Ante M. Krstulovic

Investigations of catecholamine metabolism using high-performance liquid chromatography : Analytical methodology and clinical applications

High-performance liquid chromatography, particularly in its reversed-phase mode, coupled with electrochemical or fluorometric detection, is becoming increasingly popular as an analytical tool for metabolic profiling of substances of neurochemical interest, such as catecholamines and their metabolites. During the last decade, a continued effort has been made to improve and simplify the analytical methodology for routine use in clinical laboratories where this technique is tremendously needed. New developments in column technology, reliable detectors, simplified sample cleanup procedures, and particularly better understanding of the complex physicochemical phenomena underlying the operation of electrochemical detection, have resulted in a steady and encouraging progress. The purpose of this review was to describe the current analytical methodology and recent applications of HPLC in the field of catecholamine metabolism. Although this discussion is by no means detailed and complete, it, at least, hints at the impact of this technique on biochemical investigations and its future potential in clinical laboratories.

Use of reversed-phase high-performance liquid chromatographic analysis for the determination of provitamin A carotenes in tomatoes.

The usual methods for provitamin A evaluation of foods convert the total pigment amount, determined spectrophotometrically, into vitamin A units. Since the totally inactive lycopene is the major carotenoid in the tomato, such readings result in erroneously high provitamin A values. In view of the recent development of chemically bonded, reversed-phase, microparticulate packings and their use in high-performance liquid chromatography which combines highly accurate and reproducible resolution with the speed and ease of operation, a new method using such a system was developed to isolate carotenoid pigments from tomato samples. A 15-min column separation was thus achieved, dramatically decreasing the analysis time of the classical open column chromatographic procedures, which often result in unresolved and altered fractions due to long-term exposure to oxygen, light, solvents and sometimes adsorbent. beta-Carotene and lycopene were determined and quantitated in six tomato samples. beta-Carotene, 100% vitamin A-active, was expressed in International Units of vitamin A. The newly developed method gives a more reliable evaluation of the fruit potency in vitamin A than the methods of the Association of Official Analytical Chemists currently used for food composition tables.

Plasma catecholamines in hypertension and pheochromocytoma determined using ion-pair reversed-phase chromatography with amperometric detection : Investigation of the separation mechanism and clinical methodology

The retention behavior of catecholamines (CAs) in ion-pair reversed-phase chromatography is examined. From the effects of pH, ionic strength and a secondary ion-pairing reagent (citric acid), under our chromatographic conditions, the retention behavior can be explained by assuming a mixed ion-exchange mechanism with octyl sulfate and citrate, on the column and in the mobile phase, respectively. The developed separation method was applied to the analysis of CAs in plasma samples purified by alumina adsorption and detected amperometrically. This method provides the basis for the determination of the short-term magnitude of CA response to physical and physiological interventions, as well as the baseline CA levels in essential hypertension and pheochromocytoma. The results seen for norepinephrine and epinephrine are consistent with eh funcitonal roles of these CAs as hormones or peripheral transmitters.

Use of native fluorescence measurements and stopped-flow scanning technique in the high-performance liquid chromatographic analysis of catecholamines and related compounds

Simultaneous separation of catecholamines and tryptophan metabolites has been carried out using a reversed-phase partition mode of high-performance liquid chromatography (HPLC). Compounds are detected and measured via their native fluorescence emitted with an excitation wavelength of 285 nm and the emission cut-off filter of 340 nm. Sample preparation is minimized and the assay is selective and well-suited for routine analyses. The sensitivity of the method is in the nanogram range. The identity of chromatographic peaks is confirmed by their excitation spectra, obtained by the stopped-flow fluorescence scanning method.

Diagnoisis of neural crest tumors by reversed-phase high-performance liquid chromatographic determination of urinary catecholamine metabolites

Aberrations in the metabolic pathways of catecholamines in patients with neural crest tumors result in characteristic urinary excretion patterns of their catabolites. Tumors such as pheochromocytoma, neuroblastoma and ganglioneuroma usually defy clinical diagnosis because of their rarity, small size, intraabdominal position and clinical symptoms similar to those of essential hypertension. Quantitative determination of catecholamine metabolites such as vanillylmandelic acid (VMA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) offers possibilities for reliable confirmation of diagnosis. However, previous techniques for the assessment of catabolite levels suffered from inadequate sensitivity, reproducibility or specificity, which seriously diminished their usefulness as biochemical determinants in the prognosis of these life-threatening tumors. Reported in this paper is the analysis of urinary levels of VMA and MHPG using reversed-phase high-performance liquid chromatography with electrochemical and sectrophotometric detection. We present the excretion patterns showing these metabolites in 15 control subjects, 15 patients with pheochromocytoma and 5 patients with neuroblastoma.

High performance liquid chromatographic determination of serum UV profiles of normal subjects and patients with breast cancer and benign fibrocystic changes.

High performance liquid chromatography (HPLC) was used to determine the UV profiles of serum samples taken postoperatively from 22 patients with histologically documented breast cancer, 8 patients with benign breast fibrocystic changes and 10 normal subjects. The analyses were performed on coded serum samples and after they were completed, the code was broken and the results correlated with the clinical data. Only one ml of serum was required for the HPLC analysis and identification. Detection limits for the nucleosides and bases were in the 10--20 pmol range and the injection volume of the deproteinated serum was 75 mul. The UV profiles of the normal subjects were very reproducible and similar to those of the patients with benign fibrocystic changes. The profiles of some of the cancer patients were distinctly different from the two other groups, 1-methylinosine and N2-methylguanosine, which were not detected in sera from normal subjects and patients with benign fibrocystic changes, were found in 45.5% and 22.7% of the cancer patients, respectively. Patients with the metastatic disease also showed elevated levels of guanosine and uridine. Only one false positive was found in the normal population. At present, it is not clear whether this indicates a subclinical manifestation of the disease and it must await further follow-up.

Endogenous levels of free and conjugated urinary 3-methoxy-4-hydroxyphenylethyleneglycol in control subjects and patients with pheochromocytoma determined by reversed-phase liquid chromatography with electrochemical detection.

A sensitive and specific reversed-phase liquid-chromatography (HPLC) method for the determination of urinary free and conjugated 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) was developed. Sample preparation is minimal and the method is ideally suited for routine clinical assays of this catecholamine metabolite. Reported in this paper are excretion profiles of controls subjects and patients with pheochromocytoma. Quantitative results were expressed as microgram MHPG per mg creatinine, in order to eliminate variations associated with the 24-h urine collection. The levels of free and conjugated MHPG, determined in 20 control subjects ranged between 0.04--0.11 microgram/mg of creatinine and 0.48--1.25 microgram/mg of creatinine, respectively. The corresponding levels of free and conjugated MHPG in 8 patients with pheochromocytoma were 0.29--1.21 microgram/mg of creatinine and 2.47--15.98 microgram/mg of creatinine, respectively. The described liquid-chromatographic analysis, coupled with the highly sensitive electrochemical detection, provides a simple and reliable method for metabolic profiling of catecholamine by-products at their endogenous levels. This offers an attractive possibility for the detection of neural crest lesions which may cause problems in diagnosis because of their small size, infrequent occurrence and symptoms similar to those of essential hypertension.

Plasma catecholamine assays: Calibration with spiked plasma versus aqueous solutions

Both aqueous standards and plasmas spiked with standards are used for assays of plasma constituents. In a previous study, high-performance liquid chromatographic (HPLC) assays of plasma norepinephrine, epinephrine, and dopamine performed with spiked plasma reference solutions resulted in lower thresholds and thus gave higher concentrations. To verify the hypothesis that spiked catecholamines bind to plasma proteins (like physiological catecholamines) and thus escape extraction by alumina prior to HPLC, the recovery of standards in human albumin solution was compared with similar investigations using aqueous standards. Our findings confirm that (a) albumin is responsible to a great extent for the drop in recovery and (b) calibration by extraction of aqueous standards is preferable to calibration by spiked plasma for free catecholamine assays.

Quantitative determination of 3-methoxy-4-hydroxyphenylethyleneglycol and its sulfate conjugate in human lumbar cerebrospinal fluid using liquid chromatography with amperometric detection

A sensitive and direct reversed-phase liquid chromatographic method with amperometric detection was developed for the determination of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG). The concentrations of the free and sulfate conjugate of MHPG were measured in human lumbar cerebrospinal fluid. All samples were preconcentrated by extraction with ethyl acetate. Deconjugation of the sulfate form of MHPG was achieved by enzymatic hydrolysis with sulfatase. Peaks were identified on the basis of chromatographic behavior, ratio of responses at several oxidation potentials and the stopped-flow UV spectra of the collected fractions. The free MHPG content of 20 cerebrospinal fluid samples ranged between 0.720 and 19.51 ng/ml with the mean of 5.126 +/- 4.652 (S.D.) ng/ml. The sulfate conjugate of MHPG in 12 samples of cerebrospinal fluid ranged between 0.08 and 0.850 ng/ml with the mean value of 0.2365 +/- 0.2269 (S.D.) ng/ml. Although our results correlate well with the literature values, no attempt was made to interpret the quantitative data since samples were obtained from routine, diagnostic testing of patients admitted to the medical or neurologic services at the Mount Sinai Hospital.

Rapid assay for tryptophanase using reversed-phase high-performance liquid chromatography.

A rapid and sensitive high-performance liquid-chromatographic assay for tryptophanase based upon the fluorometric measurement of the enzymatically liberated indole was developed. The total incubation time is 20 min, and the reversed-phase separation is fast (elution time of indole in 8 min) and reproducible. The sensitivity of the method is in the nanomole range. This method was tested in the assay of tryptophanase activity in E. coli, giving an average activity of 6589.6 U/g of cells. Because of its speed, high sensitivity and minimal sample preparation, this method circumvents several problems commonly encountered in standard spectrophotometric methods of analysis.