Citation Classics in Intermediary Metabolism

Among the list of 37 Citation Classics in Clinical Chemistry published in the March issue of this Journal (1) were 7 dealing with analytes of intermediary metabolism (2)(3)(4)(5)(6)(7)(8). Two each covered measurement of urea, glucose, and calcium, whereas one described the combined assay of lactate and pyruvate. All were published in the period 1962–1968, when modifications to methods for these commonly measured substances were welcome even as automation slowly took over much of their routine testing. Chaney and Marbach (2), in their paper from the Chaney Chemical Laboratory in Glendale, CA, “Modified reagents for determination of urea and ammonia” (Fig. 1), simplified the catalyzed indophenol reaction for the determination of ammonia by combining four reagents into two, each stable for 60 days or more; urea was determined after conversion to ammonia and carbon dioxide by incubation with urease for 20 min at room temperature or 5 min at 60 °C (now there’s a stable enzyme!). Although this is a labor-intensive manual procedure, most readers will probably not recall the diacetyl monoxime method it largely replaced, which required heating a serum filtrate in strong sulfuric acid in a boiling water bath for 20 min. The 1554 citations received by this paper through 1995 were well deserved. The first paper in which Chaney and Marbach describe a simplified indophenol reaction for the measurement of ammonia, Clinical Chemistry 1962;8:130–2. Marsh et al. (3) in 1965 published modifications of the direct assay of urea with diacetyl monoxime, applicable to both manual and automated (AutoAnalyzer®, Technicon Corp.) procedures (Fig. 2). By including ferric chloride and thiosemicarbazide to intensify the color development, they were able to reduce the acid concentration from 67% (v/v) sulfuric acid to 8% (v/v) sulfuric acid and 1% (v/v) phosphoric acid. Heating in a boiling water bath for 20 min was still needed in the manual procedure; the automated method used the Technicon 95 °C heating bath. Their paper received 889 citations. The first description by Marsh et al. of improved methods for the measurement of blood urea, Clinical Chemistry 1965;11:624–7. Dubowski (4) while at the University of Florida was seeking a single-reagent manual procedure for glucose as a standby procedure for the automated methods by then in widespread use. Previous color reagents, benzidine and aminobiphenyl, were unstable or carcinogenic; Dr. Dubowski adapted o-toluidine (2-aminotoluene) in glacial acetic acid on the suggestion of Scandinavian chemist O.M. Forsell (Fig. 3). This reagent is stable for many months at room temperature. In the test, it can be added directly to various types of protein-free filtrates of body fluids and the absorbance read after 20 min in a boiling water bath. Kurt Dubowski, now at the University of Oklahoma, is well known to many members as a former secretary and president of AACC. This detailed and well-documented paper is a good example of his thorough scientific style. Dubowski’s paper describing a single-reagent manual method for measurement of glucose, Clinical Chemistry 1962;8:215–35. In 1968, Kadish et al. (5) introduced a more specific assay for glucose, using glucose oxidase with polarographic detection of the oxygen consumed (Fig. 4). Co-author James Sternberg, well known for his work with instrumentation at Beckman Instruments, Inc., was doubtless the provider of the polarographic analyzer. Measurement of oxygen consumption directly rather than of hydrogen peroxide production by reaction with peroxidase greatly improved the specificity of the method, because peroxide could be lost by action of catalase in the specimen. In this procedure, any peroxide was destroyed by reduction by iodide in the presence of ammonium molybdate before it could regenerate oxygen. The procedure measured the maximum rate of oxygen loss rather than overall oxygen consumption, which allowed for rapid readouts with fewer concerns of back-diffusion. It was designed for the small laboratory or for emergency use. The first description of a polarographic method for the measurement of glucose, Clinical Chemistry 1968;14:116–31. Gerald Kessler and Morris Wolfman (6) in 1964 adapted accepted procedures for calcium and phosphate to the AutoAnalyzer (Fig. 5). Dialysis obviated preparation of a protein-free filtrate; addition of 1 mol/L HCl to the specimen assured the release of bound calcium so that total calcium would be measured. This was one of the early applications of the colorimetric metal-complexing dye, cresolphthalein complexone, to detection of calcium; the phosphate assay used the standard Fiske-SubbaRow chemistry. The first AutoAnalyzer method for the simultaneous measurement of calcium and phosphorus without preliminary sample treatment, Clinical Chemistry 1964;10:686–703. In 1967, Trudeau and Freier (7) studied the use of lanthanum chloride to simplify the assay of calcium by atomic absorption spectrophotometry (AAS) (Fig. 6).Following the results of Zettner and Seligson(9) (Fig. 7), they found that 0.1% lanthanum (w/v) suppressed the effects of anions, including protein and phosphate, on the calcium absorbance. At the same time, the arrival of the more sensitive Perkin–Elmer 303 spectrophotometer permitted use of a 1:50 dilution of serum, which avoided clogging of the burner by protein and so eliminated the need for preparation of a protein-free filtrate. Urine could be analyzed accurately upon dilution with 0.5% lanthanum. The entire procedure required only 1 to 2 min. The original paper by Zettner and Seligson on the use of atomic absorption spectrophotometry to measure serum calcium, Clinical Chemistry 1964;10:869–90. The paper in which Trudeau and Freier first describe the use of lanthanum in the measurement of calcium in biological fluids, Clinical Chemistry 1967;13:101–14. Marbach and Weil (8) in 1967 simplified the enzymatic assay of lactate and pyruvate in blood (Fig. 8), using a single enzyme (lactic dehydrogenase) and a single protein-free filtrate (in 5% metaphosphoric acid). To measure lactate, an excess of NAD was added in a glycine-hydrazine buffer at pH 9.0; to measure pyruvate, an excess of NADH was used in a Tris buffer at pH 7.5. Dr. Marbach, it might be noted, is one of only two authors whose name appears twice in the list of 37 “Classics” (1) (the other is G. Szasz). By the time of this paper, Edward Marbach had moved from the Chaney Laboratory to the University of Southern California; he appeared in the 1988 ACCC Membership Directory as a bioconsultant in Los Angeles. The first description by Marbach and Weil of a simplified assay for lactate and pyruvate, Clinical Chemistry 1967;13:314–25. Why were these seven papers so frequently recognized? We can’t be sure, but they offered sound chemistry in detailed, well-documented procedures for important metabolic constituents. And I like to think that their popularity is also a credit to the tradition of careful editing by the Clinical Chemistry staff.