Ga Vandenberg

INVESTIGATION OF POLYAMINE METABOLISM BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC AND GAS-CHROMATOGRAPHIC PROFILING METHODS

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N1-acetylspermidine and N8-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-alpha-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-alpha-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.

CATABOLISM OF POLYAMINES IN THE RAT POLYAMINES AND THEIR NON-ALPHA-AMINO ACID METABOLITES

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, only labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine, gamma-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and delta-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, gamma-amino-n-butyric acid, 1,3-diaminopropane, beta-alanine and spermine. After the injections with bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, beta-alanine, 2-hydroxyputrescine and possibly gamma-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.

MICROBIAL INFLUENCES ON URINARY POLYAMINE EXCRETION

We determined diamines, polyamines, their monoacetylated conjugates and some of their catabolites in urines of healthy persons during decontamination of the gastrointestinal tract and patients with urinary tract infections. The compounds were also measured after in vitro incubation of urines from healthy persons and patients. During decontamination the urinary excretion of total putrescine decreased by a small amount. This fall was for the greater part accountable to monoacetylated putrescine. Free putrescine levels were increased in urines of patients with urinary tract infections, decreased after therapy, and increased after incubation of the pretherapeutical samples. Total cadaverine decreased during decontamination and increased during recontamination. The changes were partly accountable to monoacetylated cadaverine. Free cadaverine levels of patients with urinary tract infections were normal and did not change after therapy. These data show that, under normal conditions, a small part of monoacetylated putrescine and a considerable part of monoacetylated cadaverine originate from the gastrointestinal tract, and that urinary tract infections lead to an increase of free putrescine. The microbial synthesis of putrescine in the gastrointestinal- and urinary tracts, should therefore be taken into account for the interpretation of urinary putrescine levels as a parameter for body cell turnover.

Mass fragmentographic identification of polyamine metabolites in the urine of normal persons and cancer patients, and its relevance to the use of polyamines as tumour markers

The mass fragmentographic identification of N-(2-carboxyethyl)-4-amino-n-butyric acid, N-(3-aminopropyl)-N1-(2-carboxyethyl)-1,4-diaminobutane, N,N1-bis(2-carboxyethyl)-1,4-diaminobutane, and delta-aminovaleric acid in acid-hydrolysed urines of a normal person and two cancer patients is described. A previous study, in which the metabolic fate of intraperitoneally injected polyamines in rats was investigated, revealed that these compounds should be considered as non-alpha-amino acid metabolites of the naturally occurring polyamines. Quantification of polyamines and their non-alpha-amino acid metabolites by gas chromatography with nitrogen--phosphorus detection showed that, relative to the parent polyamines, humans normally excrete higher quantities of polyamine catabolites in urine than rats, suggesting that humans catabolize polyamines more efficiently. As illustrated by the follow-up of the concentrations of polyamines and their catabolites in the urine of a patient with high-grade non-Hodgkin lymphoma during chemotherapy, the catabolic pressure on polyamines may be considerably increased during neoplastic diseases, since an even higher proportion of oxidized polyamine metabolites was observed. It is therefore suggested that the additional measurement of the circulating concentrations of polyamine-degrading enzymes is of importance for the correct interpretation of polyamine (metabolite) determinations for oncological purposes.

DETERMINATION OF POLYAMINES IN HUMAN-ERYTHROCYTES BY CAPILLARY GAS-CHROMATOGRAPHY WITH NITROGEN PHOSPHORUS DETECTION

A capillary gas chromatographic method with nitrogen-phosphorus detection is used to determine simultaneously 1,3-diaminopropane, putrescine, cadaverine, spermidine and spermine in human erythrocytes. The compounds are isolated by adsorption on silica and converted into their heptafluorobutyryl derivatives. We give quality-control data and (age-dependent) normal values for 48 apparently healthy controls.

Determination of pipecolic acid in urine and plasma by isotope dilution mass fragmentography.

A capillary gas chromatographic method with mass spectrometric detection for the determination of pipecolic acid in urine and plasma (or serum) has been developed. Using a quantification based on stable isotope dilution mass fragmentography the concentration of pipecolic acid was determined in urines of 34 healthy children and 8 patients with Zellwegers syndrome. The urinary pipecolic acid excretion of healthy infants decreases with age. Its concentration in urines of patients with Zellwegers syndrome was not consistently elevated. Normal values for pipecolic acid in plasma were established for 19 healthy children. Pipecolic acid concentrations in 47 urine samples (range 0.02-228.3 mmol/mol of creatinine) and 6 serum samples of Zellweger patients after oral loading with DL-pipecolic acid (range 65-1334 mumol/l) were found to correlate satisfactorily with the results obtained by an amino acid analyzer method. The major advantage of the presented method over the amino acid analyzer method concerns its greater sensitivity and its much shorter analysis time.

Catecholamine metabolism during additional administration of DL-threo-3,4-dihydroxyphenylserine to patients with Parkinson's Disease

SummaryDL-threo-3,4-dihydroxyphenylserine (DL-threo-DOPS) was administered during 10 days to 4 patients with longstanding Parkinsons disease in addition to their treatment with L-3,4-dihydroxyphenyl-L-alanine (L-DOPA)-carbidopa (Sinemet). All patients tended to improve in their symptoms freezing, all day life activity and mood. There were no improvements in rigidity, tremor, and akinesia (in general). During the DL-threo-DOPS-treatment cerebrospinal fluid (CSF), serum and urine concentrations of catecholamines were measured. The results show that DL-threo-DOPS is transported to the brain and CSF in a way comparable with L-DOPA. However, no measurable increase of 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in CSF could be demonstrated. This suggests that the synthesis of noradrenaline from DL-threo-DOPS in the brain is doubtful. In addition measurements in urine reveals that at the dose used Sinemet, prevents peripheral decarboxylation of DL-threo-DOPS into noradrenaline. Other possible metabolic pathways of DL-threo-DOPS are discussed.

Determination of polyamines and metabolites in cerebrospinal fluid by isotope dilution mass fragmentography, and a clinical application.

Capillary gas chromatography and mass fragmentography was used to determine simultaneously 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, isoputreanine and putreanine in cerebrospinal fluid. After addition of deuterium labelled analogs and acid hydrolysis, the compounds were isolated by adsorption onto silica and converted into their N-heptafluorobutyryl-methylesters. Quality control data and an application of the method are given. A patient harbouring an astrocytoma was monitored during chemotherapeutic treatment.

DIURNAL CHANGES IN PLASMA-LEVELS OF 2-PYRROLIDINONE DETERMINED BY ISOTOPE-DILUTION MASS-SPECTROMETRY

Abstract A new capillary gas chromatographic method with mass spectrometric detection for the determination of 2-pyrrolidinone was developed. Using quantification based on stable isotope dilution mass spectrometry by monitoring selected ions in the ammonia chemical ionization mode diurnal changes of 2-pyrrolidinone levels in plasma of three healthy adults were established. Although substantial fluctuations, ranging from 5 to 30 μg/l, occurred during a 24-h daytime period, no consistent picture related to clock time or food intake was obtained. Endogenous oscillations should be taken into consideration, for example when evaluating transdermal penetration of 2-pyrrolidinone, used as enhancer in topical drug formulation.