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This mini-review summarizes studies my associates and I carried out that are relevant to the topic of the present volume [i.e. glutamate dehydrogenase (GDH)] using radioactive (13)N (t(1/2) 9.96 min) as a biological tracer. These studies revealed the previously unrecognized rapidity with which nitrogen is exchanged among certain metabolites in vivo. For example, our work demonstrated that (a) the t(1/2) for conversion of portal vein ammonia to urea in the rat liver is ∼10-11s, despite the need for five enzyme-catalyzed steps and two mitochondrial transport steps, (b) the residence time for ammonia in the blood of anesthetized rats is ≤7-8s, (c) the t(1/2) for incorporation of blood-borne ammonia into glutamine in the normal rat brain is <3s, and (d) equilibration between glutamate and aspartate nitrogen in rat liver is extremely rapid (seconds), a reflection of the fact that the components of the hepatic aspartate aminotransferase reaction are in thermodynamic equilibrium. Our work emphasizes the importance of the GDH reaction in rat liver as a conduit for dissimilating or assimilating ammonia as needed. In contrast, our work shows that the GDH reaction in rat brain appears to operate mostly in the direction of ammonia production (dissimilation). The importance of the GDH reaction as an endogenous source of ammonia in the brain and the relation of GDH to the brain glutamine cycle is discussed. Finally, our work integrates with the increasing use of positron emission tomography (PET) and nuclear magnetic resonance (NMR) to study brain ammonia uptake and brain glutamine, respectively, in normal individuals and in patients with liver disease or other diseases associated with hyperammonemia., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Introduction: Normal pregnancy is associated with a local hypercoagulable state that becomes more profound in certain obstetric complications such pre-eclampsia (P-EC). Current literature on the levels of individual haemostatic factors in women with P-EC is limited and results are inconsistent. In this study we provide detailed investigation on the tissue factor (TF)-dependent pathway in women with P-EC., Materials and Methods: Enzyme-linked immunosorbent assays (ELISA) were used to measure plasma factor (F) FVII, FVIIa, TF and tissue factor pathway inhibitor (TFPI) in healthy non-pregnant women (n = 22), normal pregnant women (n = 15), and women with P-EC (n = 20). All subjects were age matched. In addition, pregnant women were matched for gestational age, parity and were all at the third trimester., Results: Plasma FVII levels were significantly higher in women with P-EC compared to the healthy non-pregnant (P<0.001) or the normal pregnant groups (P<0.001). No such significant trends were observed for plasma FVIIa, TF or TFPI levels. Plasma FVII levels can distinguish women with P-EC from healthy non-pregnant women or normal pregnant women at the third trimester, with high sensitivity (90%), specificity (80%), positive and negative predictive values (86%)., Conclusions: Plasma FVII levels are significantly elevated in women with P-EC, in the absence of comparable changes in other TF-dependent pathway factors (FVIIa, TF and TFPI). We propose the use of plasma FVII as a marker for P-EC., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Introduction: Tissue factor (TF) is the main physiological initiator of blood coagulation; it is membrane-bound on monocytes (mTF) and free in plasma (pTF). Abnormal expression of TF by monocytes has been implicated in various diseases. We therefore quantified monocytes expressing TF and pTF levels in patients with lower-limb deep venous thrombosis (DVT)., Materials and Methods: DVT was confirmed by Duplex Scan. Blood mTF levels under resting condition (baseline), after incubation without (unstimulated) and with (stimulated) lipopolysaccharide (LPS), and total mTF levels were determined by flow cytometry using two analytical methods (Histogram and Quadrant-Statistics). Plasma TF levels were measured using an enzyme-linked immunoabsorbent assay (ELISA). Results were compared with age-matched controls., Results: Histogram analysis in patients with DVT showed significantly elevated mTF levels for baseline, unstimulated and total mTF over controls. For Quadrant-Statistics, DVT patients also showed significantly raised baseline, unstimulated, stimulated and total mTF. Similarly, pTF levels were significantly raised in subjects with DVT compared to controls. Baseline mTF levels correlated with pTF levels by Histogram and Quadrant-Statistics analysis. Using the relative operating characteristic (ROC) curve, baseline mTF and pTF assays displayed sensitivity and specificity in detecting DVT. Quadrant-Statistics baseline mTF and pTF gave the best discrimination., Conclusions: The TF assays used in this study showed acceptable sensitivity and specificity and are cost-effective and practical. Therefore, they should be considered in patients with, or at risk of, DVT.

Bolus injection of [13N]ammonia into the femoral vein of pentobarbital-anesthetized rats was followed by rapid clearance from the blood and first-pass extraction of nearly 30% by the lungs. Of the label present in the lungs at 6 s after injection (about 27% of the dose), more than 20% was in metabolized form. Of the label present in the lungs at 2 min after injection (about 10% of the dose), 18-25% was in ammonia, about 75% was in glutamine (amide) and less than 1% was in glutamate and aspartate. Thus, despite the presence of significant amounts of glutamate dehydrogenase, the overwhelming route for metabolism of ammonia entering the rat lung in vivo was the glutamine synthetase reaction. Lung tissue that was removed 6 s after intravenous injection of [13N]ammonia and incubated in Krebs-Ringer glucose medium at 37 degrees C for 20 min, showed a significant increase (more than one-third), compared to unincubated lung tissue in the quantity of label in glutamine. Between 6s and 2 min after injection, during which time the total 13N content of the lungs decreased by more than 60%, the maintenance of a quasi-steady state in the concentration of labeled glutamine suggested a short-term balance between formation from extracted ammonia and loss of glutamine into the circulation. Our data support the concept that the lungs are a source of circulating glutamine in the rat. Despite the large fractional extraction of blood-borne [13N]ammonia by the lungs, only minute amounts of tracer (0.2-0.6 ppm of the injected dose) were detected in the expired air within the first 5 min after administration of [13N]ammonia to anesthetized rats, so that pulmonary excretion was not a significant pathway of ammonia elimination. The present findings emphasize the importance of the lungs in the maintenance of whole-body nitrogen homeostasis and suggest the use of [13N]ammonia and 13N-labeled amino acids as non-invasive probes in the study of normal and diseased lung metabolism.

Glutamine transaminase K (GTK), which is a freely reversible glutamine (methionine) aromatic amino acid aminotransferase, is present in most mammalian tissues, including brain. Quantitatively, the most important amine donor in vivo is glutamine. The product of glutamine transamination (i.e., alpha-ketoglutaramate; alphaKGM) is rapidly removed by cyclization and/or conversion to alpha-ketoglutarate. Transamination is therefore "pulled" in the direction of glutamine utilization. Major biological roles of GTK are to maintain low levels of phenylpyruvate and to close the methionine salvage pathway. GTK also catalyzes the transamination of cystathionine, lanthionine, and thialysine to the corresponding alpha-keto acids, which cyclize to ketimines. The cyclic ketimines and several metabolites derived therefrom are found in brain. It is not clear whether these compounds have a biological function or are metabolic dead-ends. However, high-affinity binding of lanthionine ketimine (LK) to brain membranes has been reported. Mammalian tissues possess several enzymes capable of catalyzing transamination of kynurenine in vitro. Two of these kynurenine aminotransferases (KATs), namely KAT I and KAT II, are present in brain and have been extensively studied. KAT I and KAT II are identical to GTK and alpha-aminoadipate aminotransferase, respectively. GTK/KAT I is largely cytosolic in kidney, but mostly mitochondrial in brain. The same gene codes for both forms, but alternative splicing dictates whether a 32-amino acid mitochondrial-targeting sequence is present in the expressed protein. The activity of KAT I is altered by a missense mutation (E61G) in the spontaneously hypertensive rat. The symptoms may be due in part to alteration of kynurenine transamination. However, owing to strong competition from other amino acid substrates, the turnover of kynurenine to kynurenate by GTK/KAT I in nervous tissue must be slow unless kynurenine and GTK are sequestered in a compartment distinct from the major amino acid pools. The possibility is discussed that the spontaneous hypertension in rats carrying the GTK/KAT I mutation may be due in part to disruption of glutamine transamination. GTK is one of several pyridoxal 5'-phosphate (PLP)-containing enzymes that can catalyze non-physiological beta-elimination reactions with cysteine S-conjugates containing a good leaving group attached at the sulfur. These elimination reactions may contribute to the bioactivation of certain electrophiles, resulting in toxicity to kidney, liver, brain, and possibly other organs. On the other hand, the beta-lyase reaction catalyzed by GTK may be useful in the conversion of some cysteine S-conjugate prodrugs to active components in vivo. The roles of GTK in (a) brain nitrogen, sulfur, and aromatic amino acid/kynurenine metabolism, (b) brain alpha-keto acid metabolism, (c) bioactivation of certain electrophiles in brain, (d) prodrug targeting, and (e) maintenance of normal blood pressure deserve further study.

Protein aggregates are a hallmark of Huntington's disease (HD) and other inherited neurodegenerative diseases caused by an elongated (CAG)(n) repeat in the genome and to a corresponding increase in the size of the Q(n) domain in the expressed protein. When the protein associated with HD (huntingtin) contains <35 Q repeats disease does not occur. However, an n>/=40 leads to disease. Some investigators have proposed that aggregates in the nuclei of affected cells are toxic, but other workers have suggested that the aggregates may be neutral or even protective. Whether or not they are toxic, an understanding of the processes whereby the aggregates develop may shed light on the neuropathological processes involved in the (CAG)(n)/Q(n)-expansion disorders. Q(n) domains have a tendency to non-covalently self align as 'polar zippers' rendering them less soluble, but evidence that such polar zippers occur in the aggregates in intact HD brain has so far been limited. The human brain contains at least three Ca(2+)-dependent enzymes (transglutaminases, TGases) that catalyze protein cross-linking reactions, namely TGase 1, TGase 2 (tissue transglutaminase, tTGase) and TGase 3. Q(n) aggregates have been found by several groups to be excellent substrates of tTGase. Moreover, the activity toward the Q(n) domains increases greatly as n is increased to 40 or beyond. tTGase mRNA and total TGase activity are elevated in HD brain. Moreover, some evidence suggests that Ca(2+) homeostasis is disrupted in HD brain. We propose that the combination of increased huntingtin (or huntingtin fragment containing the Q(n) domain) in the nucleus, increased the ability of the Q(n) domains to act as substrate, increased Ca(2+) levels and increased inherent TGase activity all contribute to increased cross-linking of proteins in HD brain. At first the proteasome machinery can recognize and degrade the cross-linked proteins, but over time the proteasome machinery may be overwhelmed and protein aggregates will accumulate.

The modulation of GABA release within the globus pallidus (GP) by dopamine was studied using whole-cell patch clamp recordings from visually identified neurones. In sagittal slices, single shock electrical stimulation in the striatum evoked GABA(A) inhibitory postsynaptic currents (IPSCs), which were inhibited by dopamine in a dose-dependent manner (0.3-30 microM) with an IC(50) value of 0.7 microM. The inhibition was accompanied by an increase in paired pulse facilitation, indicative of a presynaptic effect. In coronal slices, stimulation within the GP adjacent to the recording site evoked GABA(A) IPSCs which were relatively unaffected by dopamine indicating the lack of modulation of GABA release from terminals of local GP axon collaterals. No consistent changes in holding current, membrane potential, firing rate or the frequency of spontaneous IPSCs was observed.Tetrodotoxin-resistant miniature (m)IPSCs were recorded in chloride-loaded cells. Dopamine (3-30 microM) reduced the frequency of mIPSCs, but was without effect on mIPSC amplitude, confirming a presynaptic effect. The addition of the "D2 like" agonist quinpirole (3 microM), but not the "D1 like" agonist SKF 38393 (10 microM), mimicked these effects. The "D2 like" antagonist sulpiride (10 microM), while having no effect alone, blocked the action of dopamine. In contrast the dopamine D4 selective antagonist L745, 870 (1 microM) or D1 antagonist SCH 23390 (10 microM) were without effect. These results indicate that dopamine acts on presynaptic D2 receptors on striatopallidal terminals to reduce the release of GABA in the GP. Attenuation of this mechanism following the depletion of dopamine may contribute to the changes in GP neuronal activity observed in animal models of Parkinson's disease.

The different types of striatal neuron show a range of vulnerabilities to a variety of insults. This can be clearly seen in Huntington's disease where a well mapped pattern of pathological events occurs. Medium spiny projection (MSP) neurons are the first striatal cells to be affected as the disease progresses whilst interneurons, in particular the NADPH diaphorase positive ones, are spared even in the late stages of the disease. The MSP neurons themselves are also differentially affected. The death of MSP neurons in the patch compartment of the striatum precedes that in the matrix compartment and the MSP neurons of the dorsomedial caudate nucleus degenerate before those in the ventral lateral putamen. The enkephalin positive striatopallidal MSP neurons are also more vulnerable than the substance P/dynorphin MSP neurons. We review the potential causes of this selective vulnerability of striatopallidal neurons and discuss the roles of endogenous glutamate, nitric oxide and calcium binding proteins. It is concluded that MSP neurons in general are especially susceptible to disruptions of cellular respiration due to the enormous amount of energy they expend on maintaining unusually high transmembrane potentials. We go on to consider a subpopulation of enkephalinergic striatopallidal neurons in the rat which are particularly vulnerable. This subpopulation of neurons readily undergo apoptosis in response to experimental manipulations which affect dopamine and/or corticosteroid levels. We speculate that the cellular mechanisms underlying this cell death may also operate in degenerative disorders such as Huntington's disease thereby imposing an additional level of selectivity on the pattern of degeneration. The possible contribution of the selective death of striatopallidal neurons to a number of clinically important psychiatric conditions including obsessive compulsive disorders and Tourette's syndrome is also discussed.

L-[13N]Tyrosine and L-[13N]phenylalanine were synthesized using immobilized enzymes by two methods. In method 1, [13N]ammonia is converted to L-[13N]glutamate; transamination with p-hydroxyphenylpyruvate yields L-[13N]tyrosine. [13N]Tyrosine is separated from other labeled intermediates on a Poropak Q column. In method 2, phenylalanine dehydrogenase catalyzes the reversible reductive [13N]amination of either phenylpyruvate or p-hydroxyphenylpyruvate to form L-[13N]phenylalanine or L-[13N]tyrosine, respectively. The feasibility of labeling DOPA and tryptophan with 13N was also demonstrated.

Twelve male patients, with a primary complaint of premature ejaculation in a setting of chronic anxiety with prominent somatic manifestations, participated in a double-blind trial: propranolol against placebo. The study consisted of 5 X 4 week phases: run-in, propranolol or placebo--120 mg/day allocated randomly, wash-out; placebo or propranolol and run-out, in a balanced design. Anxiety was rated initially, and every 2 weeks, throughout the trial using the Hamilton Rating Scale. Sitting blood pressure and pulse were also noted. The time to coital ejaculation (every 3 days) was recorded using a stopwatch, and subjects were also required to rate "overall coital satisfaction" and "quality of erection". Neither prematurity nor other signs/symptoms of anxiety improved on the preparations, which were statistically equivalent. Moderate beta-blockade was achieved with propranolol as evidenced by a median reduction in pulse rate of 5 beats/min.

The VCD is a mechanical device that produces an erection by creating a vacuum of up to 250 mm Hg. Up to now, it has been used in patients with organic impotence (mainly diabetic) with a high level of success and minimal side effects. This study reports preliminary findings on the application of the VCD in both organic (e.g. diabetics) and psychogenic cases of impotence. All subjects were able to develop an adequate erection with the instrument making intercourse possible. However, the frequency of use dropped sharply as the study progressed and some subjects and their sexual partners expressed reservations about continuing with it. Mild numbness in the penis was experienced by several users.