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 of 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.
Acetaminophen is a commonly used analgesic, available without prescription. Several of its metabolites have heretofore been isolated from physiologic fluids and analytically characterized. In general, the separation methods are complicated, usually requiring extensive sample pretreatment, and do not measure the individual conjugated metabolites. High-resolution anionexchange separation of urinary samples from subjects receiving acetaminophen reveals eight chromatographic peaks, representing seven metabolites and the free drug itself. Metabolites separated include 2-methoxyacetaminophen, its glucuronide and sulfate conjugates, the sulfate conjugate of 2-hydroxyacetaminophen, the glucuronide and sulfate conjugates of acetaminophen, S-(5-acetamido-2-hydroxyphenyl)cysteine, and S-(5-acetamido-2-glucuronosidophenyl)cysteine. Urinary and serum concentrations of the drug and its seven metabolites were determined by high-resolution liquid chromatography as a function of time after two clinically normal men ingested 1950 mg of the drug. Concentrations in urine and serum are compared, and estimated urinary excretion rates are reported for all metabolites except S-(5-acetamido-2-hydroxyphenyl)cysteine. Serum concentrations of the glucuronide were higher than concentrations of the free drug 2 h after the drug was ingested, indicating that solvent-extraction procedures for serum will yield low estimates of total drug unless hydrolysis precedes the extraction step.
Urines, selected to demonstrate the utility of the LC system known as the "UV-Analyzer" for simultaneous investigation of a wide range of metabolites, were obtained from Parkinsonian patients being treated with l-DOPA alone or l-DOPA plus α-methyldopahydrazine, a dopa decarboxylase inhibitor. Sulfate conjugates are the major excretion products of those metabolites of l-DOPA retaining the catechol (3,4-dihydroxyphenyl) structure. Administration of the dopa decarboxylase inhibitor considerably decreased urinary excretion of 3,4-dihydroxyphenylacetic and 3,4-dihydroxymandelic acids and increased excretion of 3-methoxy-4-hydroxy-phenyl lactic acid. Urinary chromatograms obtained for several children with neurological disorders manifest by seizures, infantile autism, and mental retardation showed elevated excretion of 4-hydroxyphenylacetic (a metabolite of tyramine) and 4-hydroxyhippuric acids. Two of the more severely affected children excreted a new metabolite, identified by mass spectrometry and gas chromatography—mass spectrometry as α-methoxy-α-(3-methoxy-4-hydroxyphenyl)-acetic acid. This homolog of 3-methoxy-4-hydroxymandelic acid (VMA) was observed also in urine samples from patients with other types of diseases, including chronic lymphocytic leukemia, Lesch— Nyhan syndrome, malignant carcinoid, synovial sarcoma, multiple myeloma, and embryonic neoplasia. Its metabolic precursors are unknown.
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