associated with CAC in middle-aged asymptomatic individuals from the general population. Furthermore, CysC levels were found to be significantly lower in patients with type 1 MI compared to patients with type 2 MI and patients with elevated troponins but without MI. Thus, in two independent and clinically different populations, no association between CysC and coronary atherosclerotic manifestations could be demonstrated.

Ionized calcium measurements are influenced by albumin--should ionized calcium be corrected?

Measurement of ionized calcium (CaI) has been reported to be dependent on albumin concentration. We examined the correlation between albumin and CaI measured on different ion selective electrode analyzers and in different groups of patients in a large dataset, extracted from the laboratory information system. In 17,281 outpatients and 16,194 inpatients, significantly positive correlations were found between CaI and albumin, with changes in CaI per 10 g/L change in albumin ranging from 0.007-0.043 mmol/L and 0.017-0.028 mmol/L, respectively. Correlations were found to be significantly different when using different analyzers. In order to examine whether the difference in correlations between the analyzers were really due to different patient populations investigated on the different analyzers, data analyzed on the same type of analyzer from inpatients from four different wards (intensive care unit, medical ward, surgical ward and orthopedic ward) were examined. There was no significant difference in correlations between patients from the four wards. Although, these results points towards technical causes behind the observed differences it cannot be entirely ruled out that clinical diseases or treatment might influence albumin interaction with CaI measurements. Combining all data from both out- and inpatients, a correction formula using a change in CaI of 0.03 mmol/L per 10 g/L change in albumin, was constructed. However, the albumin influence on CaI is only a minor part of the total CaI variation and, in most situations, the relatively small effect of changes in albumin on CaI-results is most likely of no clinical importance.

Dopamine release in organotypic cultures of foetal mouse mesencephalon: effects of depolarizing agents, pargyline, nomifensine, tetrodotoxin and calcium.

Organotypic mesencephalic cultures provide an attractive in vitro alternative to study development of the nigrostriatal system and pathophysiological mechanisms related to Parkinson's disease. However, dopamine (DA) release mechanisms have been poorly characterized in such cultures. We report here endogenous DA release (assessed by high-performance liquid chromatography) in organotypic cultures of foetal mouse (E12) midbrain following single or multiple challenges (1-h incubations) with high K(+) or veratridine in the presence or absence of pargyline, nomifensine, calcium and/or tetrodotoxin (TTX). Basal (i.e. spontaneous) DA release was only detected in the presence of pargyline and nomifensine (PN), and was highly dependent on calcium and sensitive to TTX. Basal DA release increased 2.4-fold between week 3 (1st DA release experiment) and week 4 in vitro (3rd DA release experiment), DA tissue levels increased 1.6-fold and DA release expressed as a percentage of total DA (medium + tissue contents) increased from 20% to 34% during this growth period in vitro. Co-treatments with high K(+) or veratridine did not cause major changes in percentages of DA release. Tyrosine hydroxylase activity was increased by high K(+), but not by the other drug treatments. The acute (single or multiple) treatments with depolarizing agents did not affect the survival of dopaminergic neurons, but chronic low-level veratridine treatments were toxic.

Nitration of soluble proteins in organotypic culture models of Parkinson's disease.

Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

The association between uric acid levels and different clinical manifestations of coronary artery disease.

AIMS: Uric acid (UA) has been associated with the presence and severity of coronary artery disease. To further assess the role of UA role in coronary artery disease, we investigated UA levels in both healthy asymptomatic middle-aged individuals and in different subgroups of hospitalized patients with suspected or definite myocardial infarction (MI). PATIENTS AND METHODS: The severity of coronary artery calcification (CAC) was examined in asymptomatic individuals (n=1039) using a noncontrast computed tomography scan. Hospitalized patients with suspected acute MI (n=772) were grouped according to troponin I (TnI) concentrations: (i) elevated TnI concentrations (>0.03 µg/l) with subdivision according to the type of MI and other clinical conditions associated with myocardial injury, or (ii) nonelevated TnI concentrations (≤0.03 µg/l). RESULTS: UA was not associated with the severity of CAC in asymptomatic individuals when adjusting for relevant risk factors. Patients with type 2 MI and patients with myocardial injury associated with conditions of myocardial ischemia showed significantly higher UA levels (0.390 mmol/l, P=0.002 and 0.400 mmol/l, P=0.001, respectively) than patients with type 1 MI (0.329 mmol/l), after adjusting for other risk factors. CONCLUSION: UA was not correlated with the severity of CAC in asymptomatic middle-aged individuals, and patients with type 2 MI or ischemic myocardial injury were shown to have higher UA levels than type 1 MI patients. This observation is concordant with the hypothesis that UA might be involved in the pathophysiological mechanisms leading to an imbalance in the oxygen supply/demand ratio in type 2 MI and ischemic myocardial injury.

Identification of nitrotyrosine containing peptides using combined fractional diagonal chromatography (COFRADIC) and off-line nano-LC-MALDI.

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.