Donald R. Strandbergh

The turbid specimen as an analytical medium: hemoglobin determination as a model

The quantitation of chemical constituents in lipemic samples is a major problem confronting the clinical laboratory. Currently, a number of cumbersome and time-consuming methods are used to clarify samples before analysis. However, the use of enzymic hydrolysis of triglycerides along with efficient chemical removal of the formed non-esterified fatty acids is exemplified here as an excellent alternative to the current methods of clarification such as ultracentrifugation, extraction or chemical precipitation of low density and very low density lipoproteins. This method of clarifying milky serum has been used by us to assay hemoglobin in severely lipemic blood samples as an analytical model.

On-line clarification for the measurement of serum glucose in hyperlipidemic specimens

The optical aberrations due to the presence of the turbidity caused by hyperlipidemia has been eliminated in two serum glucose procedures. This has been accomplished by incorporating lipase and alpha-cyclodextrin into the two glucose reagents. The hydrolytic action of the lipase generates water soluble glycerol and insoluble fatty acids. By including the chemical scavenger alpha-cyclodextrin into the reagent the fatty acids are solubilized and thus the production of a second turbidity is avoided. Because the clearing reagents are incorporated into the glucose reagents, this is an on-line process and no additional labor is required to clear the sample-reagent mixture. Furthermore, no additional time is required as the clearing occurs in the same period of time that it takes for the indicator reactions to reach equilibrium.

Study of continuous flow automation for serum iron on comparing several sensitive reagents

Abstract Three reagents, for the determination of serum iron concentrations, are compared for their use on an AutoAnalyzer. Under identical conditions, all three reagents ferrozine, 2,4-bis (5,6-diphenyl-1,2,4-triazin-3-yl)pyridinetetrasulfonate, and ferene S, behave in a similar manner. The interference of copper is studied as well as the possibility of using various masking reagents to reduce this interference. It is demonstrated that, contrary to a previously reported manual procedure, thioglycolic acid, thiourea, and neocuproine, the masking reagents, do not all result in exactly the same statistics. As well, it is shown that the time of addition of the masking reagent (i.e., pre- or postdialysis) is not particularly important. Comparison of a manual procedure, involving the preparation of a protein-free filtrate with one of the reagents on the AutoAnalyzer, shows very good correlation. Comparison of the various iron reagents with the AutoAnalyzer suggests that the prime advantage of one over the others is sensitivity.

Stimulation of phosphatidylcholine synthesis by insulin and ATP in isolated rat adipocyte plasma membranes.

Added individually or together, insulin and/or ATP significantly and rapidly increased the concentration of phosphatidylcholine in an enriched plasma membrane preparation from rat adipocytes. The increase in phosphatidylcholine synthesis mediated by insulin or ATP was suppressed by the phospholipid methyltransferase inhibitor, S-adenosylhomocysteine. These results suggest that the activity of phospholipid methyltransferase from adipocyte plasma membranes may be increased by phosphorylation and that insulin may further increase the activity of the phosphorylated phospholipid methyltransferase by an alternative pathway.

On the use of a sensitive indicator reaction for the automated glucose oxidase-peroxidase coupled reaction

A study into the substitution of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate for phenol in the indicator reaction for an automated glucose procedure is presented. Apart from having physical properties that are more conducive to easy handling than phenol, the former material affords considerably more sensitivity than does the latter. Results obtained with either of these glucose oxidase-coupled systems demonstrate good correlation not only with each other but also with the glucose oxidase procedure of the Beckman Astra 8.

Spectral study of bichromatics: Biuret−protein and glucose−hexokinase reactions as visible−ultraviolet models for turbidity

Abstract A model was devised for the study of the analytical effectiveness of bichromatic spectral measurements when faced with a severe interference such as turbidity. This was accomplished by using the biuret reaction of serum proteins to investigate the midvisible range and the 340-nm spectral peak of the NADPH produced in a hexokinase-based glucose reaction for the ultraviolet range. Two different bichromatic pairs that are currently used for the biuret reaction were compared in which the side-wavelengths were on either the blue or the red side of the peak maximum (540–650 and 550-510 nm, respectively), while the most common ultraviolet wavelength pair (340–383 nm) was used for the glucose reaction. Single wavelength absorbance peak measurements were also included in this study in which Intralipid, a total parenteral nutrition solution, served as a controlled source of the interference, turbidity. By using single wavelength measurements for comparison to bichromatic determination, one could determine if advantages accrue to one or the other in the face of light scatter. Although Intralipid does not mimic natural hyperlipemia perfectly, choosing it allowed us to use uncontaminated preparations as a baseline to which we could compare the same specimens deliberately contaminated with known amounts of turbid substances. The concentrations obtained in this manner showed marked deviations from those values for protein and glucose known to be present in the studied turbid mixtures. These findings indicate that there may be shortcomings to bichromatics if it is used as a means of correcting for interference when the constraints inherent in such a process are not observed. The limitations of the bichromatic measurement process that should be kept in mind are discussed here.

Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.

Enzymic clearing of lipaemic serum following total parenteral nutrition: determination of neonatal bilirubin as a model

Bilirubin determinations in the newborn infant are one of the many analytical tests that can yield misleading results when the specimen is either iatrogenically or naturally lipaemic. Incorporation of a recently reported enzymic clarification system into a commercially available test kit enables one to conveniently and accurately quantify both total and direct bilirubin within the present procedural characteristics of the Bilirubin Stat Analyser Photometer. This instrument measures the former directly with bichromatic spectrophotometry and the latter with a conventional diazo-type reaction. The proposed modification of existing reagents allows one to apply the assay to samples with as much as, and possibly more than 16 g/l of triglycerides which has been introduced into the vascular circulation as intravenous total parenteral nutrition.