Karoline Vrecko

Monoaminergic neurotransmitters, their precursors and metabolites in brains of Alzheimer patients

The catecholamines dopamine (DA), noradrenaline (NA) and adrenaline (A), their aminoacid precursors tyrosine (Tyr), L-3,4-dihydroxyphenylalanine (L-DOPA), two of their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxy phenyl glycol (MHPG), serotonin (5-HT) and its precursor tryptophan (Trp), were measured by high pressure liquid chromatography (HPLC) with electrochemical detection in seven regions (globus pallidus, putamen, nucleus amygdalae, nucleus caudatus, substantia nigra, gyrus cinguli and raphe) of postmortem brains from eight histologically verified cases with Alzheimers disease (AD) and six histologically normal controls. Concentrations of L-DOPA, DA, DOPAC, NA and 5-HT were significantly reduced, while Tyr and MHPG concentrations were significantly increased in AD versus control patients. The concentrations of Trp and A in AD patients were not significantly different from controls. Furthermore, for most brain regions examined, significant negative correlations between Tyr and DA as well as between NA and MHPG levels were found. These data confirm and extend findings of monoaminergic systems disturbances in AD, emphasize the significance of dopaminergic deficit for AD and suggest that in pharmacotherapy of AD, attempts to restore deficits of the transmitter systems should be directed to the monoaminergic, in particular the dopaminergic system.

Hyperhomocysteinemia in dementia

Summary. Hyperhomocysteinemia is a strong risk factor for atherosclerotic vascular disease, and elevated serum homocysteine is correlated with vitamin B deficiency. In this pilot study, significantly elevated homocysteine levels were found in patients with Alzheimers disease as well as in patients with vascular dementia, probably indicating similar pathophysiological pathways. We found significant correlations between low folic acid concentrations as well as high homocysteine concentrations and cognitive decline. Supplementation with folic acid may be an inexpensive way to reduce elevated homocysteine levels in demented patients.

NADH stimulates endogenous dopamine biosynthesis by enhancing the recycling of tetrahydrobiopterin in rat phaeochromocytoma cells

Treatment of Parkinson patients with L-DOPA (3,4-dihydroxy-L-phenylalanine) leads to endproduct inhibition of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis and the enzyme needing tetrahydrobiopterin and iron as cofactors. To overcome this problem an alternative treatment was investigated which attempted to stimulate endogenous dopamine biosynthesis. Incubation of rat PC 12 cells with NADH (beta-nicotinamide adeninedinucleotide) leads to increased dopamine production. We investigated the possibility that this increase of dopamine biosynthesis was due to stimulation of quinonoid dihydropteridine reductase, the enzyme which recycles the inactive dihydrobiopterin to the active tetrahydrobiopterin. The experiments showed that whereas NADH is able to increase dopamine production in PC 12 cells (rat phaeochromocytoma cells, clone PC 12) up to three-fold, no influence is exerted by NADH on pteridine metabolism; neither are tetrahydrobiopterin concentrations nor the de novo-biosynthesis of pteridines from guanosine triphosphate altered by NADH. Further no influence of NADH on protein de novo synthesis of quinonoid dihydropteridine reductase was observed. However, NADH was able to directly increase the catalytic activity of this enzyme. Our results suggest that the stimulation of dopamine biosynthesis by NADH is due to more rapid regeneration of quinonoid dihydrobiopterin to tetrahydrobiopterin.

NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATP

The aim of this study was to investigate the effect of nicotinamide‐adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea‐pig ventricular cardiomyocytes. The pinacidil‐primed ATP‐dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. Membrane currents were studied using the patch‐clamp technique in the whole‐cell recording mode at 36–37°C. Adenine nucleotides were determined by HPLC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPi) IK(ATP) did not contribute to basal electrical activity. The ATP‐dependent potassium (K(ATP)) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]i. Supplementation of cardiomyocytes with 300 μg ml−1 NADH (4–6 h) resulted in a significantly reduced IK(ATP) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF−1 (n=19; P<0.05). Equimolar amounts of the related compounds nicotinamide and NAD+ did not achieve a similar effect like NADH. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg−1 protein versus control: 35.4±2.57 nmol mg−1 protein, P<0.000005). These data show that supplementation of guinea‐pig ventricular cardiomyocytes with NADH results in a decreased activation of IK(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.

Periodontitis and concentrations of the cellular immune activation marker neopterin in saliva and urine

Neopterin concentrations in diverse body fluids provide a well established indication for activation of the cell-mediated immune system. Neopterin concentrations were measured in the saliva and urine of 29 patients with varying numbers of teeth affected by periodontitis. While neopterin concentrations in urine increased slightly but not significantly in parallel with increasing numbers of affected teeth, salivary neopterin levels showed a significant and positive correlation with number of diseased teeth (linear correlation coefficient = 0.48, P = 0.012). Additionally, when the patients were grouped according to the median number of affected teeth (20), salivary specimens of subjects with one to 20 affected teeth showed significantly lower neopterin concentrations than specimens from those with more than 20 diseased teeth (P = 0.0045, Students t-test). In contrast, urinary neopterin concentrations did not differ significantly among these two groups. This pilot study suggests that salivary neopterin concentrations may reflect local immune activation even in situations where no systemic activation can be detected.

Stimulation of dopamine biosynthesis in cultured PC 12 phaeochromocytoma cells by the coenzyme nicotinamide adeninedinucleotide (NADH)

SummaryThe activity of the tyrosine hydroxylase, the enzyme which is diminished in the brains of Parkinson patients, has been measured in cultured PC 12 rat phaeochromocytoma cells. In the same way dopamine content in the medium after incubating these cells with or without NADH was assayed. The experiment shows that NADH is able to increase the activity of the tyrosine hydroxylase and dopamine — production in PC 12 cells up to 6 times.The results provide evidence that NADH is able to stimulate dopamine — biosynthesis directly.

Coagulation Changes during Presyncope and Recovery

Orthostatic stress activates the coagulation system. The extent of coagulation activation with full orthostatic load leading to presyncope is unknown. We examined in 7 healthy males whether presyncope, using a combination of head up tilt (HUT) and lower body negative pressure (LBNP), leads to coagulation changes as well as in the return to baseline during recovery. Coagulation responses (whole blood thrombelastometry, whole blood platelet aggregation, endogenous thrombin potential, markers of endothelial activation and thrombin generation), blood cell counts and plasma mass density (for volume changes) were measured before, during, and 20 min after the orthostatic stress. Maximum orthostatic load led to a 25% plasma volume loss. Blood cell counts, prothrombin levels, thrombin peak, endogenous thrombin potential, and tissue factor pathway inhibitor levels increased during the protocol, commensurable with hemoconcentration. The markers of endothelial activation (tissue factor, tissue plasminogen activator), and thrombin generation (F1+2, prothrombin fragments 1 and 2, and TAT, thrombin-antithrombin complex) increased to an extent far beyond the hemoconcentration effect. During recovery, the markers of endothelial activation returned to initial supine values, but F1+2 and TAT remained elevated, suggestive of increased coagulability. Our findings of increased coagulability at 20 min of recovery from presyncope may have greater clinical significance than short-term procoagulant changes observed during standing. While our experiments were conducted in healthy subjects, the observed hypercoagulability during graded orthostatic challenge, at presyncope and in recovery may be an important risk factor particularly for patients already at high risk for thromboembolic events (e.g. those with coronary heart disease, atherosclerosis or hypertensives).

Collagen/endogenous thrombin-induced platelet aggregation in cord versus adult whole blood.

Background: In previous studies, neonatal platelets have been shown to be hypoaggregable to various agonists when compared with adult platelets. Objectives: It was the aim of this study to investigate the aggregability of neonatal versus adult platelets when the physiological relevant agonist collagen/endogenous thrombin is used. Methods and Results: Whole blood (WB) aggregation experiments employing the impedance method revealed the same responsiveness of neonatal and adult platelets to collagen/endogenous thrombin. Maximum aggregation (13.5 ± 3.2 vs. 13.6 ± 3.2 Ω; p = 0.94), slope (5.8 ± 1.8 vs. 6.2 ± 2.6 Ω/min; p = 0.79) and lag time until the onset of platelet aggregation (38.7 ± 8.9 vs. 42.6 ± 16.5 s; p = 0.59) were similar in cord and adult WB. However, the rise in serotonin plasma levels due to platelet activation was significantly lower in neonates versus adults (227.57 ± 57.65 vs. 473.34 ± 155.75 ng/ml; p = 0.0001). Furthermore, we found a fast capability of cord plasma to generate (the efficient platelet agonist) endogenous thrombin: thrombin generation started significantly earlier in cord compared with adult plasma (215 ± 19 vs. 247 ± 21 s; p = 0.01). Moreover, thrombelastometry revealed significantly shorter coagulation times in cord versus adult WB activated with collagen/endogenous thrombin (229.8 ± 12.5 vs. 256.3 ± 25.3 s; p = 0.003). Conclusions: The efficient platelet aggregation in cord WB provoked by collagen/endogenous thrombin might help to explain the clinically observed well-functioning primary hemostasis of neonates.

The coenzyme nicotinamide adenine dinucleotide (NADH) improves the disability of Parkinsonian patients

SummaryThe coenzyme nicotinamide adenine dinucleotide (NADH) has been used in an open label trial as novel medication in 34 patients with Parkinsons disease, using an intravenous administration technique. In all patients a beneficial clinical effect was observed. 21 patients (61.7%) showed a very good (better than 30%) improvement of disability, 13 patients (38.3%) a moderate (up to 30%) improvement. Concomitant with the improvement of the disability the urine level of homovanillic acid (HVA) increased significantly in all patients (in some patients by more than a 100%). The daily “on phases” of the patients could be increased from 2 up to 9 hours in the individual patients by NADH administration.

Effect of a high fat diet on plasma lipids, lipoprotein lipase, lecithin:cholesterol acyltransferase, and insulin function in adult rabbits

Abstract The influence of a high fat diet (19% wt./wt.) vs. a standard, chow diet (2% fat) on plasma lipids, lipoprotein lipase (LPL), lecithin:cholesterol acyltransferase (LCAT), glucose, and glucose tolerance, was investigated. Both diets had a similar fatty acid pattern and a polyunsaturated:saturated (P:S) fatty acid ratio of 2.7. The high fat diet elevated plasma triglycerides, phospholipids, and cholesterol concentrations and changed the percent distribution of cholesterol and phospholipids among the lipoprotein fractions. Additionally, LCAT and plasma glucose increased, while lipoprotein lipase and its products, free fatty acids and glycerol, were not altered. Glucose tolerance was significantly inhibited in animals on the high fat diet, which also exhibited a diminished insulin secretion. As such, the high fat diet seemed to evoke a diabetogenic situation. The return to the standard chow diet appeared to normalize, to a great extent, the alterations evoked by the high fat diet.