Joseph D. Artiss

Spectrophotometric study of several sensitive reagents for serum iron

A comparison of three sensitive ligands for iron is described. All show varying degrees of enhancement of values by copper, an element that is the primary interference encountered in serum assays for iron. Two of three tested reagents, 2,4-bis(5,6-diphenyl-1,2,4-triazin-3-yl) pyridine tetrasulfonate and Ferene S are not current clinical chemistry choices whereas ferrozine is widely used. The described study presents spectral characterization of the copper interference with all three ligands. The virtual complete removal of this interference, by the masking actions of either thioglycolic acid, neocuproine or thiourea, is also described . Evidence will be presented that contradicts a previously proposed mechanism for the action of thioglycolic acid which suggests that copper is prevented from dissociating from its protein-binding site by this compound. The preparation of a protein-free filtrate as the matrix in which to generate the color reaction is described, and alternative procedures discussed in brief.

The benefits of early intervention in obese diabetic patients with FBCx: a new dietary fibre.

Obesity and diabetes have become epidemic in the US. Dietary fibres have been reported to reduce the absorption of dietary fat, prevent weight gain, and reduce blood lipid levels. In the current double‐blind study, obese patients with type 2 diabetes were recruited for a 3‐month study to examine the health effects of a new dietary fibre, FBCx™.

The Beneficial Effects α‐Cyclodextrin on Blood Lipids and Weight Loss in Healthy Humans

α‐Cyclodextrin (α‐CD) is a soluble fiber derived from corn. It has previously been reported that early intervention with Mirafit fbcx, a trademarked name for α‐CD, has beneficial effects on weight management in obese individuals with type 2 diabetes, and that it preferentially reduces blood levels of saturated and trans fats in the LDL receptor knockout mice. The current investigation involves overweight but not obese nondiabetic individuals and was intended to confirm the effects of α‐CD on both weight management and improving blood lipid levels. Forty‐one healthy adults (age: 41.4 ± 13.6 years) participated in this 2‐month, double‐blinded, crossover study. In 28 compliant participants (8 males and 20 females), when the active phase was compared to the control phase, there were significant decreases in body weight (−0.4 ± 0.2 kg, P < 0.05), serum total cholesterol (mean ± s.e.m., −0.295 ± 0.10 mmol/l, 5.3%, P < 0.02) and low‐density lipoprotein (LDL) cholesterol (−0.23 ± 0.11 mmol/l, −6.7%, P < 0.05). Apolipoprotein B (Apo B) (−0.0404 ± 0.02 g/l, −5.6%, P = 0.06) and insulin levels also decreased by 9.5% (−0.16 ± 0.08 pmol/l, P = 0.06) while blood glucose and leptin levels did not change. These results suggest that α‐CD exerts its beneficial health effects on body weight and blood lipid profile in healthy nonobese individuals, as previously reported in obese individuals with type 2 diabetes.

A method for the sequential colorimetric determination of serum triglycerides and cholesterol

A simple spectrophotometric method for the sequential determination of triglycerides and cholesterol from a single serum sample was developed. In this two-stage procedure, the triglycerides and cholesterol esters are first hydrolysed to glycerol and free cholesterol respectively, with simultaneous scavenging of the liberated free fatty acids, a technique that ensures clarity of the sample. The glycerol is subsequently reacted to result in an intense red chromogen with a peak absorption maximum at 510 nm following a series of enzymic reactions. In the second stage, addition of cholesterol oxidase leads to oxidation of free cholesterol generated from the cholesterol esters in the first stage and the free cholesterol normally present in the sample, yielding in a similar fashion the identical red chromogen whose absorbance is also measured at 510 nm. Results obtained with the proposed method demonstrate good correlation with established individual procedures for triglycerides and cholesterol.

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.

A liquid-stable reagent for lactic acid levels. Application to the Hitachi 911 and Beckman CX7.

We evaluated the use of a new lactate oxidase-based reagent for the determination of serum and plasma lactic acid levels with the Hitachi 911 (Roche Diagnostics, Indianapolis, IN) and the Beckman CX7 (Beckman Instruments, Brea, CA). Evaluation studies demonstrated on-board stability of at least 3 months and a calibration stability of more than 5 months. Within- and between-day imprecision of this reagent was less than 2% for both applications. The reagent is free of the deleterious effects of triglyceride up to levels of 1,400 mg/dL (15.8 mmol/L), bilirubin to concentrations of 24.6 mg/dL (420 mumol/L), and hemoglobin, from lysed erythrocytes, to levels of more than 0.3 g/dL (3.0 g/L). When used on the Hitachi 911 for the determination of plasma lactate concentrations, the reagent correlates with the Dade aca III (Dade International, Deerfield, IL). When applied to the Beckman CX7 for the determination of serum lactate levels, the method correlates with the Beckman method.

PROBLEMS WITH MEASUREMENTS CAUSED BY HIGH CONCENTRATIONS OF SERUM SOLIDS

There have been numerous reports of spectrophotometric and volume problems caused by elevated levels of lipids in blood. The offending lipids, primarily triglycerides, not only cause turbidity leading to optical aberrations when added to analytical reagents, but also result in short-sampling errors leading to the measurement of inaccurate volumes of sample. Numerous methods have been developed to clear the lipemia, including ultracentrifugation organic solvent extraction, chemical precipitation and, most recently, enzymic hydrolysis. Although the latter procedures eliminate the optical problems, they do not deal with the volume dilution error created by the triglycerides. In turn, corrective mathematics have been developed to compensate for the inaccurate pipetting caused by the elevated lipids in a sample; however, these empirical calculations are not truly accurate at high concentrations of total lipids. This monograph will describe the problems caused by the presence of elevated lipids and the means available for treating them.

Description of analytical problems arising from elevated serum solids.

There has always been a problem with the collection of data and interpretation of the results obtained from any biological fluid in which the solids content was increased to a great extent. Of these solids, the triglycerides of the lipids may cause a plasma (serum) to vary in appearance from opalescent to milky. This condition of the specimen and the concomitant turbidity upon its addition to reagents creates the well-documented optical aberrations of spectrophotometric measurements. In addition, the lipids, in conjunction with the proteins, can act as diluents when they are elevated, thereby decreasing what might be termed the residual true plasma volume. Thus the water content of an aliquot sampled for a particular analytical procedure is diminished, and by that means a situation is created in which a short sample is drawn. This dilution effect by the solids results in a lowering of the assay values obtained for the measured constituents of such a serum sample. An associated phenomenon of high concentrations of solids, especially proteins, is the increase in viscosity of a specimen, a condition that also causes an error of short sampling when certain peristaltic pumping devices are used. This review considers several aspects of problems encountered when dealing with a number of circumstances that are critical to the measurement of analytes in severely hyperlipemic and/or hyperproteinemic specimens. These include the problems of short sampling; the potential amelioration of the problem by corrective mathematics, extraction of the lipids, or ultracentrifugation of the true plasma from the lipids; the important need to include most analytes into our considerations; the difference in reference base values for the calculation of concentrations of lipids of serum versus other analytes; the concept of the use of ratios when the reference base values differ, numerator analyte from denominator analyte; and the problems of using serum blanks when necessary corrective action for the solids volume is neglected. Thus, in the final analysis, problems with underestimated volumes of samples used for many spectrophotometric determinations are considered here along with the other difficulties encountered when the need to measure analytes in serums with extremely high solids content presents.

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.

Determination of free, esterified, and total serum cholesterol using a sensitive color reaction

Abstract A method is described for the enzymatic determination of free and total cholesterol sequentially in either whole serum or the HDL fraction. Measurement of low levels of free cholesterol is facilitated by the substitution of sodium 2-hydroxy-3,5-dichlorobenzene-sulfonate for phenol in the peroxidase-catalyzed indicator reaction. Both free and total cholesterol may be measured in the same tube or separately. These alternatives may appeal to investigators who for various reasons may prefer to perform these determinations either sequentially or separately. The sequential approach is a means of conserving the relatively expensive enzymatic reagents.