Biological markers as an aid in the clinical management of patients with liver metastases.

Liver metastases due to the more common neoplastic diseases such as colorectal, breast, or bronchogenic carcinoma are a frequent occurrence and are associated with an ominous prognosis. Earlier detection followed by appropriate therapeutic interventions might have a decided effect on the subsequent course of disease. Controversy exists over the selection of tests with the greatest sensitivity, specificity, and potential utility. Preliminary evidence suggest that gamma-glutamyl transpeptidase and 5'-nucleotidase may be of particular significance. Four enzymes--gamma-glutamyl transpeptidase, 5'-nucleotidase, leucine aminopeptidase, and alkaline phosphatase plus carcinoembryonic antigen--were compared in the same blood samples from selected patients with breast and small cell carcinoma of the lung. Gamma-Glutamyl transpeptidase was the most sensitive test with 28/29 (97%) patients with hepatic metastases having elevated enzymatic activity in their sera. For patients with small cell carcinoma of the lung followed serially, gamma-glutamyl transpeptidase activity was increased an average of 5 months before liver metastases were detected by clinical means. Two factors are important in the interpretation of the results of gamma-glutamyl transpeptidase analysis: (1) Hepatic dysfunction due to diseases other than metastatic tumor involvement can cause a rise in enzyme levels as can (2) medications or ethanol which activate the hepatic microsomal drug metabolizing system. Of particular importance, however, is the fact that antitumor chemotherapy, even intensive and multiple agent, did not appear to effect the enzyme activity in the sera of patients with breast or small cell carcinoma of the lung. Gamma-glutamyl transpeptidase in combination with carcinoembryonic antigen may be of particular value in detecting liver metastases and in assessing subsequent response to therapy.

Induction of O-deethylase activity as an index to exposure to coal-derived products and trace environmental pollutants.

The metabolism of the synthetic substrate 7-ethoxyresorufin is a selective measure of the activity of cytochrome P-448 monooxygenase, the subset of cytochrome P-450-mediated enzymes preferentially induced by polycyclic aromatic hydrocarbons and related compounds. 7-Ethoxycoumarin metabolism, on the other hand, reflects total (nonselective) cytochrome P-450 monooxygenase activity. Either substrate yields a single, highly fluorescent product, amenable to direct, sensitive assay with the portable centrifugal analyzer. We used three assays with liver microsomes from C57/BL6 mice for the short-term bioassay of the dose-dependent effects of exposure to selected environmental toxins, including petroleums, polychlorinated biphenyls, and their oxidative degradation products.

Multiple biological markers and breast carcinoma: a preliminary study in the detection of recurrent disease after primary therapy.

The use of a selected group of biological markers in breast cancer patients who are disease free but at risk of relapse after mastectomy has potential for detecting recurrent tumor before there is clinical evidence. In this preliminary study, multiple materials were serially analyzed in the body fluids of patients without overt tumor but receiving adjuvant chemotherapy because of positive axillary nodes at surgery. The total frequency of elevated levels was determined and compared for those patients who remained disease free, for those who subsequently relapsed, and for a third group of patients with proven metastases. Frequency of elevation was directly proportional to increasing disease. Although differences in the relative frequency of individual materials was observed, the same trends with increasing tumor burden were found. The results suggest that the serial measurement of biological markers has potential for indicating the presence of occult disease. A nucleus of biological markers to be considered should include carcinoembryonic antigen, urinary hydroxyproline/creatinine ratio for bone lesions, and gamma-glutamyltranspeptidase for liver involvement.

Serum protein-bound carbohydrates for following the course of disease in patients with metastatic breast carcinoma.

Serum protein-bound carbohydrates, L-fucose, sialic acid, D-galactose, and D-mannose, were measured as potential biologic markers in patients with breast cancer with the use of high-resolution anion exchange separation in combination with a sensitive cerate oxidimetric fluorescence detector system. For 22 randomly selected patients with proved metastatic breast cancer, both L-fucose and sialic acid levels were above the normal range in 21 patients (95%). In contrast, D-mannose was increased in the sera of 9 patients (41%), and D-galactose in 6 (27%). By comparison, the level of carcinoembryonic antigen (CEA) was elevated in 14 patients (64%). The levels for serially determined serum protein-bound carbohydrates paralleled objective changes of response or progression in 5 patients with measurable disease. As might be expected, the degree and pattern of elevation for the individual carbohydrates varied for each patient studied. Combinations of serum protein-bound carbohydrates, particularly L-fucose and sialic acid, and, in addition, CEA, appear to have promise as potential biomarkers for following the course of the disease in patients with metastatic breast cancer.

Automated system for fractionation of blood samples.

We describe a prototype system for preparing multiple fractions of blood components (plasma, washed erythrocytes, and hemolysates) by use of automated techniques. The procedure is based on centrifugal separation and sample transfer by induced pressure differentials in a blood-processing vessel (rotor) that has been designed to process 24 samples simultaneously. Erythrocytes are sedimented against the outer walls of individual sample chambers, and plasma is siphoned, by imposition of either a slight positive or negative pressure, into individual reservoirs in a collection ring. Cells are washed in situ; samples of washed cells, either packed or in saline solution, can be recovered. Cellular hemolysates are automatically prepared and transferred to individual, commercially available collection vials, ready for immediate analysis or for storage in liquid nitrogen. The system has potential application in any biomedical area in which many samples are to be expeditiously analyzed and in which one or more of the blood fractions will be used. A separate unit has been designed and developed for the semiautomated cleaning of the rotor.

Chromatographic separation and continuously referenced, on-line monitoring of creatine kinase isoenzymes by use of an immobilized-enzyme microreactor.

We describe a new concept in continuously referenced monitoring of the isoenzyme activities of creatine kinase (EC 2.7.3.2) after liquid-chromatographic separation. After separation on a diethylaminoethyl-Sephacel column, the three isoenzymes of creatine kinase undergo a series of coupled enzyme reactions, ultimately resulting in the formation of ultraviolet-detectable NADPH. A major advantage of this detection system is the immobilized-enzyme microreactor (2 X 17 mm), which may be removed and stored refrigerated when not in use. A split-stream configuation allows self-blanking of endogenous ultraviolet-absorbing constituents in authentic sera samples, which would otherwise make definitive diagnosis and quantitation difficult or impossible. This system is applicable to the automated analysis of creatine kinase isoenzymes in the clinical laboratory.

Separation and analysis of arylsulfatase isoenzymes in body fluids of man.

Soluble arylsulfatase (EC 3.1.6.1) is present in the body fluids of man in the form of two isoenzymes, arylsulfatase A and B, which reportedly are useful biochemical markers for certain types of malignancy. However, rapid assay of the individual isoenzymes is extremely difficult; procedures based on differential inhibition or activation of the isoenzymes in a mixture yield only semiquantitative results. A feature of these isoenzymes is their inhibition by some common anions (notably phosphate) at physiologic concentrations. The isoenzymes can be separated by anion-exchange chromatography, the B isoenzyme being eluted in the void volume and the A isoenzyme and the anionic inhibitors retarded. Lead is used to sequester phosphate, enabling measurement of A in the salt-eluted fraction. Using this technique, we have found significant elevations of B in the sera of patients with colorectal cancer. The potential of rapid, chromatographic separation coupled with continuous monitoring for arylsulfatase activity is discussed.

Evaluation with the centrifugal fast analyzer of a chemical activation procedure for creatine kinase MB isoenzyme.

The differential activation method for the determination of the "myocardial" isoenzyme of creatine kinase (MB) is based on the computed difference in activity of serum aliquots activated by the combination of dithiothreitol and glutathione (skeletal plus myocardial creatine kinase) and by glutathione alone (skeletal creatine kinase). The unique ability of the Centrifugal Fast Analyzer to perform analyses in parallel is used to precisely measure (CV = 0.1-1.5%) the activity of the two chemically activated aliquots. Statistical considerations concerning the reliability of this difference estimation are discussed with respect to both the precision of the rate measurements and to the relative amount of isoenzyme MB present. Another potential source of error in the analysis of the two differently activated aliquots, namely variation in lag phases, is circumvented by use of a linear-search FOCAL software package. This program searches the data for a linear segment of maximum slope, automatically rejecting those data that appear in lag or depletion regions of the curve representing the progress of the reaction. Correspondence of the results obtained with those of comparison techniques (chromatography and electrophoresis) are discussed.

Composition, associated tissue methyltransferase activity, and catabolic end products of transfer RNA from carcinogen-induced hepatoma and normal monkey livers.

This investigation was designed to explore transfer RNA (TRNA) methyltransferase activity, urinary excretion levels of tRNA degradation products, and tRNA base composition in normal monkeys and in those with hepatocellular carcinomas induced by N-nitrosodiethylamine. After the development of the tumor, 24-hr urine specimens were collected, the monkeys were sacrificed, and the livers were removed for tRNA isolation and methyltransferase activity studies. The tRNA methyltransferase activity and capacity and the urinary excretion levels for selected tRNA degradation products (pseudouridine, N2,N2-dimethylguanosine, 1-methylinosine, 7-methylguanine, and beta-aminoisobutyric acid) were elevated for the hepatoma-bearing monkeys when compared to those with normal liver. The isolated tRNA pools were analyzed by high-resolution liquid chromatography, and similar base compositions were found for the hepatoma-bearing and normal monkeys. With the use of methyl-deficient Escherichia coli tRNA as the methyl receptor and the analytical procedure for tRNA anlysis, the methylating ability of the tRNA methyltransferases in hepatoma and normal liver extracts was determined. The hepatoma methyltransferase homogenates were found to produce increased levels of 7-methylguanine, N2,N2-dimethylguanine, and thymine, while the normal liver extracts gave higher levels of N2-methylguanine. These differences were not apparent in the base composition of the tRNA pools. The increased urinary excretion and higher methyltransferase activity of the hepatoma-bearing monkeys without an apparent increase in the methylated base content of their tRNA suggest increased tRNA tf individual isoaccepting tRNA's would be missed by analyzing the tRNA pools. The variations in the individual tRNA methyltransferase activities of the hepatoma and normal liver homogenates indicate a difference in the methlation of their tRNA's.

Metabolic response of humans to ingestion of nicotinic acid and nicotinamide.

The identification of nicotinamide-N1-oxide as a metabolite in the urine of a schizophrenic patient prompted a study of the relative metabolism of nicotinic acid and nicotinamide in mental patients and healthy volunteers. Metabolites quantified included N1-methyl-2-pyridone-5-carboxamide, N1-methyl-4-pyridone-3-carboxamide, N1-methylnicotinamide, nicotinuric acid, and nicotinamide-N1-oxide. More of most of these metabolites evidently was excreted after nicotinamide ingestion than after nicotinic acid. At the highest doses (3000 mg/day), the relative proportions of these metabolites in the urine were changed. There were only slight difference between healthy individuals and mental patients in the quantities of metabolites excreted, and no statistically significant trends were noted.

Protein-bound carbohydrates in breast cancer. Liquid-chromatographic analysis for mannose, galactose, fucose, and sialic acid in serum.

We describr high-resolution chromatographic analysis for protein-bound sialic acid in serum, with use of a cerate oxidimetric detector. Values for sera from normal women averaged 680.5 mg/liter, with a coefficient of variation of 23%. Including data obtained by previously developed chromatographic procedures for protein-bound mannose, galactose, and fucsoe, we assessed sera from breast-cancer patients whose malignancy had been categorized as either stable, responsive, or progressive (based on clinical observations spaced from two to five months apart). All of 12 responsive patients had decreases of protein-bound fucose averaging 34.5% (SD, 16.1) and all of 10 patients with progressive disease had increases averaging 38.3% (SD 21.5). Changes in fucose averaged less than 6.7% (SD, 4.9) for eight patients with clinically stable breast cancer. Changes in protein-bound mannose, galactose, and sialic acid did not correlate as well as did fucose with the clinical disease status of the patients.

High-resolution liquid chromatographic analysis of methylated purine and pyrimidine bases in transfer RNA.

Methylated and major purine and pyrimidine bases were separated and quantified by high-resolution liquid chromatography after hydrolyzing transfer ribonucleic acids (tRNAs). Separation was accomplished by eluting the hydrolyzed samples from an anion-exchange column with a concentration gradient of ammonium acetate at pH 9.2. Isolated sample of tRNA were hydrolyzed to the free bases with a trifluoroacetic acid-formic acid mixture of 200 degrees. Detection limits of 100-200 ng/ml were measured for the methylated bases; analytical data are reported for ten methylated bases plus the four major bases of calf liver and rat liver tRNA.

Liquid-chromatographic analysis for neutral carbohydrates in serum glycoproteins.

We describe a sensitive, reproducible procedure of analysis for the six neutral carbohydrates in glycoproteins, by high-resolution anion-exchange chromatography. As many as 16 neutral carbohydrates can be separated by elution with a concentration gradient of boric acid (pH 7, 67 to 672 mmol/liter). The carbohydrates are detected with a cerate oxidimetric detector system, which monitors the fluorescence of Ce3+ produced by the reaction of the eluted constituents with Ce4+. Sensitivity to 1 nmol of fucose is demonstrated. Analytical methods and results are presented for mannose, fucose, and galactose in serum glycoproteins for both normal women and those with metastatic breast cancer. We briefly discuss the possibility of separating and analyzing for the three neutral carbohydrates in serum glycoproteins in 4 h by isocratic (constant eluent concentration) elution from a chromatographic column.

Comparative serum and urine analyses by dual-detector anion-exchange chromatography.

A high-pressure anion-exchange chromatographic system has been modified to provide measurement of large numbers of molecular constituents in serum and for direct comparison to similar measurement in urine. Operating parameters have been adopted which greatly extend the range of elution for strongly retained anionic constituents and limit resolution of early-eluting basic and neutral compounds which were of less interest in this study. Dual monitoring by UV absorption and fluorescence produced by cerate oxidation provides both sensitive and wide-ranging detection capability. Comparative serum and urine chromatograms for a clinically normal subject, a subject after ingesting the drug 4-hydroxyacetanilide, and an infant suffering from extreme acidosis, illustrate the potential usefulness of this analysis in studying the origin, transport, in vivo reactions, and disposition of metabolites.