A mass spectrometric multicenter study supports classification of preeclampsia as heterogeneous disorder

The diagnostic value of affinity-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis to distinguish preeclampsia (PE) from matched controls was tested in a multicenter setting.

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.

Analbuminemic Nagase rats: blood pressure response to dietary salt challenge

BACKGROUND: The role of albumin on blood pressure response to different salt challenges is not known. Therefore, we studied the blood pressure response of analbuminemic Nagase rats (NAR) to different salt challenges. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme regulating the glucocorticoid access to the mineralocorticoid receptor, an enzyme that is decreased in humans with salt sensitive hypertension and other diseases with abnormal renal salt retention, was assessed during salt challenges. METHODS: Blood pressure was measured continuously by an intra-arterial catheter and a telemetry system in NAR (n = 8). NAR were set successively for 7 days on a normal (0.45% NaCl), high (8% NaCl), low (0.1% NaCl) and normal salt diet again, to assess salt related response in mean systolic (SBP) and diastolic blood pressure (DBP). 11beta-HSD2activity was assessed by measuring the urinary (THB + 5alpha-THB)/THA ratio with gas chromatography - mass spectrometry. RESULTS: Mean SBP and DBP increased with high salt intake (normal salt vs. high salt: SBP: 114 +/- 1 vs.119 +/- 3 mm Hg, p < 0.01; DBP: 84 +/- 1 vs. 88 +/- 3 mm Hg; n = 8; p < 0.01). Urinary (THB +5alpha-THB)/THA ratio increased during the high-salt period when compared to the normal-salt period (high salt vs. normal salt: 0.52 +/- 0.10 vs. 0.37 +/- 0.07; p = 0.05) indicating decreased 11beta-HSD2activity. CONCLUSION: Analbuminemic Nagase rats express increased blood pressure and reduced 11beta-HSD2 activity in response to a high-salt diet.

Application of headspace solid phase microextraction to qualitative and quantitative analysis of tobacco additives in cigarettes

Cigarettes may contain up to 10% by weight additives which are intended to make them more attractive. A fast and rugged method for a cigarette-screening for additives with medium volatility was developed using automatic headspace solid phase microextraction (HS-SPME) with a 65 microm carbowax-divinylbenzene fiber and gas chromatography-mass spectrometry (GC-MS) with standard electron impact ionisation. In three runs, each cigarette sample was extracted in closed headspace vials using basic, acidic and neutral medium containing 0.5 g NaCl or Na2SO4. Furthermore, the method was optimized for quantitative determination of 17 frequently occurring additives. The practical applicability of the method was demonstrated for cigarettes from 32 brands.

Cerebellar cortical degeneration with selective granule cell loss in Bavarian mountain dogs

Three Bavarian mountain dogs aged between 18 and 20 months, not related to each other, were presented with chronic signs of cerebellar dysfunction. On sagittal T2-weighted magnetic resonance imaging brain images, the tentative diagnosis of cerebellar hypoplasia was established based on an enlarged cerebrospinal fluid space around the cerebellum and an increased cerebrospinal fluid signal between the folia. Post-mortem examination was performed in one dog and did show an overall reduction of cerebellar size. On histopathologic examination, a selective loss of cerebellar granule cells with sparing of Purkinje cells was evident. Therefore, the Bavarian mountain dog is a breed where cerebellar cortical degeneration caused by the rather exceptional selective granule cell loss can be seen as cause of chronic, slowly progressive cerebellar dysfunction starting at an age of several months.

Increased airway smooth muscle mass in children with asthma, cystic fibrosis, and non-cystic fibrosis bronchiectasis

RATIONALE: Structural alterations to airway smooth muscle (ASM) are a feature of asthma and cystic fibrosis (CF) in adults. OBJECTIVES: We investigated whether increase in ASM mass is already present in children with chronic inflammatory lung disease. METHODS: Fiberoptic bronchoscopy was performed in 78 children (median age [IQR], 11.3 [8.5-13.8] yr): 24 with asthma, 27 with CF, 16 with non-CF bronchiectasis (BX), and 11 control children without lower respiratory tract disease. Endobronchial biopsy ASM content and myocyte number and size were quantified using stereology. MEASUREMENTS AND MAIN RESULTS: The median (IQR) volume fraction of subepithelial tissue occupied by ASM was increased in the children with asthma (0.27 [0.12-0.49]; P < 0.0001), CF (0.12 [0.06-0.21]; P < 0.01), and BX (0.16 [0.04-0.21]; P < 0.01) compared with control subjects (0.04 [0.02-0.05]). ASM content was related to bronchodilator responsiveness in the asthmatic group (r = 0.66, P < 0.01). Median (IQR) myocyte number (cells per mm(2) of reticular basement membrane) was 8,204 (5,270-11,749; P < 0.05) in children with asthma, 4,504 (2,838-8,962; not significant) in children with CF, 4,971 (3,476-10,057; not significant) in children with BX, and 1,944 (1,596-6,318) in control subjects. Mean (SD) myocyte size (mum(3)) was 3,344 (801; P < 0.01) in children with asthma, 3,264 (809; P < 0.01) in children with CF, 3,177 (873; P < 0.05) in children with BX, and 1,927 (386) in control subjects. In all disease groups, the volume fraction of ASM in subepithelial tissue was related to myocyte number (asthma: r = 0.84, P < 0.001; CF: r = 0.81, P < 0.01; BX: r = 0.95, P < 0.001), but not to myocyte size. CONCLUSIONS: Increases in ASM (both number and size) occur in children with chronic inflammatory lung diseases that include CF, asthma, and BX.

Age-dependent decrease in 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in hypertensive patients

BACKGROUND: The prevalence of arterial hypertension lacking a defined underlying cause increases with age. Age-related arterial hypertension is insufficiently understood, yet known characteristics suggest an aldosterone-independent activation of the mineralocorticoid receptor. Therefore, we hypothesized that 11beta-HSD2 activity is age-dependently impaired, resulting in a compromised intracellular inactivation of cortisol (F) with F-mediated mineralocorticoid hypertension. METHODS: Steroid hormone metabolites in 24-h urine samples of 165 consecutive hypertensive patients were analyzed for F and cortisone (E), and their TH-metabolites tetrahydro-F (THF), 5alphaTHF, TH-deoxycortisol (THS), and THE by gas chromatography-mass spectroscopy. Apparent 11beta-HSD2 and 11beta-hydroxylase activity and excretion of F metabolites were assessed. RESULTS: In 72 female and 93 male patients aged 18-84 years, age correlated positively with the ratios of (THF + 5alphaTHF)/THE (P = 0.065) and F/E (P < 0.002) suggesting an age-dependent reduction in the apparent 11beta-HSD2 activity, which persisted (F/E; P = 0.020) after excluding impaired renal function. Excretion of F metabolites remained age-independent most likely as a consequence of an age-dependent diminished apparent 11beta-hydroxylase activity (P = 0.038). CONCLUSION: Reduced 11beta-HSD2 activity emerges as a previously unrecognized risk factor contributing to the rising prevalence of arterial hypertension in elderly. This opens new perspectives for targeted treatment of age-related hypertension.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Prematurity is related to high placental cortisol in preeclampsia

Fetal growth is compromised in animal models with high cortisol availability. In healthy pregnancies, the fetus is protected from high circulating cortisol levels by the placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which is reduced in preeclampsia. We hypothesized increased placental cortisol availability in preeclampsia as missing link to fetal growth restriction and prematurity. Placental tissue was obtained from 39 pregnant women dichotomized normotensive (n = 16) or preeclamptic (n = 23). Placental steroid hormone metabolites were analyzed by gas chromatography-mass spectrometry. Apparent 11beta-HSD2 enzyme activity was calculated as substrate to product ratio. Estradiol and pregnandiol positively correlated with gestational age. Cortisol was virtually absent in 93.8% of controls, yet detectable in 79.3% of preeclamptic samples resulting in an odds ratio (OR) of 0.019 (95% CI 0.002-0.185) for the presence of placental cortisol. Apparent 11beta-HSD2 activity directly correlated with birth weight (R2 = 0.16; p < 0.02) and gestational age (R2 = 0.11; p < 0.04) ensuing a reduced risk of premature delivery (OR 0.12; 95% CI 0.02-0.58). We conclude that normotensive pregnancies are characterized by an almost completely inactivated placental cortisol. In line with our hypothesis, reduced 11beta-HSD2 activity in preeclampsia is unable to abolish placental cortisol, a finding clearly associated with prematurity and low birth weight.

High protein intake reduces intrahepatocellular lipid deposition in humans

BACKGROUND: High sugar and fat intakes are known to increase intrahepatocellular lipids (IHCLs) and to cause insulin resistance. High protein intake may facilitate weight loss and improve glucose homeostasis in insulin-resistant patients, but its effects on IHCLs remain unknown. OBJECTIVE: The aim was to assess the effect of high protein intake on high-fat diet-induced IHCL accumulation and insulin sensitivity in healthy young men. DESIGN: Ten volunteers were studied in a crossover design after 4 d of either a hypercaloric high-fat (HF) diet; a hypercaloric high-fat, high-protein (HFHP) diet; or a control, isocaloric (control) diet. IHCLs were measured by (1)H-magnetic resonance spectroscopy, fasting metabolism was measured by indirect calorimetry, insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and plasma concentrations were measured by enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry; expression of key lipogenic genes was assessed in subcutaneous adipose tissue biopsy specimens. RESULTS: The HF diet increased IHCLs by 90 +/- 26% and plasma tissue-type plasminogen activator inhibitor-1 (tPAI-1) by 54 +/- 11% (P < 0.02 for both) and inhibited plasma free fatty acids by 26 +/- 11% and beta-hydroxybutyrate by 61 +/- 27% (P < 0.05 for both). The HFHP diet blunted the increase in IHCLs and normalized plasma beta-hydroxybutyrate and tPAI-1 concentrations. Insulin sensitivity was not altered, whereas the expression of sterol regulatory element-binding protein-1c and key lipogenic genes increased with the HF and HFHP diets (P < 0.02). Bile acid concentrations remained unchanged after the HF diet but increased by 50 +/- 24% after the HFHP diet (P = 0.14). CONCLUSIONS: Protein intake significantly blunts the effects of an HF diet on IHCLs and tPAI-1 through effects presumably exerted at the level of the liver. Protein-induced increases in bile acid concentrations may be involved. This trial was registered at www.clinicaltrials.gov as NCT00523562.

MR angiography of infrapopliteal arteries in patients with peripheral arterial occlusive disease by using Gadofosveset at 3.0 T: diagnostic accuracy compared with selective DSA

To prospectively compare the diagnostic accuracy of steady-state, high-spatial-resolution magnetic resonance (MR) angiography of the lower leg, performed with a blood pool contrast agent, with selective digital subtraction angiography (DSA) as the reference standard in patients with symptomatic peripheral arterial disease.