Oxidative stress: Determination of 4-hydroxy-2-nonenal by gas chromatography/mass spectrometry in human and rat plasma.

The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography-mass spectrometry (GC-MS) method based on derivatization of HNE with pentafluorobenzyl-hydroxylamine-HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d11) as internal standard. This excellent stable and precise GC-MS method was carefully validated for HNE, and showed good linearity (r(2) = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4-6.1% and between-day precisions were 5.2-10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5-250 nmol/L) of HNE. To examine the versatility of this modified GC-MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats. Taken together, the GC-MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.

microRNA expressions in CD4+ and CD8+ T-cell subsets in autoimmune thyroid diseases.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) are the most common autoimmune thyroid diseases (AITD). MicroRNAs (miRNAs) critically control gene-expression and play an important role in regulating the immune response. The aim of this study was to prove significant variations of key immunoregulatory miRNAs in peripheral blood mononuclear cells (PBMCs) and in CD4+ and CD 8+ T-cells of AITD patients. Selected miRNAs were amplified by a semiquantitative SYBR Green PCR from PBMCs and purified CD4+ and CD 8+ T-cells of 59 patients with GD, HT, and healthy controls. Both GD and HT showed significantly decreased miRNA 200a_1 and miRNA 200a2* in CD4+-T-cells (mean relative expression 12,57 in HT vs. 19.40 in control group (CG), p=0.0002; 12,10 in GD vs. 19.40 in CG, p=0.0002) and in CD8+-T-cells (13.13 in HT vs. 18,12 in CG, p=0.02; 11.66 in GD vs. 18.12 in CG, p=0.0002). GD and HT showed significantly decreased miRNA 155_2 and miRNA 155*_1 in HT in CD8+-T-cells (10.69 in HT vs. 11.30 in CG, p=0.01; 10.40 in GD vs. 11.30 in CG, p=0.005). This study confirms significant variations of miRNA200a and miRNA155 in patients suffering from GD and HT in vivo in CD4+ T-cells and CD8+ T-cells. These data may help to better understand the gene regulations in the causative cells causing these autoimmune processes. They extend our very limited knowledge concerning miRNAs in thyroid diseases.

Treatment with the nitric oxide donor SNP increases triiodothyronine levels in hyper- and hypothyroid Sprague-Dawley rats.

Nitric oxide pathway might play a crucial role in the pathophysiology of thyroid dysfunctions. This study aimed to investigate the impact of nitric oxide (NO) on hypothyroid and hyperthyroid Sprague-Dawley rats under controlled diet. Furthermore, the effects of the nitric oxide donor sodium nitroprusside (SNP) on thyroid dysfunctions were also assessed. Sprague-Dawley rats (n=107) were subdivided into normal diet and high-fat diet (HFD) groups and grouped into controls, hypothyroid, hyperthyroid, and SNP treated groups. Hypothyroidism was induced through propylthiouracil, whereas hyperthyroidism by triiodothyronine (T3). After 12 weeks of T3 treatment, serum nitric oxides (NOX), endogenous asymmetric dimethylarginine (ADMA), body weight and food intake were analyzed. Hypothyroid rats showed decreased serum T3 levels, hyperthyroid rats increased T3 compared to controls. Diet had no impact on T3. Thyroid dysfunctions were accompanied by changes in calorie intake and body weight. Serum NOX was significantly reduced in normal diet hypothyroid rats. SNP administration compensated the decrease and markedly increased T3. NO synthase inhibitor ADMA levels were significantly higher in the HFD control group than in the normal diet controls. ADMA was declined in both hypothyroid groups and increased in normal diet hyperthyroid rats. An association of thyroid dysfunctions with reduced bioavailability of NO and alterations of ADMA levels could be established. Treatment with the NO donor SNP resulted in an increase of serum T3 levels. These results demonstrate that the NO pathway is implicated in thyroid dysfunctions, which may be of clinical relevance.

[Development of Cushing's syndrome during phytotherapy]. / Entwicklung eines Cushing-Syndroms während einer Phytotherapie.

UNLABELLED: HISTORY AND INITIAL FINDINGS: In a 75-year-old woman with unclear weight gain and typical signs of Cushing's syndrome, a pituitary microadenoma and hyperplasia of the left adrenal gland were diagnosed. She was referred for preoperative diagnostics. Her clinical appearance suggested hypercortisolism. INVESTIGATIONS: The lab test suggested external glucocorticoid application. Basal ACTH and cortisol were low. DIAGNOSIS, TREATMENT AND FURTHER COURSE: The patients' phytotherapeutics received from a masseuse were analyzed in a special lab. The analysis showed that the pills were enriched with cortisone and hydrocortisone and were causal for the development of Cushing's syndrome and the symptoms of secondary adrenal insufficiency. CONCLUSION: Symptoms of Cushing's syndrome develop during chronic exposure to glucocorticoids. The development of Cushing's syndrome depends on the patient's sensitivity and on the duration and dose of the glucocorticoid application. Clinical and laboratory studies precede imaging.

Measurement of total and free malondialdehyde by gas-chromatography mass spectrometry--comparison with high-performance liquid chromatography methology.

Malondialdehyde (MDA) is considered to be a biomarker for enzymatic degradation and lipid peroxidation of polyunsaturated fatty acids. Usually, MDA determination from different biological materials is performed by reaction with thiobarbituric acid (TBA) followed by high-performance liquid chromatography (HPLC) analysis and fluorometric detection. As this method lacks specificity and sensitivity, we developed a gas chromatography-mass spectrometry (GC-MS) method based on derivatization of MDA with 2,4-dinitrophenylhydrazine. Representative ions in negative ion chemical ionization (NICI) mode were recorded at m/z 204 for MDA and at m/z 206 for the deuterated analogon (MDA-d2) as internal standard. This stable and precise GC-MS method showed good linearity (r² = 0.999) and higher specificity and sensitivity than the HPLC method and was validated for both total MDA (t-MDA) and free MDA (f-MDA). Within-day precisions were 1.8-5.4%, between-day precisions were 4.8-9.2%; and accuracies were between 99% and 101% for the whole calibration range (0.156-5.0 µmol/L for t-MDA and 0.039-0.625 µmol/L for f-MDA). Although comparison of t-MDA levels from GC-MS and HPLC results using Passing-Bablok regression analysis as well as Bland-Altman plot showed a correlation of the data, a tendency to increased results for the HPLC values was detectable, due to possible formation of unspecific products of the TBA reaction.

MicroRNA expressions in PMBCs, CD4+, and CD8+ T-cells from patients suffering from autoimmune Addison's disease.

Autoimmune Addison's disease (AD) is a rare but potentially life threatening disease. The exact etiology of the immune response to the adrenal gland is still unknown. MicroRNAs (miRNAs) critically control gene-expression and play an important role in regulating the immune response. The aim of this study was to determine key immunoregulatory miRNAs influencing autoimmune adrenal insufficiency. For this purpose selected miRNAs were amplified by a semiquantitative SYBR Green PCR from blood mononuclear cells and after purification from CD4+ and CD 8+ cells of 6 patients with autoimmune adrenal insufficiency and 10 healthy controls. In CD4+ T-cells miRNA 181a*_1 (18.02 in AD vs. 11.99 in CG, p=0.0047) is significantly increased whereas miRNA 200a_1 (12.48 in AD vs. 19.40 in CG, p=0.0003) and miRNA 200a_2* (8.59 in AD vs. 17.94 in CG, p=0.0160) are significantly decreased. miRNA 200a_1 (12.37 in AD group vs. 18.12 in control group, p=0.001) and miRNA 200a_2* (10.72 in AD group vs. 17.84 in control group, p=0.022) are also significantly decreased in CD8+ T-cells. This study could show for the first time a significant change of three defined miRNAs in PBMCs, CD4+, and CD8+ T-cells of autoimmune AD patients in vivo. These data may help to better understand the cause of the autoimmune processes leading to autoimmune AD. They extend our very limited knowledge concerning miRNAs in autoimmune Addison's disease.

HLA-A2 phenotype may be protective against Graves' disease but not against Hashimoto's thyroiditis in Caucasians.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) are the most common autoimmune thyroid diseases (AITDs) affecting up to 5% of the general population. In Caucasians HT has a prevalence of up to 4.60% and GD a prevalence of 1-2%. The aim of this study was to investigate the association between HLA-A2 and the AITDs GD and HT among Caucasians. HLA alleles of 33 patients with GD and 75 patients with HT were determined by serological typing. The frequency of HLA A2 was significantly reduced in GD (p=0.033) but not in HT (p=n.s.) as compared to control samples. In individuals positive for HLA-A2 odds ratio for protection from GD was found to be 2.8. This study supports the hypothesis that genetic predisposition to GD is not restricted to MHC class II molecules. The significant negative association between HLA A2 and GD supports the hypothesis that MHC class I genes may be relevant for the protection from GD. In contrast the nonsignificant results for HT indicate that this association may not apply to AITDs in general.

Evidence for an exercise induced increase of TNF-α and IL-6 in marathon runners.

Regular physical activity of moderate intensity improves cardiovascular risk factors including low-grade inflammation. However, acute vigorous exercise such as marathon running results in marked increases of circulating pro-inflammatory markers. Up to now, the origin of this pro-inflammatory boost is still debated equivocally. We analyzed the change of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and leptin from pre- to immediately post-race in 15 male runners (age 43 ± 10.9 years and body mass index 24.5 ± 2.7 kg/m(2) ) both on the protein level in the plasma and on the messenger ribonucleic acid (mRNA) level in blood mononuclear cells (BMNC). We observed a significant increase of IL-6 (prerace 2.08 ± 0.10 ng/L and postrace 40.14 ± 24.85 ng/L, P < 0.001) and TNF-α (prerace 8.14 ± 1.38 ng/L and postrace 12.40 ± 3.15 ng/L, P < 0.001) and a decrease of leptin (prerace 1.64 ± 2.64 µg/L and postrace 0.80 ± 1.70 µg/L, P = 0.04) serum levels after the marathon race. Furthermore, TNF-α, IL-6, and leptin were expressed (mRNA level) in BMNC. However no significant differences in mRNA levels were seen before and after the run in these cells. We found an up-regulation of TNF-α and IL-6 in the plasma during vigorous exercise. This increase is not attributable to BMNC. We assume a local production in, or release from, exercised tissues.

Reduced urinary glutamate levels are associated with the frequency of migraine attacks in females.

BACKGROUND AND PURPOSE: Recent evidences indicate that glutamatergic homeostasis disorders are implicated in the pathogenesis of migraine. In particular, plasma and cerebrospinal fluid glutamate levels seem to be altered in migraine patients. However, the impacts of glutamate on migraine and especially on aura symptoms, alterations in the frequency of migraine attacks as well as investigations on glutamate on migraine-related metabolic dysfunctions, like hyperinsulinaemia, and an atherogenic lipid profile remain elusive to date. The aim of the present study was to investigate the impact of glutamate on migraine and related metabolic dysfunctions. METHODS: We investigated the urinary glutamate levels of female migraineurs (n = 48) in the interictal phase and healthy controls (n = 48). Parameters of the insulin- and lipid metabolism, inflammatory parameters and anthropometric parameters were additionally determined. RESULTS: Urinary glutamate levels of female migraineurs were significantly decreased with respect to the control group. Logistic regression revealed an odds ratio of 4.04 for migraine. We found a significant correlation with the time-period of patients' last attack and a significant inverse correlation with the annual frequency of migraine attacks. Other parameters of the insulin- and lipid metabolism, anthropometric and inflammatory parameters showed no significant correlation with glutamate levels. CONCLUSION: We show here that female migraineurs exhibit decreased urinary glutamate levels which are associated with a 4.04-fold higher risk for migraine and correlated with patients' frequency of migraine attacks.

Hypercortisolism caused by ritonavir associated inhibition of CYP 3A4 under inhalative glucocorticoid therapy. 2 case reports and a review of the literature.

Recent in vitro and in vivo studies have shown a potent inhibition of cytochrome P450 CYP3A4 through human immune deficiency virus (HIV) protease inhibitors (PIs). The PI ritonavir is described as the most potent compound within these CYP3A4 inhibitors. We present 2 cases who developed the sequelae of glucocorticoid excess following ritonavir therapy and inhalative glucocorticoid treatment: A 60-year-old HIV positive man developed the typical symptoms of Cushing's syndrome and a 52-year-old HIV positive man developed severe osteoporosis.

[Kallmann syndrome]. / Kallmann-Syndrom.

The Kallmann syndrome is a very rare congenital association of gonadotropin-releasing hormone deficiency and hyposmia or anosmia. Clinically it is characterized by low serum concentrations of testosterone and inadequate low levels of luteinizing hormone and follicle-stimulating hormone as well as incomplete sexual maturation, lack of secondary sexual features (facial and body hair growth, deepening of the voice), micropenis and sometimes even cryptorchidism. The reduced or absent sense of smell is typical for the Kallmann syndrome and distinguishes this syndrome from other causes of hypogonadotropic hypogonadism. Additional findings may include synkinesia, hearing loss, unilateral renal aplasia, brachy- or syndactyly, agenesis of corpus callosum, cleft palate and dental agenesis. A 19-year-old man presented to our male infertility clinic with delayed sexual maturation, eunuchoid habitus, micropenis, cryptorchidism, erectile dysfunction and absence of ejaculation, anemia and osteoporosis as well as low serum concentrations of luteinizing hormone, follicle-stimulating hormone and testosterone in combination with hyposmia.

Oxidative stress is associated with migraine and migraine-related metabolic risk in females.

BACKGROUND AND PURPOSE: Oxidative stress is discussed to be implicated in the pathophysiology of migraine. However, data are in part controversial and the possible underlying mechanisms remain elusive to date. The aim of this study was to investigate the oxidative stress status of female patients with migraine and its implications on migraine-related metabolic alterations. METHODS: Oxidative stress markers malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), carbonylated proteins, parameters of associated nitric oxide stress, inflammation, lipid- and glucose-metabolism were determined in the interictal phase in female patients with migraine and controls. RESULTS: We found significantly increased HNE levels in female migraineurs compared with controls. Logistic regression analyses of HNE revealed an odds ratio for migraine of 4.55. HNE showed significant correlations with the nitric oxide pathway, the insulin- and the lipid-metabolism. CONCLUSIONS: We show here that increased oxidative stress is associated with migraine and contributes to migraine-related metabolic risk like nitrosative stress, an atherogenic lipid profile and hyperinsulinemia. Our data suggest that oxidative stress may represent a key event in the pathophysiology of migraine and a suitable therapeutic target.

Increased matrix metalloproteinase activity is associated with migraine and migraine-related metabolic dysfunctions.

OBJECTIVE: Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are discussed to be involved in the pathophysiology of migraine. Moreover, MMPs may also be involved in migraine-related metabolic alterations like an atherogenic lipid profile and hyperinsulinemia. The aim of this study was to investigate the impact of MMPs and TIMPs on migraine with and without aura and related metabolic dysfunctions. METHODS: MMP activity, six MMPs and three TIMPs, parameters of the insulin and lipid metabolism as well as anthropometric parameters were determined in 124 non-obese subjects. RESULTS: We found highly significant increased MMP activity in migraine patients independent of aura symptoms, which was associated with migraine with an odds ratio of 7.57. Interestingly, none of the determined MMPs and TIMPs showed significant different serum levels between migraine patients and healthy controls. We found significant correlations between MMP activity and parameters of the insulin and lipid metabolism, like Homeostasis Model Assessment index (HOMA index), cholesterol, triglycerides, and oxidized LDL. CONCLUSION: We show here that increased MMP activity is tightly associated with migraine and migraine-related hyperinsulinemia and atherogenic lipid alterations. Our findings represent a new pathophysiological mechanism, which may be of clinical relevance, especially in regard to therapeutic approaches using MMP inhibitors.

Lipid profile in normal weight migraineurs - evidence for cardiovascular risk.

BACKGROUND: Recent studies suggest that migraine is associated with metabolic disorders. In particular, migraine may be associated with cardiovascular risk; however, an association of migraine with cardiovascular risk factors like hypercholesterolemia has been proposed, but previous studies have yielded in part conflicting results. The aim of the present study is to evaluate the lipid profile in normal weight migraine patients. METHODS: One hundred thirty-six probands participated in this study. The study group was divided into normal weight migraineurs and control groups, including normal weight controls, obese and overweight controls and migraineurs. Various parameters of the lipid metabolism and inflammatory parameters were investigated. RESULTS: We found significant increased cholesterol, low density lipoprotein cholesterol (LDL-C) and oxidized LDL-C in normal weight migraineurs. Increased oxidized LDL-C was associated with a 7.93-fold increased risk for migraine. Alterations in the lipid profile were not accompanied by increased inflammatory parameters. CONCLUSIONS: We show here that normal weight migraineurs exhibit independent of aura symptoms an atherogenic lipid profile, which shares common features with obesity-related lipid alterations. Our data suggest that migraine is associated with a higher risk for cardiovascular disease and its clinical consequences.

Hyperinsulinaemia in migraineurs is associated with nitric oxide stress.

There is growing evidence that alterations in the insulin and glucose metabolism may be involved in the pathogenesis of migraine. Nitric oxide (NO) stress has been associated with migraine. However, the role of NO on the insulin and glucose metabolism in migraineurs has remained elusive to date. The aim of the present study was to investigate the insulin and glucose metabolism in migraineurs and to determine possible interactions with the NO pathway. One hundred and twenty non-obese probands participated in this study, including 48 migraineurs and 72 healthy volunteers. Various parameters of the NO pathway, glucose metabolism as well as body measurement parameters were determined. We found a highly significantly increased insulin and Homeostasis Model Assessment (HOMA)-index in migraine patients, whereas fasting glucose was decreased. Logistic regression revealed an odds ratio of 5.67 for migraine, when comparing the lowest with the highest quartile of HOMA. Multivariate analysis showed that HOMA, waist-to-length ratio and nitrite as parameters of NO stress were highly significantly correlated. We show here that hyperinsulinaemia is associated with migraine and, furthermore, is correlated with increased NO stress. These findings represent a new pathophysiological mechanism that may be of clinical relevance.

Increased nitric oxide stress is associated with migraine.

Nitric oxide (NO) has been implicated in migraine attacks, but the role of NO in migraine remains unclear. We here hypothesize that increased NO in the headache-free period is associated with migraine. One hundred and thirty probands participated in this study. Various parameters of the NO pathway, such as nitrate, nitrite, arginine, citrulline, nitrosylated proteins, asymmetric dimethylarginine, symmetrical dimethylarginine, expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase and two polymorphisms of eNOS were investigated. We found significant increased nitrate and decreased nitrite levels in migraineurs in the headache-free period. Nitrate and nitrite levels showed a significant inverse correlation. Logistic regression revealed an odds ratio of 3.6 for migraine. Other parameters of the NO pathway were neither altered in migraineurs nor correlated with nitrate. We show here that migraine patients suffer under sustained increased nitrosative stress in the headache-free period, which is associated with a 3.6-fold higher risk for migraine.

The effects of intracanopy lighting on cowpea production.

Utilizing a combination of above-canopy and intracanopy lighting may prove highly beneficial in a Lunar Controlled Ecological Life Support System (LCELSS) as a means of increasing volumetric efficiency of plant growth. Intracanopy lighting was not found to be detrimental to plant tissue and production per plant when cowpeas were grown using sand culture. Specifically, intracanopy lighting did not adversely influence leaf area, dry mass, or wet mass production. Although no significant differences were found when 25% of the lighting was placed intracanopy, this is important because it indicates that there is potential in saving space for controlled growth systems. The use of intracanopy lighting would allow plant trays to be "stacked" closer, thereby increasing volumetric plant density.