Advances in catecholamine and metabolite measurements for diagnosis of pheochromocytoma.

Assessment of catecholamine production and excretion is important in the laboratory detection of pheochromocytoma, a rare but curable cause of hypertension. Advances in catecholamine and metabolite methodologies have enhanced the diagnostic acumen by increasing analytical sensitivity and eliminating many of the interferences observed with earlier methods. Estimation of urinary catecholamines metanephrine and vanillylmandelic acid is routinely used in the biochemical detection of pheochromocytoma and in monitoring the completeness of tumor excision as well as the possibility of recurrence. Traditional spectrophotometric and fluorometric methods for urinary catecholamines and their metabolites are being replaced by highly sensitive and selective chromatographic methods. The ability to quantify individual catecholamines and metanephrines by high-performance liquid chromatography is of particular value for detecting familial forms of the tumor that may secrete epinephrine. Plasma norepinephrine and epinephrine measurements are of additional diagnostic value in determining recent catecholamine release and response to clonidine suppression. For either urine or plasma measurements, appropriate patient preparation, sample collection, and method validation along with an understanding of the variable pattern of catecholamine secretion and metabolism in pheochromocytoma are essential. Advances in laboratory methodology and reference intervals for catecholamines for clinical interpretation are reviewed.

Relationships between the proton nuclear magnetic resonance properties of plasma lipoproteins and cancer.

We conducted a comprehensive investigation of the origin of nuclear magnetic resonance (NMR) lineshape variability of plasma lipids among healthy individuals and those with cancer. The methyl and methylene resonances of lipid in human plasma, whose linewidths have been reported to correlate with the presence of malignancy, are composed of the overlapping resonances of "mobile" protons from the major lipoproteins (very-low-, low-, and high-density lipoproteins). We tested two hypotheses for the origin of the narrower plasma linewidths observed for cancer patients: (a) malignancy-associated differences in the spectral properties (chemical shift, lineshape) of one or more of the lipoproteins, and (b) differences in the fraction of lipoprotein lipid giving rise to detectable NMR signal. Analysis of the concentrations of lipoprotein lipid and of 500 MHz NMR spectra of the lipoprotein constituents in greater than 100 plasma samples failed to provide support for either hypothesis. Although linewidths were found to be significantly narrower for the cancer group, the difference is entirely attributable to differences in the concentrations of the lipoproteins.

Concentrations of morphine and codeine in serum and urine after ingestion of poppy seeds.

We measured morphine and codeine in commercially available poppy seeds and in serum and urine samples from healthy adults who had ingested these poppy seeds. Four brands of black poppy seeds, examined by gas chromatography-mass spectroscopy (GC-MS) with deuterated internal standards, contained from 17 to 294 micrograms of morphine and 3 to 14 micrograms of codeine per gram of seeds. Morphine was detected by GC-MS in hydrolysates of serum as late as 24 h after ingestion, with a maximum mean concentration of 100 ng/mL (range 82-131) measured 2 h after the subjects ingested 25 g of seeds. Opiates were detectable (greater than 300 micrograms/L) in urine by enzyme-multiplied immunoassay (EMIT; Syva Co.) and by radioimmunoassay screening procedures for as long as 48 h after ingestion. The identity and quantities of morphine and codeine in poppy seed extracts and in hydrolysates of serum and urine were confirmed by GC-MS. Therefore a positive finding of morphine or codeine in blood and urine may sometimes be due to ingestion of poppy seeds.

Angiotensin-converting enzyme in cultured endothelial cells. Synthesis, degradation, and transfer to culture medium.

Cultured endothelial cells from swine aorta possess the ecto-enzyme angiotensin-converting enzyme (E.C.3.4.15.1) in cell-associated form and also release it in soluble form into the culture medium. Using antibody to purified converting enzyme from swine kidney and incorporation of 3H-leucine, we examined the synthesis, degradation, and release of enzyme into the medium. 3H-leucine is incorporated into cellular converting enzyme, and later appears in the enzyme in the culture medium. The amount of cell-associated enzyme activity remains constant, and the amount of activity in the medium increases linearly during this period. These data show unequivocally that the appearance of enzyme activity in the cell culture medium is accompanied by synthesis of new enzyme protein. In pulse-chase experiments, the radioactivity disappeared from the cellular enzyme in two kinetic components with apparent half-lives of 1-2 hours and 20 hours, respectively. The appearance of the radiolabel in the medium enzyme corresponds very closely in rate and amount to the slow disappearance from the cells. There was no apparent uptake of labeled medium enzyme by the cells. The data suggest that the pool of active enzyme on the cell surface is the immediate precursor of the medium enzyme. The effects of culture conditions on the turnover of angiotensin-converting enzyme were also examined. The incorporation of 3H-leucine into both cell and medium enzyme was greater when cells were maintained in medium containing serum than when they were maintained in serum-free medium. The rate of degradation of the cellular enzyme was similar under the two culture conditions.

Angiotensin converting enzyme in cultured endothelial cells and growth medium. Relationships to enzyme from kidney and plasma.

We have previously reported that cultured cells from swine aorta possess angiotensin converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) and release it into serum-free culture medium. The present work compares enzyme from these two sources, and from swine kidney and serum, with respect to antibody and lectin binding. Purified enzyme from swine kidney, and the activity in swine serum, cultured endothelial cells and culture medium bind similarly to rabbit antibodies prepared against the kidney converting enzyme. Enzyme from each of these sources was allowed to bind to an immobilized lectin (Ricinus communis), which binds to terminal galactose residues of glycoproteins. Increasing concentrations of galactose were used to remove enzyme from the lectin column and the distribution of enzyme activity in the galactose eluates was determined. The elution pattern was similar for kidney and endothelial cell enzyme, and different from the pattern found for both serum and medium enzymes. Neuraminidase treatment of either serum or medium enzyme altered the distribution of activity eluted to that found for endothelial cell or kidney enzymes. The effects of neuraminidase suggest that the difference in lectin binding between cell and medium enzyme reflects differences in the number of terminal sialic acid residues that cover galactose residues.

Enzyme activities in endothelial cells and smooth muscle cells from swine aorta.

In populations of cultured arterial endothelial and smooth muscle cells grown under the same conditions, we have measured the total activity per cell of 10 enzymes commonly used as "markers" for subcellular organelles: NADH: ferricyanide reductase, NADH:cytochrome c reductase (rotenone insensitive). NADPH:cytochrome c reductase, alpha-glucosidase, 5'-nucleotidase, alkaline phosphodiesterase I, cytochrome oxidase, monoamine oxidase, cathepsin D, and N-acetyl-beta-glucosaminidase. Significant differences between the cell types were found for 7 of the 10 enzymes tested. The total activity of 5'-nucleotidase in cultured smooth muscle cells was 17 times that of cultured endothelial cells. Comparison of the activities in the two cell types freshly collected and in culture showed that the difference in 5'-nucleotidase in cultured cells is due principally to loss of activity from endothelial cells, suggesting that this activity is regulated differently in the two cell types. In both cell types cathepsin D activity rose during culture.

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.