Maleic acid is a biomarker for maleylacetoacetate isomerase deficiency; implications for newborn screening of tyrosinemia type 1.

Dried blood spot succinylacetone (SA) is often used as a biomarker for newborn screening (NBS) for tyrosinemia type 1 (TT1). However, false-positive SA results are often observed. Elevated SA may also be due to maleylacetoacetate isomerase deficiency (MAAI-D), which appears to be clinically insignificant. This study investigated whether urine organic acid (uOA) and quantitative urine maleic acid (Q-uMA) analyses can distinguish between TT1 and MAAI-D. We reevaluated/measured uOA (GC-MS) and/or Q-uMA (LC-MS/MS) in available urine samples of nine referred newborns (2 TT1, 7 false-positive), eight genetically confirmed MAAI-D children, and 66 controls. Maleic acid was elevated in uOA of 5/7 false-positive newborns and in the three available samples of confirmed MAAI-D children, but not in TT1 patients. Q-uMA ranged from not detectable to 1.16 mmol/mol creatinine in controls (n = 66) and from 0.95 to 192.06 mmol/mol creatinine in false-positive newborns and MAAI-D children (n = 10). MAAI-D was genetically confirmed in 4/7 false-positive newborns, all with elevated Q-uMA, and rejected in the two newborns with normal Q-uMA. No sample was available for genetic analysis of the last false-positive infant with elevated Q-uMA. Our study shows that MAAI-D is a recognizable cause of false-positive TT1 NBS results. Elevated urine maleic acid excretion seems highly effective in discriminating MAAI-D from TT1.

Online detection of an emotional response of a horse during physical activity.

The objective of this research was to develop a non-invasive method to detect an emotional response of a horse to novelty during physical activity. Two horses performed 20 trials each, in which the horse's heart rate (HR) and physical activity were continuously measured. The relationship between the horse's physical activity and HR was described by a mathematical model allowing online decomposition of the horse's HR into a physical component and a component containing information about its emotional state. Exposure to the novel object resulted in an increase in the emotional component of HR, which allowed automatic detection of an emotional response of the horse in 33/40 trials. In the remaining seven trials no stable model could be built or data were missing. The results show that model-based decomposition of HR can be a useful tool for quantification of certain aspects of temperament.

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Demonstration of neuronal and extraneuronal uptake of circulating norepinephrine in the forearm.

Disturbances in peripheral norepinephrine release or removal by neuronal and extraneuronal uptake may have pathogenetic significance in cardiovascular disease states. We investigated the mechanisms of removal of norepinephrine in the forearm of healthy subjects under basal conditions, using measurements of arterial and venous plasma norepinephrine concentrations, blood pressure, heart rate, and forearm blood flow. The specific inhibitor of neuronal uptake, desipramine, was infused intra-arterially into the brachial artery of five subjects. Net norepinephrine overflow from the forearm increased markedly, revealing considerable local release of norepinephrine. Six other subjects received four intra-arterial infusions of norepinephrine, 1.18 pmol/kg/min, with various doses of desipramine and the extraneuronal uptake-inhibiting drug hydrocortisone. The forearm extraction rate for circulating norepinephrine decreased with increasing doses of desipramine (from 69.4 +/- 3.0 [SEM] to 35.3 +/- 8.4%; p less than 0.001). Increasing doses of hydrocortisone during continued inhibition of neuronal uptake resulted in decreased forearm extraction of norepinephrine (from 63.3 +/- 4.9 to 40.6 +/- 4.4%; p less than 0.01). In six other subjects who received the highest dose of hydrocortisone without concomitant inhibition of neuronal uptake, forearm extraction of norepinephrine decreased from 57.1 +/- 4.9 to 51.5 +/- 4.7% (p less than 0.05). These results suggest that neuronal uptake contributes markedly to the removal of circulating and endogenously released norepinephrine in the forearm. For circulating norepinephrine, a corticosteroid-sensitive mechanism of extraneuronal uptake was also demonstrated. These results indicate that neuronal and extraneuronal uptake can be estimated separately in this vascular bed. Similar organ-specific studies in patients may reveal disturbances in mechanisms of norepinephrine removal.

Norepinephrine removal and release in the forearm of healthy subjects.

The relevance of local removal and release of norepinephrine (NE) for antecubital venous plasma NE concentration was studied in 22 healthy subjects. Arterial and venous plasma NE and forearm blood flow were measured during intra-arterial infusion of two doses of NE, intra-arterial NE infusion with two doses of sodium nitroprusside, intravenous infusion of NE with intra-arterial infusion of four doses of sodium nitroprusside, and lower body negative pressure of -20 mm Hg for 15 minutes. The venous plasma NE concentration-time curves during the infusions of the two doses of NE indicated first-order kinetics for forearm extraction: forearm NE extraction rate during the low dose infusion was 67 +/- 4.1% (SEM) and correlated with basal forearm blood flow (r = -0.64, p less than 0.03, n = 12). Local sodium nitroprusside-induced vasodilatation during the intra-arterial and intravenous NE infusions was accompanied by dose-dependent decreases in forearm extraction rates for NE and epinephrine. During lower body negative pressure, taking into account the high basal forearm extraction rate for NE, local and systemic release of NE was indicated by increases in arterial and venous plasma and the venous-arterial plasma NE concentration difference (p less than 0.05 for all). These data show that removal of NE from forearm circulation is a process with a high extraction ratio obeying first-order kinetics and that this extraction process inversely relates to forearm blood flow. Thus, antecubital venous plasma NE is likely to be derived mainly from local release and not from the arterial plasma NE input.

Does regional norepinephrine spillover represent local sympathetic activity?

Regional spillover of norepinephrine (NE), based on isotope dilution and single-compartment steady-state kinetics, is considered one of the best parameters for estimating organ sympathetic activity. However, the effects of local changes in clearance of NE on the spillover have not yet been investigated. We studied local NE kinetics and clearance in the forearm of 10 healthy subjects using intra-arterial infusions of NE, tritiated NE, the neuronal uptake inhibitor desipramine, and tyramine, which competes with NE for the neuronal uptake carrier. Before and during complete blockade of neuronal uptake by desipramine the venous concentration-time curves for tritiated NE and for NE released by tyramine were biexponential, consistent with the presence of (at least) two compartments for circulating tritiated NE and for locally released NE. The time constants for tyramine-induced release of NE and, in the same subjects during desipramine infusion, for tritiated NE were almost equal at the same level of forearm blood flow. This argues against possible diffusion or transport differences for NE to and from the circulation and the synapse. The regional intrinsic clearance capacity (a measure of the maximal ability of an organ to irreversibly remove drug by all pathways in the absence of any flow limitations) for NE decreased in the forearm by 65% (p less than 0.01) during neuronal uptake blockade by desipramine; the forearm clearance decreased by 59% (p less than 0.001), whereas the spillover rate of NE increased from 33 +/- 5 to 63 +/- 11 pmol.min-1 (p less than 0.05). Nitroprusside-induced increments in blood flow increased the spillover of NE from 18 +/- 4 to 35 +/- 6 pmol.min-1 (p less than 0.01); the clearance of circulating NE also increased (by 58%, p less than 0.05), and the intrinsic clearance capacity remained unchanged. This demonstrates that regional spillover of NE is markedly influenced by local changes in clearance and flow. The new parameter plasma appearance rate of NE is proposed. Although also derived from isotope dilution, this parameter may better approximate the regional entry of NE into the blood pool than spillover. This is corroborated by the nonsignificant changes of plasma appearance rate of NE during our desipramine and nitroprusside infusions.

The effect of a converting enzyme inhibitor upon renal damage in spontaneously hypertensive Fawn Hooded rats.

OBJECTIVE: To determine the effect of angiotensin converting enzyme inhibition (CEI) upon renal function and the incidence of glomerulosclerosis in spontaneously hypertensive Fawn Hooded rats (FHR). DESIGN: Male FHR were treated with captopril from the age of 5 months when mild hypertension, proteinuria and glomerulosclerosis are present, and sacrificed at 12 months of age. Renal function was determined in separate groups of FHR at 6 months of age. METHODS: Proteinuria, body weight and systolic blood pressure were determined at regular intervals. Blood pressure was measured by the tail-cuff method. Kidneys were prepared for histological examination by standard methods. Renal function was determined by inulin clearance and urinary kallikrein by an amydolitic assay. RESULTS: In untreated FHR blood pressure, proteinuria and glomerulosclerosis increased with time. Captopril normalized blood pressure and stabilized proteinuria at pretreatment levels. At the end of the study, the incidence of glomerulosclerosis was significantly lower and comparable with the incidence at 5 months. Glomerular volume did not show a correlation with the incidence of glomerulosclerosis. Hemodynamic studies showed a significant increase of glomerular filtration rate in captopril-treated rats. No statistically significant effect was seen on renal plasma flow or filtration fraction. Urinary excretion of kallikrein was increased in captopril-treated rats. CONCLUSIONS: CEI is effective in protecting the kidney from structural damage in hypertensive FHR even when treatment is started under conditions of established glomerular injury. The protection given by captopril is probably related to intrarenal effects.

Binding of [3H]flunitrazepam and [3H]spiperone to melanoma cell membrane preparations.

Binding of [3H]flunitrazepam and [3H]spiperone to membrane preparations isolated by high speed centrifugation of hamster, rabbit and human melanoma cell homogenates was analyzed. All melanoma cell types expressed a high density of specific binding sites for [3H]flunitrazepam (3-4 pmol/mg protein) with a high affinity (Kd about 30 nM). This binding was independent of melanin content of cells and could be classified, based on competition experiments, as a Ro 5-4864-like binding type. Specific [3H]spiperone binding to these cell lines clearly revealed at least two types of binding sites: a low affinity, high capacity type of binding site (Kd greater than 100 nM, Bmax about 50 pmol/mg protein) and a high affinity, low capacity binding site (Kd less than 1 nm, Bmax 30 fmol/mg protein). Binding of spiperone to the low affinity, high capacity site appeared displaceable by NM 113 and dependent on melanin content of the cells and probably represents binding to melanin. Analysis of drug binding to melanoma membrane cell preparations and correlation with drug effects should include the possible involvement of binding to melanin.

Monitoring the specific activities of dopamine and its metabolites in striatum and olfactory tubercle after intravenous administration of l-[(3)H]tyrosine: Complex relations, indicating more than two compartments.

It is still a matter of debate whether in dopaminergic nerve endings dopamine (DA) is present in different functional and/or metabolic compartments. To investigate this, DA metabolism was studied in vivo by measuring the specific activity of DA and its metabolites after intravenous administration of l-[3,5-(3)H]tyrosine (200 ?Ci/rat) to freely moving animals. The incorporation of (3)H into DA and metabolites was determined in striatum and olfactory tubercle at 5, 10, 20, 40, 60 and 80 min after [(3)H]tyrosine administration. In both structures the level of [(3)H]tyrosine initially declined monoexponentially, but deviated from that pattern later on. The curves representing the formation in time of [(3)H]DA and [(3)H]metabolites were very similar in both structures, although as a whole, the levels in the olfactory tubercle were higher. The relative patterns of the specific activities of DA and those of its metabolites, a possible clue to DA compartmentation, neither indicated a clearcut metabolic one-compartment, nor a two-compartment system. The flow of radioactivity through DA metabolism could in fact only be explained by assuming more complex metabolic relations.

A procedure to measure the specific activities of dopamine and its metabolites in rat striatum, based on HPLC, electrochemical detection and liquid scintillation counting.

A procedure to determine the specific activities (s.a.) of the putative neurotransmitter dopamine (DA) and its metabolites in rat striatum is described. For this purpose 200 mu Ci L-[3,5-3H]tyrosine was injected via a jugular vein cannula into freely moving rats. Contents and radioactivity of striatal tyrosine, DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) were determined by means of HPLC, electrochemical detection (ECD) and liquid scintillation counting. In the phase system applied DA, DOPAC, 3-MT and HVA can be separated in a single run after direct injection of striatal supernatants. The selectivity of the phase system was sufficient to analyse the supernatant of two rat striata over a 150 X 4.6 mm column, even when DA turnover was strongly stimulated. Tyrosine levels and radioactivity were measured by re-analysis of the front peak with divergent mobile phase parameters. In order to demonstrate the applicability of the present procedure, the s.a. of DA and its metabolites, as found 20 min after [3H]tyrosine administration, and the effect of haloperidol thereupon, are presented.

Repeated administration of HA-966 and haloperidol to rats: similar tolerance to striatal dopamine accumulation after HA-966 challenge, but dissimilar effects on striatal [3H]spiperone binding.

Repeated administration of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) to rats induces tolerance, as shown by a decreased, drug-stimulated accumulation of dopamine (DA) in the striatum. In the present study we compared the adaptive response of the striatal dopaminergic system to repeated administration of HA-966 with the adaptive response observed after repeated haloperidol. These treatments deprive dopamine (DA) receptors from their agonist and cause a blockade of DA receptors, respectively. Tolerance to HA-966 was not accompanied by a change in the specific binding of [3H]spiperone to striatal membranes. This is in contrast to the well-documented up-regulation of DA receptors that occurs with tolerance to haloperidol. Repeated haloperidol pretreatment also diminished DA accumulation following a challenge dose of HA-966, to a similar extent as that caused by repeated pretreatment with HA-966. These similar effects of pretreatment with HA-966 or haloperidol on the response to the HA-966 challenge are in line with, and strengthen, the idea that an increased sensitivity of presynaptic DA receptors is responsible for the decreasing effect of HA-966 after its repeated administration. Haloperidol and HA-966 clearly have different effects on postsynaptic DA receptors, as is shown by their differential effects on striatal [3H]spiperone binding.

Localization of rat striatal monoamine oxidase activities towards dopamine, serotonin and kynuramine by gradient centrifugation and nigro-striatal lesions.

Subfractionation of the crude synaptosomal-mitochondrial fraction of rat striatum in a continuous sucrose gradient in a zonal rotor led to the following results. The distribution pattern of monoamine oxidase (MAO) activity towards dopamine (DA) was very similar to the pattern of MAO activity towards serotonin (5HT), but differed from the pattern of MAO activity towards kynuramine (KYN). As 5HT is specifically deaminated by MAO-A while KYN is a common MAO substrate, this supports earlier suggestions that in rat striatal preparations DA is deaminated preferentially by MAO-A. The patterns of the MAO activities towards DA and 5HT were clearly dissimilar, despite considerable overlap, to the patterns of tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) activity, both marking the presence of striatal dopaminergic synaptosomes. The peak activities were separated and all patterns were symmetrical without showing a shoulder. This indicates that rat striatal MAO activity towards DA and 5HT is not specifically or for the greater part localized in dopaminergic terminals. We also investigated the effects of electrolytic and 6-hydroxydopamine lesions of the substantia nigra, both causing extensive degeneration of striatal dopaminergic terminals as appeared from the large decrease of striatal TH and DD activity. However, neither type of lesion induced a reduction of the MAO activity towards any of the substrates used. It is concluded that the amount of MAO activity towards DA and 5HT (probably MAO-A activity) present in dopaminergic terminals is very low compared with the total activity of this enzyme in rat striatal tissue.

Subcellular fractionation of striatum: sedimentation properties of dopaminergic synaptosomes.

Linear sucrose density gradient centrifugation of a crude synaptosomal-mitochondrial preparation of rat striatum was performed at 82,500g for 7.5, 15 and 30 min and 1, 4 and 20 h. After centrifugation various marker enzyme activities were measured throughout the gradients, viz. tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) as markers of dopaminergic synaptosomes, lactate dehydrogenase (LDH) as a general synaptosomal marker and monoamine oxidase (MAO) as a mitochondrial marker. At all centrifugation times the distribution patterns of TH and DD activity coincided almost perfectly. Notable differences were found between the sedimentation properties of these TH/DD-containing particles and LDH-containing particles: TH and DD were symmetrically distributed in the gradient much sooner than LDH, at all centrifugation times the top of the TH and DD curves was lying deeper in the gradient than the highest LDH activity, and TH and DD became enriched in the gradients to a much greater extent than LDH. It is concluded that rat striatal dopaminergic synaptosomes form a relatively homogeneous population of particles sedimenting faster into the gradients than the bulk of striatal synaptosomes does. This distinct sedimentation behaviour of the dopaminergic synaptosomes can be usefully applied for analytical purposes.

Neuronal and extraneuronal uptake of norepinephrine in the human forearm.

The effect of the neuronal uptake (U1) inhibiting drug desmethylimipramine (DMI) and the extraneuronal uptake (U2) inhibiting drug hydrocortisone (HC) upon the forearm extraction (FE) of norepinephrine (NE) was investigated in six healthy male subjects. Four infusions of NE 0.2 ng/kg per min were given intra-arterially (i.a.) concomitantly with three doses of DMI and subsequently with three doses of HC combined with one dose of DMI. All doses of DMI and the highest two doses of HC decreased the FE of NE significantly. As a result of local U1 and possibly also U2 inhibition, the venous-arterial plasma NE difference over the forearm increased between the start of the first infusion and the start of the last infusion, indicating an important degree of local neuronal release of NE in basal conditions. It is concluded that the extensive removal of NE by U1 and U2, and the local release of NE limit the use of peripheral venous NE concentration as a measure of overall sympathetic activity.

Effect of propofol on peripheral vascular resistance during cardiopulmonary bypass.

Twenty-eight patients undergoing elective coronary artery bypass surgery were allocated randomly to receive either propofol 2 mg kg-1 or an equivalent volume of its vehicle during cardiopulmonary bypass with constant pump flow. Peripheral vascular resistance (PVR) was calculated from perfusion pressure and pump flow. After propofol, PVR decreased from 1767 (SD 415) dyn s cm-5 to a minimum of 1263 (283) dyn s cm-5 at 2 min, and remained significantly less than the control value until 12.5 min after administration of propofol. In the group given the vehicle, PVR did not change significantly. In a second study in 10 patients, venous blood samples were withdrawn before and 2, 4, 6, 8, 10, 20 and 30 min after injection of propofol 2 mg kg-1 during cardiopulmonary bypass, for measurement of blood concentrations of propofol. Concentrations were greater than predicted by a computer simulation based on published pharmacokinetic data. The decrease in PVR may be an important factor in the hypotension caused by propofol during induction of anaesthesia.

Tritium isotope effect in reversed-phase high-performance liquid chromatographic analysis of dopamine and dihydroxyphenylacetic acid.

A tritium isotope effect has been demonstrated in the high-performance liquid chromatographic analysis of dopamine and its acidic metabolite dihydroxyphenylacetic acid. The chromatographic system consisted of tributyl-n-phosphate, bound to a ChromSpher C8 column, as stationary phase, and a citrate buffer, containing the ion-pairing agent perchlorate, as the mobile phase. For detection we used continuous electrochemical monitoring (for the total amount of solutes) and discontinuous liquid scintillation counting (for radiolabelled molecules) of the column effluent. [3H]Dopamine and [3H]dihydroxyphenylacetic acid were biosynthesized by incubation of homogenates of striatal tissue from rat brains with 3H-labelled L-tyrosine. The tritium-labelled compounds were eluted before the corresponding unlabelled analogues. The capacity factor reduction increased with the number of tritium atoms incorporated in the molecules: for single, double and triple tritium-labelled dopamine the separation factors amounted to 1.015, 1.028 and 1.033, respectively. No isotope separation was observed for 7-14C-labelled dopamine and dihydroxyphenylacetic acid. The isotope effect observed is ascribed to a decrease in lipophilicity following tritium substitution for hydrogen.

Determination of dopamine and its acidic metabolites in brain tissue by HPLC with electrochemical detection in a single run after minimal sample pretreatment.

A method, based on reverse-phase liquid-liquid chromatography, has been developed for the determination, in a single run, of dopamine (DA) and its acidic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), combined with electrochemical detection (ECD). If applied to brain tissue, sample pretreatment can be reduced to centrifugation, filtration and adjustment of pH and perchlorate concentration prior to introduction into the liquid chromatograph. The relation between the perchlorate (counterion) concentration of the mobile phase and the retention (k') of the amines is linear, as is the relation between the H+ concentration of the mobile phase and the retention of the acidic metabolites. This flexible phase system, combined with a simple and therefore reproducible sample pretreatment, warrants a high throughput of samples. The procedure offers good possibilities for routine analysis of catecholamines and their acidic metabolites in the picogram range. Some typical examples of the behaviour of this phase system and the electrochemical detector are presented and discussed.

Beta 1-adrenoceptor selectivity of single oral doses of bisoprolol and atenolol.

The hemodynamic and biochemical properties of bisoprolol, a new highly beta 1-adrenoceptor selective blocker, were compared with those of atenolol. The studies were performed in eight healthy volunteers and consisted of two separate parts. In the first part the effects of oral doses of 20 mg bisoprolol, 100 mg atenolol, and placebo, given in a double-blind randomized order, on the hemodynamic and biochemical changes induced by multiple doses of intravenous isoproterenol and bicycle exercise were investigated. Blood pressure and heart rate and the plasma concentrations of magnesium, potassium (K+), glucose, lactate, renin activity, norepinephrine, and epinephrine were determined. In the second part the effects of the same oral doses of bisoprolol and atenolol on changes in forearm blood flow (FBF) induced by intra-arterial (i.a.) infusions of isoproterenol and epinephrine were investigated. After both beta blockers approximately 10-times higher doses of isoproterenol i.v. were needed to obtain similar hemodynamic responses as after placebo. The hemodynamic and biochemical changes during isoproterenol i.v. and bicycle exercise were almost identically influenced by bisoprolol and atenolol. However, after bisoprolol, isoproterenol lowered plasma K+ more (p less than 0.02) and the exercise-induced increase in renin activity was less (p less than 0.02) than after atenolol. The effect of isoproterenol i.a. on FBF was attenuated to the same extent by bisoprolol and atenolol. The vasodilatory component of epinephrine i.a. was reduced only by atenolol (p less than 0.05). It is concluded that bisoprolol and atenolol are selective beta 1 antagonists. In the doses used, bisoprolol showed only slightly more potency and selectivity for beta 1 adrenoceptors than atenolol.