HPTLC-FLD-SERS as a facile and reliable screening tool: Exemplarily shown with tyramine in cheese

The serious cytotoxicity of tyramine attracted marked attention as it induced necrosis of human intestinal cells. This paper presented a novel and facile high performance thin-layer chromatography (HPTLC) method tailored for screening tyramine in cheese. Separation was performed on glass backed silica gel plates, using methanol/ethyl acetate/ammonia (6/4/1 v/v/v) as the mobile phase. Special efforts were focused on optimizing conditions (substrate preparation, laser wavelength, salt types and concentrations) of surface enhanced Raman spectroscopy (SERS) measurements directly on plates after derivatization, which enabled molecule-specific identification of targeted bands. In parallel, fluorescent densitometry (FLD) scanning at 380</400 nm offered satisfactory quantitative performances (LOD 9 ng/zone, LOQ 17 ng/zone, linearity 0.9996 and %RSD 6.7). Including a quick extraction/cleanup step, the established method was successfully validated with different cheese samples, both qualitatively (straightforward confirmation) and quantitatively (recovery rates from 83.7 to 108.5%). Beyond this application, HPTLC-FLD-SERS provided a new horizon in fast and reliable screening of sophisticated samples like food and herb drugs, striking an excellent balance between specificity, sensitivity and simplicity.     

Head-twitch response induced by tyramine

The intracerebroventricular (IC) administration of tyramine (TyA) induced a characteristic head-twitch response in mice pretreated with safrazine, a monoamine oxidase inhibitor. Safrazine-pretreated mice exhibited similar head-twitches following the IC treatment of serotonin (5-HT). The maximum dose of 5-HT which did not elicit head-twitches significantly potentiated TyA-induced head-twitches. Antiserotonergic drugs such as morphine and dimethothiazine antagonized TyA-induced head-twitches. A serotonergic denervator, 5,6-dihydroxytryptamine, potentiated head-twitch induced by TyA or 5-HT. Both TyA-induced and 5-HT-induced head-twitches were inhibited by dopamine and noradrenaline, while catecholaminergic denervators such as reserpine and 6-hydroxydopamine, and diethyldithiocarbamic acid, a dopamine--hydroxylase inhibitor, increased the TyA response. These results indicate that head-twitches induced by TyA may be mediated via the serotonergic system and may inhibit the catecholaminergic system.     

A kinetic study of the release of noradrenaline by tyramine

Summary Dog saphenous vein strips obtained from untreated or reserpine-pretreated animals were incubated with 1,4 mol/l ³H-(–)noradrenaline for 60 min after inhibition of the noradrenaline-metabolizing enzymes and of extraneuronal uptake. At the end of the incubation period the strips were perifused during 200 min. Some strips were exposed to tyramine from the 100th to the 200th min of perifusion. A compartment analysis of spontaneous or tyramine-induced efflux of ³H-noradrenaline was made. The spontaneous efflux of strips obtained from untreated animals had a long half time (t/2=269 min), and most of the ³H-noradrenaline which accumulated in the strips did not participate in the efflux (bound fraction, representing 85% of tissue activity at the 100th min of perifusion). The strips were exposed to five concentrations of tyramine, ranging from 0.49–3,240 mol/l. At all concentrations, tyramine mobilized only one noradrenaline compartment. The increase of tyramine concentration decreased the bound fraction, which became negligible for the highest concentration of tyramine. The half time of ³H-efflux induced by tyramine decreased from 278–106 min when the tyramine concentration was increased from 0.49–40 mol/l. However, further increases of the concentration of tyramine up to 3240 mol/l did not result in a significantly lower half time.Strips obtained from reserpine-pretreated animals were characterized by a low accumulation of ³H-noradrenaline (403 ng/g vs. 1,374 ng/g for strips obtained from unpretreated animals, at the 100th min of perifusion), the efflux had a half time of only 122 min and the bound fraction was smaller than in strips obtained from unpretreated animals (bound fraction representing 47% of tissue activity at the 100th min of perifusion). Reserpine-pretreated strips were exposed to 40 mol/l tyramine. Under these experimental conditions there was no significant bound fraction. The ³H-efflux induced by tyramine decayed according to a two-compartment model, with half times of 8 and 50 min.The results support the view that in preparations from unpretreated animals the size of the noradrenaline pool available for release by tyramine is dependent on the concentration of the latter amine. Furthermore, the half time of the efflux evoked by tyramine in strips with intact vesicular stores is dependent on a rate-limiting process, probably of vesicular location.Results presented to the 4th Meeting on Adrenergic Mechanisms (Porto 1980). This work was supported by a grant from Instituto Nacional de Investigação Cientifica (Centro de Farmacologia e Biopatologia Química da Universidade do Porto)     

OA-2500S手性柱拆分3种氨基酸对映体

目的建立了柱前衍生结合手性固定相HPLC法拆分了天冬酰胺(Asn)、蛋氨酸(Met)、色氨酸(Trp)3种氨基酸对映体。方法利用4-氯-7-硝基-2,1,3-苯并噁二唑(NBD-Cl)柱前衍生,采用SumichiralOA-2500S色谱柱,以甲醇-乙腈(50∶50,含5mmol.L-1柠檬酸)为流动相,在470nm波长下检测。结果在最佳色谱条件下,3种氨基酸对映体均达到了基线分离。结论方法灵敏度高,重现性好。     

“Supramaximal” enhancement of the inotropic effect of noradrenaline by tyramine

Summary The positive inotropic effect of noradrenaline on the guinea-pig papillary muscle is potentiated in the presence of 1×10^–5 M tyramine, the concentration of noradrenaline that is necessary to produce a half maximal increase in force of contraction being reduced to about one third. There is no alteration of the maximal inotropic effect since the concentration-effect curve of noradrenaline is simply shifted to the left.In the presence of 3×10^–3 M tyramine, which by itself increases contractility by a dual mechanism (an indirect sympathomimetic and a direct postsynaptic one which is not induced by stimulation of adrenergic -receptors), noradrenaline (1×10^–5 M) produces an additional inotropic effect leading to a force of contraction which surmounts the maximum of the normal concentration-effect curve of noradrenaline by about 30%.The supramaximal isometric contraction curve of the papillary muscle produced by the combined effects of 3×10^–3 M tyramine and 1×10^–5 M noradrenaline differs from the contraction curve in the presence of 1×10^–5 M noradrenaline alone in having a steeper ascending slope and a slower relaxation phase. The mean velocity of force development (S ₁) exceeds the maximum value of the normal concentration-effect curve of noradrenaline by about 50%. There is no increase in the maximum of the mean velocity of relaxation (S ₂).The relaxation time of the supramaximal contraction curve as well as the duration of its action potential are the result of the opposing influences of the two substances, noradrenaline shortening and tyramine prolonging both action potential and relaxation time.     

Determination of aflatoxin M1 levels in Iranian white and cream cheese

A screening survey on the occurrence of aflatoxin M1 (AFM1) was accomplished on 210 cheese samples composed of white cheese (116 samples) and cream cheese (94 samples) purchased from popular markets in central part of Iran (Esfahan and Yazd provinces). The quantitative analysis of AFM1 levels in the samples was performed by using the competitive enzyme-linked immunosorbent assay (ELISA) technique. Aflatoxin M1 at measurable level (50 ng/kg) was detected in 161 (76.6%) samples, consisting of 93 (80.1%) white and 68 (72.3%) cream cheese samples. The concentration of AFM1 in the samples ranged from 52.1 to 785.4 ng/kg. Comparing to legal regulation (250 ng/kg) accepted by some of the countries, 24.2% of the samples exceeded the accepted limit. Among these, the AFM1 levels in 28.4% of white and 19.1% of cream cheese samples were not in accordance with the safety limit. The results indicated that contamination of the samples with AFM1 in such a level appear to be a potential hazard for public health. This paper represents the data of the first survey on the occurrence of AFM1 in cheeses consumed in central part of Iran.     

Differences in the metabolic fate of noradrenaline released by electrical stimulation or by tyramine

Summary Strips of canine saphenous vein were loaded with ³H-noradrenaline (1.4 M) and perifused with Krebs solution and either subjected to field stimulation or exposed to tyramine 40 M. ³H, noradrenaline and its metabolites were determined in the perifusion fluid. Stimulation caused an increase predominantly in noradrenaline, followed by DOPEG, whereas tyramine released DOPEG in larger amounts than noradrenaline. Tyramine had more sustained effects than stimulation. Cocaine (1.6 M) drastically reduced DOPEG efflux due to stimulation, but had no effects on the pattern of release by tyramine. It is concluded that tyramine releases noradrenaline which is deaminated before it reaches the synaptic gap, whereas after stimulation deamination of the transmitter occurs after re-uptake.Results presented in part to the 8th Annual Meeting of the Portuguese Pharmacological Society (Coimbra, December 1977) and to the 3rd International Symposium on Vascular Neuroeffector Mechanisms (Louvain-Antwerpen, July 24–26, 1978). This work was supported by a grant from Instituto Nacional de Investigação Científica (FmPl)     

Analysis of the action of tyramine on blood pressure in the rat

In urethane anaesthetized rats, tyramine, 1 mg/kg, i.v. produced a considerable rise in blood pressure. In rats pretreated with reserpine, 10 mg/kg, injected i.p. 16–24 hr before, the pressor response was still quite large.The residual response after reserpinization was almost abolished by adrenalectomy or by pithing. The findings suggest that the pressor action of tyramine in the rat is due in part to a central or centrally mediated stimulation of the nerve supply to the adrenal medulla.     

A postsynaptic effect of tyramine in ventricular muscle

Summary Tachyphylaxis and susceptibility to pretreatment with reserpine demonstrate that tyramine increases contractility of the guinea-pig papillary muscle in concentrations up to 10^–4 M by means of an indirect sympathomimetic effect.In higher concentrations (up to 3×10^–3 M) tyramine causes an increase in force of contraction which is characterized by a slowing of relaxation of the isometric contraction curve. Pretreatment with reserpine reveals that this positive inotropic effect is composed of an indirect sympathomimetic and a direct post-synaptic effect; the latter is not subject to tachyphylaxis and produces by itself (at 3×10^–3 M) an increase in force of contraction which amounts to about 50% of the maximum of the indirectly produced inotropic effect.The postsynaptically-induced positive inotropic effect of tyramine is not antagonized by propranolol (5×10^–6 M). It is unlikely, therefore, that it is brought about by stimulation of adrenergic -receptors.The height of the isometric contraction curve in reserpine-pretreated muscles is increased by tyramine as a result of an increase in the rate of force development despite a marked concentration-dependent increase in relaxation time (t ₂).The direct inotropic effect of tyramine is correlated with a prolongation of the duration of the action potential (AP) which amounts to 70% at 3×10^–3 M. In addition to its influence on the duration of the AP, tyramine slows the rate of depolarization and decreases the membrane resting potential.     

Uptake of tyramine into synaptic vesicles of the caudate nucleus

Summary Synaptic vesicles isolated from the caudate nucleus of the pig were preincubated with reserpine, serotonin or tyramine at concentrations which caused a 90% inhibition of the ATP-Mg²⁺-dependent uptake of ¹⁴C-dopamine.The preincubated vesicles were sedimented by centrifugation and resuspended in drug-free buffer. The vesicles were incubated with ¹⁴C-dopamine in the presence or in the absence of ATP-Mg²⁺; at the end of the incubation period they were separated from the incubation medium by centrifugation. The inhibitory effect of reserpine on the ATP-Mg²⁺-dependent uptake of ¹⁴C-dopamine was slightly diminished, that of serotonin reduced while the inhibitory effect of tyramine was abolished. Hence, tyramine seems to be more easily removable than the other substances.In another series of experiments the vesicles were incubated with ³H-tyramine and immediately separated by filtration through membrane filters. Addition of ATP-Mg²⁺ enhanced the uptake of ³H-tyramine. The Km of the ATP-Mg²⁺-dependent uptake of ³H-tyramine was 1.4×10^–7 M, the Vmax 29.2 pmoles/mg protein/min. At 0°C the uptake of ³H-tyramine in the absence of ATP-Mg²⁺ was reduced, that in the presence of ATP-Mg²⁺ abolished. Incubation of the vesicles with ³H-dopamine revealed K _m and V _max values similar to those previously found when the vesicles were isolated from the incubation medium by centrifugation. Incubation in the presence of reserpine inhibited the ATP-Mg²⁺-dependent uptake of ³H-tyramine (IC₅₀ 1.4×10^–8 M) and ³H-dopamine (IC₅₀ 4.1×10^–8 M). The results demonstrate that tyramine is taken up into the vesicles by a process which is dependent on ATP-Mg²⁺ and temperature.Failure of previous attempts to prove an ATP-Mg²⁺-dependent uptake of tyramine when the vesicles were separated by centrifugation seems to be due to release of the accumulated tyramine during the centrifugation procedure.This work was supported by the Deutsche Forschungsgemeinschaft     

Characteristics of tyramine induced release of noradrenaline: Mode of action of tyramine and metabolic fate of the transmitter

Summary Dog saphenous vein strips were perifused and exposed to seven concentrations of tyramine, ranging from 1.5 M to 1.08 mM during 2.5 min. Fractional release of tritium (determined during and after the exposure to tyramine) was directly proportional to the concentration of tyramine. However, the percentage contribution by noradrenaline, normetanephrine and dihydroxymandelic acid to total release of radioactivity increased with tyramine concentration, whereas that by dihydroxyphenylglycol decreased. Inhibition of neuronal uptake by cocaine (8 M) resulted in a marked reduction of the release of noradrenaline by tyramine, but did not affect the metabolic pattern of the released noradrenaline. In strips obtained from animals pretreated with reserpine and iproniazid and exposed to U-0521 and hydrocortisone, electrical stimulation caused no release of noradrenaline, but tyramine induced a small and transient efflux of noradrenaline. In another experimental series, strips obtained from untreated animals were subjected to electrical stimulation or tyramine during 40 min. Apart from differences in the metabolic pattern of noradrenaline released by either process, it was found that efflux due to tyramine was stable during the whole period of exposure, whereas noradrenaline release gradually and markedly decreased during the electrical stimulation period.The results show that in this preparation tyramine releases noradrenaline by a mechanism which differs from that leading to the release by electrical stimulation, namely by a non-exocytotic mechanism, the transmitter being subject to oxidative deamination when diffusing through the axoplasm, before it reaches the synaptic gap.Results presented in part to the 9th Annual Meeting of the Portuguese Pharmacological Society (Porto, December, 1978) Work supported by a grant from Instituto Nacional de Investigação Cientifica (FmP1)     

高效液相色谱法测定阿瑞匹坦中同分异构体

目的：建立高效液相色谱法测定阿瑞匹坦的同分异构体。方法采用高效液相色谱法,色谱柱为 Waters XBridgeTM Shield RP18（250 mm ×4．6 mm,5μm）,检测波长：210 nm,流速：1．0 mL·min－1,柱温：30℃,进样量：20μL。结果阿瑞匹坦的几个同分异构体均能很好的分离。结论该方法分离阿瑞匹坦的手性同分异构体的杂质方法简便、准确、可靠,灵敏度高。     

Release of neuropeptide Y (NPY) induced by tyramine in the isolated vas deferens of rat

Summary The effect of tyramine on the isolated vas deferens of rats was investigated. Tyramine induced a dose-dependent contraction which was blocked by phentolamine and disappeared in adrenergic denervated tissues. In the presence of an antiserum to neuropeptide Y (NPY), the contraction induced by concentrations of tyramine greater than 10 M was markedly increased. In addition to inducing the release of ³H-norepinephrine (NE), tyramine evoked a concentration-dependent efflux of NPY-like immunoreactivity (NPYLI) from synaptosomal preparations. This action was not modified either by the removal of calcium ion from the medium or by the pretreatment with tetrodotoxin (0.5 M). Desipramine suppressed the NPY-LI release induced by tyramine apparently by the inhibition of the uptake of tyramine is suggested by the significant positive correlation between the reduction of ⁴C-tyramine uptake and the inhibition of NPY-LI release induced by desipramine (r = 0.946). Therefore, we suggest that tyramine does induce the release of NPY from rat vas deferens, in addition to effecting NE secretion. Send offprint request to J. T. Cheng at the above address     

高效液相色谱法测定酪胺含量

目的：建立快速、准确测定酪胺含量的高效液相色谱方法。方法：采用安捷伦HPLC1200系统,色谱柱采用Agilent Eclipse XDB—C18填料（250mm×4．6mm,5um）,流动相：0．02mol／L磷酸氢二钠-乙腈（85：15）用10％磷酸调节pH值为8．3,流速：1．0ml／min;在225nm波长处检测。结果：酪胺在8．0～160ug·ml-1 的浓度范围内呈线性关系,r=0．9999。     

HPLC法测定人血浆中华法林药物浓度

目的：建立HPLC法测定血浆中华法林浓度方法,并用此方法监测本院术后患者华法林血浓度。方法：采用ZORBAXEclipseXDB-C8色谱柱（150mm×4.6mm,5μm）,流动相：0.1%醋酸：甲醇（35∶65）,流速：1.0ml·min-1;检测波长：308nm;柱温：30℃;进样量：20μl。二极管阵列检测器检测。结果：线性范围为0.1～5.12μg·ml-1,提取回收率为85.0%～102.3%,RSD为1.2%～3.9%（n=6）。日内、日间RSD均小于5%,重复性好。结论：该方法准确、简便、精密度高,可用于华法林血药浓度监测。     

The effect of adrenaline,tyramine and guanethidine on two-way avoidance conditioning and on pseudoconditioning

Adrenaline (0.5 mg/kg) or guanethidine (10 mg/kg) given i.p. depressed performance of pseudoconditioned shuttle responses by rats. In a previous paper it had been shown that tyramine (5 mg/kg) had an opposite effect (Izquierdo, 1974a). Pre-trial administration of any of the three drugs also depressed two-way avoidance conditioning. Following posttrial administration, only guanethidine had a deleterious effect on reténtion. Since none of these drugs is believed to reach the brain in significant amounts following systemic injection, the present results suggest that peripheral factors may influence both conditioned and pseudoconditioned shuttle behavior.     

The effects of alaproclate on the pupillary responses to tyramine,phenylephrine and pilocarpine in depressed patients

Nine depressed patients were treated with alaproclate, a selective 5-HT uptake inhibitor, for 3 weeks in a dose of 400 mg daily. The pupillary responses to tyramine, phenylephrine, and pilocarpine eye drops were measured on consecutive days before, after 1 week and after 3 weeks of treatment. The tyramine-induced mydriasis was unaffected by alaproclate, suggesting that it does not significantly inhibit the reuptake of noradrenaline. The pilocarpine-induced miosis and the phenylephrine-induced mydriasis were both enhanced after 1 week but not after 3 weeks of treatment. This suggests that alaproclate acutely increases the responsiveness of postsynaptic muscarinic and ₁ adrenoceptors.     

Augmentation of the indirect sympathomimetic action of tyramine by cardioactive steroids is a consequence of elevated intracellular sodium

Summary The augmentation by the cardioactive steroid acetylstrophanthidin of neurotransmitter release evoked by tyramine, and the dependence of the augmentation upon Na⁺, has been investigated in dog saphenous vein rings in which monoamine oxidase activity and uptake₂ had been inhibited with pargyline and corticosterone respectively. High extracellular Na⁺ (Na_o; 263 mmol/1) reduced basal efflux of ³H-compounds from the rings and also reduced tyramine-evoked efflux. Low Na_o (25 mmol/1) increased basal efflux of ³H but reduced tyramine-evoked efflux. The increment in basal ³H-efflux caused by low Na_o was cocaine-sensitive. A presumed increase in intracellular Na⁺ (Na_i), produced by preincubating rings with acetylstrophanthidin in normal (143 mmol/1) or high Na_o, augmented ³H-efflux evoked by subsequent incubation with tyramine in normal Na_o Pre-incubating rings with acetylstrophanthidin in low Na_o, conditions which would not be expected to increase Na_i, did not cause augmentation of the subsequent tyramine-evoked ³H-efflux. An increase in Na_i, produced either as above or by pre-incubating rings in high Na_o alone, reduced subsequent neuronal ¹⁴C-tyramine uptake. Low Na_o present only during incubation reduced neuronal ¹⁴C-tyramine uptake, but high Nao present only during incubation did not increase neuronal ¹⁴C-tyramine uptake from that measured in normal Na_o The data are consistent with the following hypotheses: that tyramine uptake is dependent upon the prevailing inwardly directed Na⁺-gradient, that consequent noradrenaline efflux is Na⁺-gradient dependent and that the enhancement by acetylstrophanthidin of tyramine-evoked ³H-efflux is a consequence of the raised Na_i caused by Na⁺,K⁺-ATPase inhibition.Send offprint requests to D. A. Powis     

HPLC法测定犬血浆中罗格列酮的浓度

目的:建立HPLC法测定犬血浆中罗格列酮浓度.方法:取犬血浆0.5mL,加内标奎宁,混匀,然后加入3mL乙醚,涡旋振荡、离心,分出有机相,40℃水浴氮气流吹干,用200L流动相溶解残渣,取10L上清液行HPLC分析.流动相为10mmol·L-1KH2PO4-乙腈(60∶40),三乙胺调pH=6.5,流速0.8mL*min-1,色谱柱为NovaPake C18柱(150mm×3.9mm,4.0μm),柱温20℃.荧光激发波长为247nm,荧光发射波长为367nm.结果:犬血浆中杂质不干扰样品的测定,罗格列酮浓度在1.0～1 000.0ng*mL-1范围内线性关系好(r=0.999 9);最低检测浓度为1.0ng*mL-1;方法的平均相对回收率大于90%;平均提取回收率高于80%;日内和日间变异系数均小于12.0%.结论:所建立方法准确可靠,符合生物样品分析要求.