Assay for aromaticl-amino acid decar☐ylase based on fluorescence derivatization with 1,2-diphenylethylenediamine

Methods based on fluorimetry and high-performance liquid chromatography (h.p.l.c.) for highly sensitive assay of aromaticl-amino acid decar?ylase are described. Dopamine formed enzymatically from a substrate,l-dihydroxyphenylalanine (l-DOPA), after chromatography on a small column of a cation-exchanger, Toyopak SP, is converted to a fluorescent compound by reaction with 1,2-diphenylethylenediamine. The derivative is measured by direct spectrofluorimetry or by reversed-phase h.p.l.c. with fluorimetric detection. The limits of detection for dopamine formed enzymatically in the direct and h.p.l.c. methods are 15 and 1 pmol per assay tube, respectively. The enzyme in rat liver, kidney, brain, heart, adrenal medula and serum can be precisely assayed.     

Assay for tyrosine hydroxylase by high-performance liquid chromatography with fluorescence detection

A sensitive assay method for tyrosine hydroxylase in rat brain and adrenal medulla by high-performance liquid chromatography with fluorescence detection is described. L-DOPA formed enzymatically from the substrate L-tyrosine and alpha-methyldopa (internal standard), after clean-up with small cartridges of an activated alumina and a cation exchanger, Toyopak IC-SP M, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for L-DOPA formed enzymatically is 2 pmol per assay tube.     

Determination of phenylethanolamine N-methyltransferase by high-performance liquid chromatography with fluorescence detection

A highly sensitive assay method for phenylethanolamine N-methyltransferase in rat adrenal medulla and brain is described which employs high-performance liquid chromatography with fluorescence detection. Epinephrine formed enzymatically from the substrate norepinephrine and isoproterenol (internal standard), after chromatography on a small cartridge of a cation exchanger, Toyopak SP, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine, a selective fluorescence derivatization reagent for catechol compounds. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for epinephrine formed enzymatically is 0.66 pmol per assay tube.     

Assay for monoamine oxidases A and B by high-performance liquid chromatography with fluorescence detection

A sensitive method for the assay of monoamine oxidases A and B is described which employs high-performance liquid chromatography with fluorescence detection. Rat brain mitochondria were used as a preparation of the enzymes. p-Sulfamoylbenzaldehyde and benzaldehyde formed enzymatically from p-sulfamoylbenzylamine (the substrate of monoamine oxidase A) and benzylamine (the substrate of monoamine oxidase B), respectively, are converted simultaneously into fluorescent compounds with 2,2'-dithiobis(1-aminonaphthalene). These compounds are separated by reversed-phase chromatography on mu Bondapak CN. The limits of detection for p-sulfamoylbenzaldehyde and benzaldehyde formed enzymatically are 30 and 10 pmol per assay tube, respectively.     

Occurrence of aromatic L-amino acid decarboxylase in human plasma and its assay by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method using fluorescence detection for assessing the activity of aromatic L-amino acid decarboxylase in human plasma is described. Dopamine, formed enzymatically from L-DOPA, and isoproterenol (internal standard) are chromatographed on a small ion-exchange cartridge (Toyopak SP) and derivatized with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on an Ultrasphere ODS column. The detection limit for dopamine formed enzymatically is 0.6 pmol per 500 microliter of enzyme reaction mixture. Aromatic L-amino acid decarboxylase in human plasma is very similar to that in rat kidney, with respect to optimum conditions for the enzyme reaction and gel chromatographic behaviour.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Fluorimetric assay for monoamine oxidases A and B by means of p-sulfamoylbenzylamine and benzylamine

p-Sulfamoylbenzylamine and p-hydroxybenzylamine are shown to be substrates for monoamine oxidase A and for monoamine oxidases A and B, respectively. This was established by studies on the selectivity of benzylamine derivatives as substrates using monoamine oxidase inhibitors. A sensitive fluorimetric method for the separate assay of monoamine oxidases A and B is described. p-Sulfamoylbenzaldehyde and benzaldehyde formed enzymatically from p-sulfamoylbenzylamine and benzylamine, a known substrate for monoamine oxidase B, are quantified by means of the selective fluorimetric determination of aromatic aldehydes with 2,2′-dithiobis(1-aminonaphthalene). The limits of detection for the p-sulfamoylbenzaldehyde and benzaldehyde formed enzymatically are, respectively, 300 and 150 pmol per assay tube. Monoamine oxidases A and B in rat brain mitochondrial tissue and human platelets were assayed.     

An ultramicro assay for human plasma prekallikrein activity by phosphorimetry

A highly sensitive method for the phosphorimetric assay of prekallikrein in human blood plasma is described. Prekallikrein is converted to kallikrein (active form) by reaction at 0°C with actin. p-Nitroaniline, formed enzymatically from H-d-prolyl-l-phenylalanyl-l-arginyl-p-nitroanilide, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanol. The method is precise and highly sensitive, requiring as little as 0.25 μl of human blood plasma. The limit of detection for p-nitroaniline formed enzymatically is 5 pmol.     

Coulometric titration of D(+)-glucose using its enzymatic oxidation.

A definitive method is described for the indirect assay of milligram quantities of D(+)-glucose by coulometric titration. D(+)-Glucose was aerobically oxidized by glucose oxidase in an acetate buffer solution (pH 5.1). Subsequently, the enzymatically formed hydrogen peroxide was titrated coulometrically with electrogenerated hypobromite in sodium bromide-sodium tetraborate medium of pH 8.6, with biamperometric end-point detection. Parameters affecting the enzymatically catalyzed oxidation and coulometric titration were evaluated. The optimized conditions for the oxidation of up to 20 mg of D(+)-glucose include the addition of 4500 U of glucose oxidase and stirring over a 10-min interval at 25 degrees C. Under proposed conditions, the assay values of several commercial D(+)-glucose reagents were somewhat lower than the guaranteed minimum values, with RSDs (n = 5) of 0.071 - 0.106%.     

Application of high-performance liquid chromatography to the study of biogenic amine-related enzymes

The application of high-performance liquid chromatography to the study of biogenic amine-related enzymes is reviewed. Biogenic amines include catecholamines (dopamine, norepinephrine and epinephrine), indoleamines (serotonin and melatonin), imidazoleamines (histamine), polyamines (putrescine, spermidine and spermine) and acetylcholine. Three particular aspects are covered. The first aspect is the assay of enzyme activities of biogenic amine-related enzymes, such as tyrosine hydroxylase, tryptophan hydroxylase, aromatic L-amino acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. The introduction of highly sensitive assays of biogenic amines with electrochemical detection or fluorescence detection have made possible the non-isotopic assay of these activities, replacing the previously used radioisotopic methods. The second aspect is the purification of these enzymes. Since biogenic amine-synthesizing enzymes are generally unstable, rapid and efficient purification of these enzymes is very useful. The third aspect is the assay of biogenic amines (for example, acetylcholine and polyamines) using post-column derivatization with biogenic amine oxidases and electrochemical detection.     

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.     

Determination of renin activity in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

A column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the determination of the renin activity in human plasma. The method is based on the quantification of the enzymatically produced angiotensin I. Angiotensin I liberated from a synthetic substrate (tridecapeptide of human angiotensinogen) and [Val5]-angiotensin I as an internal standard are converted into fluorescent derivatives by reaction with benzoin. The derivatives are separated from various interfering substances by column-switching HPLC using three reversed-phase columns. The limit of detection (signal-to-noise ratio = 3) of the renin activity is 2.7 pmol of angiotensin I formed per h per ml of plasma, which corresponds to approximately 820 fmol of angiotensin I injected. The column-switching method in combination with pre-column derivatization for the fluorimetric detection permits the sensitive and selective determination of the enzymatically formed angiotensin I. Hence low activities of renin in normal human plasma are readily measured.     

Simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue by high-performance liquid chromatography with electrochemical detection

A simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue was developed by using high-performance liquid chromatography with electrochemical detection. These compounds are analysed in a single chromatographic run within 30 min with a simple sample clean-up procedure. The detection system consists of two electrochemical detector cells aligned in series: a glassy-carbon electrode for catecholamines and serotonin, and a platinum electrode for acetylcholine and choline. For the detection of the latter compounds, they were converted enzymatically into hydrogen peroxide through a column reactor with immobilized acetylcholinesterase and choline oxidase. A column of boronic acid gel was placed just ahead of the immobilized enzyme column to remove catecholamines, which caused interfering responses on the platinum electrode. Two equivalent analytical columns and a column switching were employed to speed up the serotonin assay. Simultaneous determination of these major neurotransmitters in rat brain regions was successfully carried out with the system described.     

Phosphorimetric assay for β-glucuronidase in biological materials

A highly sensitive phosphorimetric method for the assay of β-glucuronidase in biological samples is described. p-Nitrophenol, formed enzymatically from p-nitrophenyl β-D-glucuronide, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanolic potassium hydroxide. The method is rapid, precise, and very sensitive, requiring as little as 0.5–5 μl of human serum or urine, or 0.3–3.0 μg of protein of rat tissue. The limit of detection for the p-nitrophenol formed is 20 pmol.     

Phosphorimetric microassay for succinyltrialanyl-p-nitroanilide-hydrolyzing enzyme activity in human serum

A phosphorimetric assay of succinyltrialanyl-p-nitroanilide-hydrolyzing enzyme activity in human serum is described. p-Nitroaniline formed enzymatically is extracted with ether and determined phosphorimetrically in an ether/ethanol glass. The method is precise and very sensitive, requiring as little as 5 μl of human serum. The limit of detection is 10 pmol.     

Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the assay of angiotensin-converting enzyme in human serum and for the separation of angiotensins and their analogues after pre-column fluorescence derivatization with benzoin. Angiotensin II, formed enzymatically from angiotensin I, is converted into a fluorescent derivative which is then separated isocratically from the substrate and biological substances in the enzyme reaction mixture on a reversed-phase column (TSK gel ODS-120T). The lower limit of detection for angiotensin II is 0.66 pmol per enzyme assay tube. The method is simple and sensitive, and requires as little as 5 microliter of human serum. Angiotensin analogues can also be separated and quantified by the chromatographic technique, and thus this method permits the use of the analogues of angiotensin I as substrates.     

Dopamine Detection Using the Selective and Spontaneous Formation of Electrocatalytic Poly(dopamine) Films on Indium?Tin Oxide Electrodes

In this study, we report the simple and sensitive electrochemical detection of dopamine in the presence of excess ascorbic acid. The detection is based on the spontaneous formation of electrocatalytic poly(dopamine) films on bare indium? tin oxide (ITO) electrodes. The poly(dopamine) films are formed by immersing ITO electrodes in a solution of dopamine and ascorbic acid for 10 min. Afterwards, the electrocatalytic oxidation of hydrazine is measured using modified electrodes. The electrooxidation current of hydrazine increases with increasing dopamine concentration. This method allows a detection limit of 1 nM for dopamine in the presence of 100 µM of ascorbic acid.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

Fluorimetric assay for measuring Dns-His-Lys-Arg-His-Lys cleaving enzyme using high-performance liquid chromatography

A rapid and sensitive assay for the determination of Dns-His-Lys-Arg-His-Lys cleaving enzyme activity is reported. This assay is based on fluorimetric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys-Arg-His-d-Lys, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This assay is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys at concentrations as low as 7 pmol, and yields highly reproducible results and requires less than 9.0 min per sample for separation and quantitation. The optimum pH for Dns-His-Lys-Arg-His-Lys cleaving enzyme activity was 7.5-8.0. The Michaelis constant (K_m) and the maximum velocity (V_max) values were 33.3 μM and 47.07 pmol/(μg h), respectively with the use of enzyme extract obtained from bovine pituitary. By using this assay, axonal transport of this enzyme activity was observed 48 h after double ligations of rat sciatic nerves. The high sensitivity and selectivity of this assay would be useful for clarification of the physiological role of this enzyme.     

Assay for guanase in blood serum by flow injection analysis with fluorescence detection

A sensitive assay for guanase activity in human serum (10 μl) is described. Xanthine, formed enzymatically from the substrate guanine, is determined in a flow system in which columns of immobilized xanthine oxidase, uricase and horseradish peroxidase are connected in that sequence in the flow line. Hydrogen peroxide formed in the enzymatic conversion of xanthine is measured fluorimetrically by reaction with 3-(p-hydroxyphenyl)-proprionic acid in the system. Linear calibration graphs are obtained for 0.52-500 pmol of xanthine in the 20-μl sample injected. This method permits the assay of guanase activity in the sera of healthy persons and patients with hepatitis.