E Epstein

Postulated flaw in densitometry

Abstract An effort was made to explain the reported large discrepancies between comparative spectrophotometric measurements for two separated hemoglobins. This is the simplest quantification requirement following electrophoretic separation involving either integrating densitometry or elution followed by spectrophotometry. A graphic simulation of several exaggerated separation conditions with known areas and assigned absorbances was postulated from which the results obtained with inadequate slit geometry were calculated. The relative concentrations found were always in marked nonlinear disagreement with the linear theoretical values present. From this one could infer that true relationships between the volume relationships of separated zones are not realized if one neglects one dimension of a three dimensional measurement. Comparison between elution followed by spectrophotometry and direct spectrophotometry (densitometry) showed a smaller difference in means than did the work which stimulated this study (12). There is no way in which the differences in these two findings can be easily defined. However, one could infer that neglect of a zone dimension during the measurement process could be considered one source of such a measurement discrepancy. But even if nonproportional measurement of different zones to obtain relative areas of each does not describe the total problem, it should still be an important factor to keep in mind. A further study of other factors such as translucence or opacity with its Lambert effect on measurement, and the related severe turbidity factor in solution measurements is in progress.

Association of high-molecular-mass and electrophoretically atypical alkaline phosphatases

Polyacrylamide gel electrophoresis of alkaline phosphatase may yield abnormally migrating fractions; these include high-molecular-mass alkaline phosphatase, which remains at the gel origin, and immunoglobulin-alkaline phosphatase complexes, which have a mobility approximately 1/3 that of liver isoenzyme. We performed a retrospective study of 19 patients whose sera exhibited atypical alkaline phosphatase fractions, defined as bands whose mobility was slower than bone, liver, or intestinal alkaline phosphatase; 17 had a mobility approximately 1/3 that of liver isoenzyme and 16 also exhibited gel origin enzyme activity or high-molecular-mass bands. The strong association of the atypical and high-molecular-mass alkaline phosphatases suggests that they may be structurally related, both consisting of either immunoglobulin-enzyme complexes or membrane-alkaline phosphatase complexes. This hypothesis is supported by (1) one serum available for investigation containing alkaline phosphatase-immunoglobulin complexes in both abnormally migrating fractions, but on detergent treatment showing no evidence of membrane-bound enzyme; (2) detergent treatment of serum from patients with only high-molecular-mass alkaline phosphatase creating bands with a mobility of approximately 1/3 that of the liver isoenzyme.

A simple prototype procedure for cerebrospinal fluid cholesterol determinations

Abstract A preliminary procedure for CSF cholesterol is described which can be considered as a prototype method for the special case in which a biological sample is so dilute with respect to all of its constituents that it can be used as the sole diluent for the reactants used to measure the desired constituent in the specimen. Since it would be comparatively simple to generate a fluorophore in place of the chromophore and thereby increase the sensitivity of the procedure, one could easily alter the limits of the determination and/or the size of the sample needed. This is contemplated as a technique which should result from the prototype procedure described. Avenues for the determination of other constituents such as triglycerides or biuret reactive compounds may possibly be accomplished by using a similar determinative approach. Again, in the specific case of cholesterol one may have to resort to detection devices other than colorimetric in order to achieve strong signals for measurement. Fluorescence appears to be a best first approach to attempt in these circumstances for a final procedure based on the suggested prototype.

Spectrophotometric study of drug interferences in an aqueous 17-ketosteroid reaction☆

Abstract A spectrophotometric study of two reported drug interferences in an aqueous Zimmermann reaction for 17-ketosteroids has been described. One drug, Tegretol, did not interfere spectrally with the 17-ketosteroid determination because its Zimmermann reaction spectrum was displaced far enough toward the ultraviolet so there was no significant superimposition of its spectrum with that of 17-ketosteroids. The other drug, Cephalothin, proved to be no problem analytically, because, even though it was Zimmermann-reactive, it was unextractable from acid hydrolyzed urines and therefore could not interfere in the color-forming step.

Effect of insulin, S-adenosylhomocysteine, phospholipase C, n-butanol and Triton X-114 on alkaline phosphatase from isolated rat adipocyte plasma membranes

Alkaline phosphatase activity has been described in aqueous extracts of rat adipose tissue [l], in rat adipocyte plasma membrane fractions [2] and more recently in human adipose tissue [3]. Studies with hepatocytes have shown this to be a membrane-bound enzyme covalently linked to the polar head group of phosphatidylinositol (PI) [4]; this interaction is supported by the finding that PI-specific phospholipase C releases alkaline phosphatase from hepatocyte plasma membranes [5,6]. It has also been proposed that the butanol-extraction of alkaline phosphatase from plasma membranes is caused by an autolytic process derived from activation of a plasma membrane phospholipase, phospholipase D [7] or PI-specific phospholipase C [8]. Insulin stimulates phospholipid methylation in rat adipocyte plasma membranes [9,10] and releases a low molecular weight, acid-stable mediator which activates mitochondrial pyruvate dehydrogenase [ll]. Inhibition of insulin-stimulated phospholipid methylation by S-adenosylhomocysteine results in a significant increase in activity of the pyruvate dehydrogenase activator [12]. Saltiel et al [13] have suggested that insulin modulates the activity of both pyruvate dehydrogenase and low k, CAMP phosphodiesterase by activation of a PI-specific phospholipase C in plasma membranes, hydrolysis of a novel PI-glycan and release of a modulator of insulin action. Using polyacrylamide gel electrophoresis (PAGE), we describe the effect of phospholipase C, n-butanol, Triton X-114, insulin and S-adenosylhomocysteine on

Spectrophotometric study of both the Zimmermann reaction and the application of a corrective measure for irrelevant absorption

Abstract A spectrophotometric study of the Zimmermann reaction carried out in several solvent matrices has been described. The advantages of an aqueous system over those containing pyridine are detailed. Purification devices such as batch chromatography and color extraction used commonly in 17KS procedures have been examined, and the results obtained are shown as evidence that they are not always effective if the actions and reactions of the interference mimic those of 17KS. Finally, the study of mathematical corrections for irrelevant absorption resulted in values that indicated that the concept of such corrective action was not always easily applicable to the determination.

Alkaline phosphatase in the assessment of choledocholithiasis before surgery

The pre-operative diagnosis of choledocholithiasis, or common bile-duct stones, is important in patients with cholelithiasis. Intraoperative cholangiography or choledochoscopy followed by exploration of the common bile duct could be limited to those patients with stones, if an adequate pre-operative diagnosis could be made. Many clinicians use pre-operative total alkaline phosphatase level alone or in combination with bilirubin level to determine the presence or absence of choledocholithiasis. Predictive value theory was used to analyze data reported by others to assess the value of alkaline phosphatase level alone or in combination with bilirubin level in identifying patients with choledocholithiasis. The authors conclude that alkaline phosphatase level is not useful either alone or in combination with bilirubin level in determining the presence of common bile duct stones in patients with cholelithiasis.

The Zimmermann reaction and a correction for irrelevant absorption

Abstract Two similar modifications of a previously described aqueous Zimmermann reaction for 17-ketosteroids were studied to see whether the Allen correction for irrelevant background absorption could be accurate when pure steroids were compared against a single steroid as a calibrator. Dehydroisoandrosterone was selected as the standard because most procedures use it for that purpose. In one procedure in which varied spectral shapes, wide-ranging peak maximum values, and molar absorptivities were evident, accuracy of recovery was poor. In a second procedure, in which the primary variation was in band width rather than in either fine structures or molar absorptivities, the results were more accurate though still inaccurate as compared to peak measurement calculations. It would seem that this correction could only be valid for those procedures in which calibrator and analyte yield spectra which show better uniformity than the two procedures described here.

Comparative chromogenicities and discrepant spectra obtained between a heterogeneous and homogeneous Zimmermann reaction

Abstract A spectrophotometric study is shown for two modifications of a 17-KS procedure in which nearly all aqueous reaction media were used. Although the apparent differences in matrices of reaction between the two procedures appear to be minute, a small volume of ethanol for one is substituted for the same volume of methanol in the other, the final alkalinity of the latter procedure is stronger, and the reaction temperature was increased, wide differences in spectral results are found between the two methods. The ethanolic, lower alkalinity procedure is a homogeneous, single-phase reaction while the methanolic, higher-alkalinity procedure is a heterogeneous system in which a precipitation of detergent occurs and the precipitate becomes an integral part of the color reaction. The rate of formation of the ethanolic procedure is variable for the different 17-KS but it is constant in the methanolic procedure. The chromogenicities of color formation between the two procedures differ in that the methanolic system provides uniform and predictable spectra with more equivalent molar absorptivities. Lastly, the methanolic system shows a much greater sensitivity owing, apparently, to a more complete final reaction. This inability to form full color for several compounds in the homogeneous system can be related in some way to the large variations in ability to form that color owing to matrix composition.