The Na+K+-ATPase Inhibitor Marinobufagenin and Early Cardiovascular Risk in Humans: a Review of Recent Evidence.

PURPOSE OF REVIEW: This review synthesizes recent findings in humans pertaining to the relationships between marinobufagenin (MBG), a steroidal Na+/K+-ATPase inhibitor and salt-sensitivity biomarker, and early cardiovascular risk markers. RECENT FINDINGS: Twenty-four-hour urinary MBG strongly associates with habitual salt intake in young healthy adults (aged 20-30 years). Furthermore, in young healthy adults free of detected cardiovascular disease, MBG associates with increased large artery stiffness and left ventricular mass independent of blood pressure. These findings in human studies corroborate mechanistic data from rat studies whereby stimulation of MBG by a high salt intake or MBG infusion increased vascular fibrosis and cardiac hypertrophy. Twenty-four-hour urinary MBG may be a potential biomarker of early cardiovascular risk. Adverse associations between MBG-which increases with salt consumption-and early cardiovascular risk markers support the global efforts to reduce population-wide salt intake in an effort to prevent and control the burden of non-communicable diseases.

Intracerebral hemorrhage in cocaine users.

BACKGROUND AND PURPOSE: Cocaine is a cause of intracerebral hemorrhage (ICH), but there are no large studies that have characterized the location, pathology, and outcome of patients with cocaine-associated ICH. METHODS: We performed a retrospective analysis of all patients admitted to our stroke service from 2004 to 2007 who had nontraumatic ICH and urine drug screens positive for cocaine and compared them with similar patients who had negative drug screens for cocaine. RESULTS: We identified 45 patients with cocaine-associated ICH and 105 patients with cocaine-negative ICH. There were no significant differences in age or gender, but there was a significantly higher incidence of black patients in the cocaine-positive group. Cocaine-associated ICH patients had higher admission blood pressures, significantly more subcortical hemorrhages, and higher rates of intraventricular hemorrhage compared to patients with cocaine-negative ICH. Cocaine-positive patients had worse functional outcome, defined as modified Rankin Scale score >3 at the time of discharge (OR, 4.90; 95% CI, 2.19-10.97), and were less likely to be discharged home or to inpatient rehabilitation. Patients with cocaine-associated ICH were nearly 3-times more likely to die during their acute hospitalization when compared to cocaine-negative patients. CONCLUSION: Recent cocaine ingestion is associated with hemorrhages that occur more frequently in subcortical locations, have a higher risk of intraventricular hemorrhage, and have a poor prognosis compared to patients with cocaine-negative, spontaneous ICH.

Determination of 8-epi PGF(2alpha) concentrations as a biomarker of oxidative stress using triple-stage liquid chromatography/tandem mass spectrometry.

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples.

Plasma and Urine Levamisole in Clinical Samples Containing Benzoylecgonine: Absence of Aminorex.

Aminorex has been reported as a metabolite of levamisole in man, but data on the aminorex concentrations in clinical samples are scant. We thus measured levamisole, aminorex and benzoylecgonine in urine, and levamisole and aminorex in plasma using achiral liquid chromatography-high resolution mass spectrometry. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standard (benzoylecgonine-D3, 25 µg/L) (450 µL). For plasma, sample (200 µL) and Tris solution (2 mol/L, pH 10.6, 100 µL) were added to a 60.5 × 7.5 mm i.d. glass test tube. Internal standard solution (ketamine-D4, 200 µg/L) (10 µL) was added and the tube contents vortex-mixed (5 s). Butyl acetate:butanol (9 + 1, v/v; 200 µL) was added and after vortex-mixing (30 s) and centrifugation (13,680 × g, 4 min), the extract was evaporated to dryness and reconstituted in 10 mmol/L aqueous ammonium formate containing 0.1% (v/v) formic acid (150 µL). Prepared samples and extracts (100 µL) were analyzed using an AccucoreTM Phenyl-Hexyl column (2.6 mm a.p.s., 100 × 2.1 mm i.d.) maintained at 40°C. MS detection was in positive mode using heated electrospray ionization (ThermoFisher Q-ExactiveTM). Intra- and inter-assay accuracy and precision were ±20%, and ≤11%, respectively, for all analytes in both matrices. Lower limits of quantitation were 0.1 and 1 µg/L (all analytes) in plasma and urine, respectively. Of 100 consecutive urine samples submitted for drugs of abuse screening containing benzoylecgonine, levamisole was detected in 72 (median 565, range 4-72,970 µg/L). Levamisole was also measured in eight plasma samples (median 10.6, range 0.9-64.1 µg/L). A number of metabolites of levamisole (4-hydroxylevamisole, levamisole sulfoxide, levamisole glucuronide, and hydroxylevamisole glucuronide) were tentatively identified in urine. Neither aminorex, nor any of its reported metabolites were detected in any sample.

Endogenous bufadienolide mediates pressor response to ethanol withdrawal in rats.

An endogenous natriuretic and vasoconstrictor Na/K-ATPase inhibitor, marinobufagenin (MBG), is implicated in NaCl-induced hypertension and in ethanol addiction. In rats, MBG suppresses voluntary alcohol intake, while immunization against MBG induces alcohol-seeking behavior. Since alcohol withdrawal is associated with elevation of blood pressure (BP) and renal sodium retention, we hypothesized that MBG mediates pressor response to ethanol withdrawal. In male Sprague-Dawley rats, forced ethanol intake (20% v/v, 2.8+/-0.2 g/day for 7 days) did not affect BP and MBG excretion. Ethanol withdrawal was associated with a 21 mm Hg increase in BP, a 10% decrease in hematocrit, and a three-fold increase in renal MBG excretion. In vivo administration of anti-MBG antibody to rats prevented withdrawal-induced BP elevation. Therefore, MBG mediates pressor response to ethanol withdrawal, and may link mechanisms of ethanol dependence and hypertension.

Vascular effects of the bufodienolides.

The bufodienolides are natriuretic steroids, which also have the capacity to cause vasoconstriction, and are cardiac inotropes. Their mechanism of action appears to be related to their ability to inhibit Na+/K+ ATPase. The actions of one of these compounds, marinobufagenin (MBG), have been investigated in a rat model of preeclampsia, an example of volume expansion-mediated hypertension. The urinary excretion of MGB is increased in this model. Furthermore, this increment in its excretion occurs prior to the development of hypertension and proteinuria. The animals also demonstrate intrauterine growth restriction. Studies of the effect of MBG on cytotrophoblast cells reveal that MGB inhibits the migration, proliferation and invasion of these cells. We propose that MGB is an important etiologic factor in at least some forms of preeclampsia and that the level of its excretion in the urine may prove to be of diagnostic value.

Unusual finding on bronchoscopy: trauma patient identified as a body stuffer.

We present the case of a trauma patient whose persistently abnormal chest radiography led to exploratory bronchoscopy. After the discovery of a foreign body in the right lower lobe bronchus, an attempted retrieval resulted in accidental perforation of a cocaine bag and release of the drug, which may have been the cause of the patient's subsequent pneumonitis.

Effects of ferrous sulphate and non-ionic iron-polymaltose complex on markers of oxidative tissue damage in patients with inflammatory bowel disease.

BACKGROUND: Iron deficiency is a common complication of inflammatory bowel disease. Oral iron therapy may reinforce intestinal tissue injury by catalyzing production of reactive oxygen species. AIM: To compare the effects of ferrous sulphate and non-ionic iron-polymaltose complex on markers of oxidative tissue damage and clinical disease activity in patients with inflammatory bowel disease. METHODS: Forty-one patients with inflammatory bowel disease and iron deficiency were randomized to treatment with ferrous sulphate 100 mg twice a day or iron-polymaltose complex 200 mg once a day for 14 days. RESULTS: Following ferrous sulphate, plasma malondialdehyde increased (P = 0.02), while urine 8-isoprostaglandin F(2alpha) and plasma antioxidants did not change significantly. Iron-polymaltose complex did not change plasma malondialdehyde, urine 8-isoprostaglandin F(2alpha) or plasma antioxidants. Comparing the two treatments, changes in plasma malondialdehyde tended to differ (P = 0.08), while urine 8-isoprostaglandin F(2alpha) and plasma antioxidants did not differ. Neither ferrous sulphate nor iron-polymaltose complex altered clinical disease activity indices. CONCLUSIONS: Ferrous sulphate increased plasma malondialdehyde, a marker of lipid peroxidation. Comparing treatment with ferrous sulphate and iron-polymaltose complex, changes in plasma malondialdehyde tended to differ. Clinical disease activity was unchanged after both treatments.

Measurement of urinary 8-Epi-prostaglandin F2alpha, a novel index of lipid peroxidation in vivo, by immunoaffinity extraction/gas chromatography-mass spectrometry. Basal levels in smokers and nonsmokers.

8-Epi-prostaglandin F2alpha (8-epi-PGF2alpha) is an F2-isoprostane recently identified as a marker of free radical-catalyzed lipid peroxidation in vivo and potential mediator of oxidative damage. Currently, endogenous 8-epi-PGF2alpha is measured by gas chromatography-mass spectrometry after lengthy sample preparation. We extracted and purified 8-epi-PGF2alpha in one step from biological samples on immunoaffinity columns prepared with an anti-8-epi-PGF2alpha antiserum, raised in our laboratory. Quantitation was done by stable-isotope dilution gas chromatography/negative-ion chemical ionization mass spectrometry, with selected ion recording. Carboxylate anions of the pentafluorobenzyl ester trimethylsilyl ether derivative of 8-epi-PGF2alpha and [2H4]8-epi-PGF2alpha were monitored (m/z 569 and 573). Basal urinary excretion of 8-epi-PGF2alpha can be accurately and rapidly measured by this method. Under normal conditions rats (n = 30) excreted 2.18 +/- 0.68 ng/24 h. In healthy nonsmoking young volunteers, urinary excretion of 8-epi-PGF2alpha, measured three times on alternate days, was fairly constant (CV 2-10%). Nonsmokers excreted significantly less 8-epi-PGF2alpha than age-matched smokers (8.08 +/- 2.3 vs. 18.40 +/- 4.77 ng/h/1.73 m2; n = 6; p < 0.005), as reported by others using different methods.

Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS.

BACKGROUND: Oxidant stress is a purported mechanism of tissue damage in patients with ARDS and acute lung injury (ALI). Isoprostanes, prostanoid compounds primarily formed nonenzymatically via lipid peroxidation, are precise markers of in vivo oxidant stress. Plasma levels of metabolites of 8-iso-prostaglandin-F2alpha (8-iso-PGF2alpha) correlate with outcome in patients with ARDS. OBJECTIVE: To examine exhaled breath condensate levels of 8-iso-PGF2alpha as a noninvasive quantification of pulmonary oxidant stress in patients with, or at risk for, ARDS/ALI. METHODS: Breath condensate was collected from 22 patients with, or at risk for, ARDS/ALI by placing Tygon tubing submerged in an ice bath in line with the expiratory limb of the ventilator circuit. Ten patients without lung disease, who were intubated while undergoing minor surgical procedures, served as control subjects. Between 1 and 3 mL of condensate was collected over a 30- to 60-min period, then immediately frozen and stored at -70 degrees C until analysis. The 8-iso-PGF2alpha was purified and derivatized, then quantified by stable isotope dilution in conjunction with gas chromatography/mass spectrometry. RESULTS: The mean level of exhaled 8-iso-PGF2alpha in the patients with ALI/ARDS, 87+/-28 pg/mL, was significantly higher than the mean in the normal group, 7+/-4 pg/mL (p = 0.007). The 8-iso-PGF2alpha levels were greater than two standard deviations above the mean of the normal group in 12 of 22 patients with or at risk for ARDS/ALI. CONCLUSIONS: These results provide further evidence that lipid peroxidation does occur in patients with ARDS/ALI. The measurement of exhaled isoprostanes provides a novel, noninvasive method to quantify oxidant stress in such patients.

Oxidative stress and baroreflex sensitivity in healthy subjects and patients with mild-to-moderate hypertension.

Decreased baroreflex sensitivity (BRS) is a prognostic marker in essential hypertension. Animal experiments suggest that decreased BRS is related to increased oxidative stress. Our study was aimed at testing whether oxidative stress, estimated by isoprostane 15-F(2t)-IsoP urinary levels, is correlated to BRS variation in healthy subjects as well as in patients suffering from essential hypertension. Urinary 15-F(2t)-IsoP levels and BRS were evaluated in two groups of subjects: healthy volunteers (n=64) and patients with untreated mild-to-moderate hypertension (n=33). Data were analysed in 61 and 31 subjects, respectively, BRS analysis being impossible in three and two subjects, respectively. 15-F(2t)-IsoP levels were measured using gas chromatography/mass spectrometry. BRS was measured using the sequence method [PS+/RR+ and PS-/RR-] and crossspectral analysis (CSP) (MF gain) at rest, lying down. No significant correlation was found between basal urinary 15-F(2t)-IsoP levels and BRS (sequence method and CSP) in either healthy controls or hypertensive patients. Our study shows that oxidative stress is not involved in interindividual variations of BRS in healthy subjects and patients suffering from mild-to-moderate hypertensionJournal of Human Hypertension (2004) 18, 517-521. doi:10.1038/sj.jhh.1001684 Published online 12 February 2004

Isoprostanes in atherosclerosis.

Isoprostanes (IP) are a new family of compounds formed during oxidation injury. 8-epi-prostaglandin (PG) F2alpha, a vasoconstrictory and mitogenic substance, is increased in hyperlipidemia in blood and urine as well as at the vascular level in the intima, in particular along foam cells. Similarly, cigarette smoking is associated with an immediate increase in 8-epi-PGF2alpha and a quick drop after quitting. Also diabetes and even the more a combination of risk factors (for the development of atherosclerosis) results in increased 8-epi-PGF2alpha in various compartments. Others, such as sex, age, hypertension and obesity were of minor influence. These findings further indicate, that in-vivo oxidation injury as reflected by increased IP may play a relevant role in atherogenesis. IP may serve as useful markers to assess oxidation injury at a local level.

Urinary excretion of vasoactive markers following estrogen replacement therapy in postmenopausal women.

OBJECTIVE: The effect of estradiol on the renal excretion of vasoactive substances was studied in postmenopausal women. The following markers surrogating an estrogenic effect on the cardiovascular system were measured: prostacyclin and thromboxane, cGMP, which reflects systemic nitric monoxide production, serotonin and relaxin. METHODS: The effect of estradiol was compared using two clinical forms of administration, transdermal and oral, in two groups of 20 postmenopausal women each. The treatment was carried out for two and four weeks, respectively. Nocturnal urine was collected over 8 hours before and after estradiol treatment. The quantity of markers excreted during the experiment was determined. RESULTS: Excretion of prostacyclin and thromboxane, calculated as prostacyclin/thromboxane ratio, was increased using both forms of administration. Both forms of treatment brought about only slight non-significant changes in renal cGMP excretion compared with values before treatment. The production of serotonin and relaxin was only increased using transdermal treatment. CONCLUSION: The resulting data show that estradiol replacement in postmenopausal women is able to increase renal excretion of various vasoactive substances implying a vasodilative effect of estrogen. This was seen, both in transdermal and oral administration, transdermal application having a more pronounced effect on the markers than oral administration.

Lipid peroxidation and cell death mechanisms in rats and human cells induced by chloral hydrate.

Chloral hydrate (CH) is widely used as a sedative and hypnotic in pediatric medicine. It is also a by-product of water chlorination and a metabolite of trichloroethylene. We examined the toxicological effects and cell death mechanisms of CH in rats and human Chang liver cells and lymphocytes. Monitoring of urinary 8-epi-PGF2alpha and serum levels of TNF-alpha served as index of lipid peroxidation and cytokine stimulation. The results indicated that a single intraperitoneal injection of 100 mg/kg CH in rats led to a nearly five-fold increase in urinary 8-epi-PGF2alpha on day 1, and a mild decrease on day 2 and day 3. The same treatment also induced significantly higher amounts of serum TNF-alpha on day 2 (about seven-fold). When the rats were treated with CH and vitamin E simultaneously, the amount of urinary 8-epi-PGF2alpha and serum TNF- were significantly lower than that in the rats treated with CH alone. CH caused a greater cytotoxic effect in human Chang liver cells than in comparison with lymphocytes. After treatment with CH, apoptosis features were observed in human lymphocytes, but not Chang liver cells. CH-induced cell damage in lymphocytes may offer signals for the induction of caspases activation. Further studies are needed to evaluate the relationship between caspases activation and the cleavage of other death substrates during postmitotic apoptosis in human lymphocytes.

Cardiovascular changes during subgingival debridement.

During dental hygiene sessions, systolic and diastolic blood pressure and heart frequency were registered continuously with a finger manometer to investigate the possible cardiovascular effects of root planing/scaling. Ultrasonic subgingival debridement was performed for an average period of 10.6 +/- 2.1 min. Analysis of variance showed significant changes of systolic and diastolic blood pressure, heart rate and the rate pressure product during debridement. The increases in systolic and diastolic blood pressure both correlated significantly with the length of the debridement. After completion of the dental hygiene session, the urinary excretion of adrenaline was increased. The data from this study suggest that painful stimuli during ultrasonic subgingival debridement have extensive cardiovascular effects.

Beta-blockers for chest pain associated with recent cocaine use.

BACKGROUND: Although beta-blockers prevent adverse events after myocardial infarction, they are contraindicated when chest pain is associated with recent cocaine use. Recommendations against this use of beta-blockers are based on animal studies, small human experiments, and anecdote. We sought to test the hypothesis that beta-blockers are safe in this setting. METHODS: We performed a retrospective cohort study of consecutive patients admitted to the San Francisco General Hospital, San Francisco, California, with chest pain and urine toxicologic test results positive for cocaine, from January 2001 to December 2006. Mortality data were collected from the National Death Index. RESULTS: Of 331 patients with chest pain in the setting of recent cocaine use, 151 (46%) received a beta-blocker in the emergency department. There were no meaningful differences in electrocardiographic changes, troponin levels, length of stay, use of vasopressor agents, intubation, ventricular tachycardia or ventricular fibrillation, or death between those who did and did not receive a beta-blocker. After adjusting for potential confounders, systolic blood pressure significantly decreased a mean 8.6 mm Hg (95% confidence interval, 14.7-2.5 mm Hg) in those receiving a beta-blocker in the emergency department compared with those who received their first beta-blocker in the hospital ward (P = .006). Over a median follow-up of 972 days (interquartile range, 555-1490 days), after adjusting for potential confounders, patients discharged on a beta-blocker regimen exhibited a significant reduction in cardiovascular death (hazard ratio, 0.29; 95% confidence interval, 0.09-0.98) (P = .047). CONCLUSION: beta-Blockers do not appear to be associated with adverse events in patients with chest pain with recent cocaine use.

Effect of acute cocaine use on vasospasm and outcome in aneurysmal subarachnoid hemorrhage.

BACKGROUND: Although acute cocaine use has been correlated with aneurysmal subarachnoid hemorrhage, its effect on vasospasm and outcome is controversial. We investigated the effect of acute cocaine use on response to vasospasm treatment and neurologic outcome in patients with aneurysmal subarachnoid hemorrhage. METHODS: Data from 600 patients with aneurysmal subarachnoid hemorrhage admitted to the University of Illinois Medical Center in Chicago between June 2002 and July 2007 were retrospectively analyzed. Patients who were positive for cocaine on urine toxicology or admitted to cocaine use within 72 hours of admission were compared with control patients with no history of cocaine use. Patients with unknown or remote history were excluded. RESULTS: Of the 600 patients with aneurysmal subarachnoid hemorrhage, 27 (5%) were excluded. Thirty-one patients (5%) acutely used cocaine before admission. Cocaine users were younger than control (45.1 vs 54.1; P ≤ .0003), and were more likely to smoke tobacco, drink alcohol, and have renal dysfunction. There was no significant difference in Hunt-Hess or Fisher grade. In univariate and multivariate analyses, there was no difference in unfavorable short-term outcome (modified Rankin scale > 3), incidence of symptomatic or radiologic vasospasm, stroke, or death. The number of interventional procedures for the treatment of vasospasm did not differ between the two groups. CONCLUSIONS: There is no significant difference in incidence of symptomatic vasospasm or neurologic outcome between cocaine users and nonusers. The severity of the vasospasm and the response to treatment, as indicated by the number of vasospasm interventions, did not differ between the two groups.

Oxidative damage to biological macromolecules in Prague bus drivers and garagemen: impact of air pollution and genetic polymorphisms.

DNA integrity was investigated in the lymphocytes of 50 bus drivers, 20 garagemen and 50 controls using the comet assay with excision repair enzymes. In parallel, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 15-F(2t)-isoprostane levels in the urine and protein carbonyl levels in the plasma were assessed as markers of oxidative damage to DNA, lipids and proteins. Exposure to carcinogenic polycyclic aromatic hydrocarbons (cPAHs) and volatile compounds was measured by personal samplers for 48 and 24h, respectively, before the collection of biological specimens. Both exposed groups exhibited a higher levels of DNA instability and oxidative damage to biological macromolecules than the controls. The incidence of oxidized lesions in lymphocyte DNA, but not the urinary levels of 8-oxodG, correlated with exposure to benzene and triglycerides increased this damage. Oxidative damage to lipids and proteins was associated with exposure to cPAHs and the lipid peroxidation levels positively correlated with age and LDL cholesterol, and negatively with vitamin C. The carriers of at least one variant hOGG1 (Cys) allele tended to higher oxidative damage to lymphocyte DNA than those with the wild genotype, while XPD23 (Gln/Gln) homozygotes were more susceptible to the induction of DNA strand breaks. In contrast, GSTM1 null variant seemed to protect DNA integrity.

Cocaine detection in postmortem samples following therapeutic administration.

Cocaine is one of the most widely abused drugs and one that is frequently encountered in forensic toxicology laboratories. Most often, the detection of cocaine would lead toxicologists and forensic pathologists to believe that the drug was used illicitly; however, cocaine is an effective local anesthetic and vasoconstrictor and is used clinically in surgeries of the eye, ear, nose, and throat. Therefore, it is important to note that the presence of cocaine and its metabolites in forensic samples cannot always be attributed to abuse and that a thorough investigation and review of medical records is warranted before an informed conclusion can be made. In this case report, a 54-year-old male died three days after an altercation in which he suffered multiple injuries. In addition to natural disease and injuries documented at autopsy, cocaine and its metabolites were detected in the decedent's urine, and a review of surgical records showed that earlier on the day of death, he was administered cocaine clinically during a procedure to repair nasal bone fractures. If not for this comprehensive investigation and review of surgical records, the assumption of cocaine abuse might have otherwise been made and the cause and manner of death incorrectly established.