Icteric Serum Research Articles

Specificity of the thiobarbituric acid reaction: its use in studies of lipid peroxidation.

The thiobarbituric acid (TBA) reaction, quantified by colorimetry or fluorimetry, is the method most widely used for studying lipid peroxidation in both laboratory animals and in humans with disorders. However, concerns regarding its analytical specificity have often been expressed, because TBA reacts with a wide variety of chemical species to produce a pink to red color. In this study, we reacted TBA with various saturated and unsaturated aldehydes (both directly and in the presence of sucrose, fructose, and glucose), substituted pyrimidines, 2-deoxyribose, and N-acetylneuraminic acid. We also studied the TBA reaction with bilirubin, biliverdin, icteric serum, and serum containing hemolyzed erythrocytes, comparing the absorption spectra of these reaction products with that for malondialdehyde (MDA). The reaction products were also analyzed for MDA by high-performance liquid chromatography (HPLC). Although the TBA reaction with some of these compounds may not be important in biological studies, others could lead to misinterpretations of increased lipid peroxidation. Use of HPLC to quantify MDA is recommended because of its high analytical sensitivity and specificity, especially in the study of lipid peroxidation in human subjects.

Evaluation of an automated fluorescence polarization immunoassay for vancomycin.

An automated fluorescence polarization immunoassay for the determination of vancomycin levels in serum was evaluated. The vancomycin assay is a homogeneous competitive inhibition immunoassay based on changes in fluorescence polarization that occur with antibody binding. This assay was compared with a liquid chromatographic assay and an agar well diffusion bioassay method by using clinical serum specimens and controls. Linear regression analysis of the data obtained on clinical specimens by the three methods resulted in correlation coefficients of 0.97 for the fluorescence polarization immunoassay versus the liquid chromatographic assay (n = 60), 0.90 for the fluorescence polarization immunoassay versus the bioassay (n = 57), and 0.90 for the liquid chromatographic assay versus the bioassay (n = 57). Repetitive analysis of control sera containing 7, 35, and 75 micrograms of vancomycin per ml by the fluorescence polarization immunoassay yielded coefficients of variation of 3.0, 1.7, and 2.3, respectively. No interference was demonstrated in spiked hemolytic, lipemic, or icteric sera, and the assay was free of matrix effects. The automated fluorescence polarization immunoassay system offers a rapid, efficient, and accurate method for monitoring vancomycin serum levels for both toxicity and efficacy.

Biliprotein in adult icteric serum--demonstrated by extension of the alkaline methanolysis procedure.

We confirmed that the alkaline methanolysis procedure of Blanckaert (Biochem. J. 185: 115-128, 1980) converts the sugar conjugates of bilirubin (Bc) into their corresponding methyl and dimethyl esters, which can be extracted into chloroform along with underivatized unconjugated bilirubin (Bu). By this procedure, we accounted for Bu nearly quantitatively, but only 76-83% of total Bc. By pretreating samples containing Bu and (or) Bc with a caffeine/benzoate reagent, we improved the analytical recovery of Bc to 85-93% without affecting the Bu. When the method (+ caffeine/benzoate) was applied to adult icteric serum, a variable fraction (20-75%) of the original total bilirubin (based on diazo reactivity) remained with the protein pellet, which is routinely discarded in the original methanolysis procedure. In this pellet we demonstrated the occurrence of a strongly protein-bonded bilirubin fraction (biliprotein) similar to the recently described "delta" fraction (Clin. Chem. 28: 629-637, 1982). The analytical and clinical implications of our findings are discussed.

Indomethacin and bilirubin-albumin binding.

We studied the effects of indomethacin on the bilirubin binding of albumin using the hydroxybenzene azobenzoic acid (HBABA) dye binding method, the estimation of unbound bilirubin concentration (UBC) by the horseradish peroxidase assay and the whole blood fractionation (WBF) technique. The addition of up to 2 micrograms/ml of indomethacin, which is the level obtained with an 0.2 mg/kg dose, did not decrease the mean HBABA dye binding capacity of 29 icteric sera or increase the mean UBC of 11 icteric sera. There was also no additional displacement of bilirubin in 5 icteric sera measured by the WBF technique. However, the addition of 10-40 micrograms/ml of drug, which is the level that would be achieved if a 2 mg/kg dose of indomethacin were administered, resulted in a significant reduction of HBABA in 15 icteric sera (81.74-57.3%, p less than 0.005) and a significant increase of UBC in 11 icteric sera (6.17-37.2 nM/l, p less than 0.001). Therefore, no displacement of bilirubin from albumin occurred in the presence of usual therapeutic level of indomethacin; higher levels displaced bilirubin.

An evaluation of methods to monitor infants receiving intravenous lipids

Nephelometric measurement of light-scattering index and visual estimates of turbidity have been advocated to monitor serum Intralipid levels. This study describes a simple modified fluorometric method for accurately measuring lipid particles in serum and examines the reliability of such estimates compared with other chemical measurements. Ten percent IL was diluted with either saline or serum to various concentrations (0 to 250 mg/dl). The LSI showed an excellent correlation with known IL standard solutions in saline or serum (R = 0.99) and with triglyceride concentrations (R= 0.98). One hundred nine blood samples were obtained from 35 patients (28 neonates) receiving IL. An elevated TG, free fatty acid, or cholesterol level could not be reliably predicted from the LSI. The ability of clinical personnel to visually grade the degree of turbidity was evaluated by having them assign a turbidity score of 0 to 4+ to 39 hematocrit tubes which contained clear, hemolyzed, or icteric serum, each of which had IL concentrations varying from 0 to 292 mg/dl. The 15 tubes of identical IL concentration of 99 mg/dl were graded from 0 to 4+ by two of the observers, from 1 to 4+ by three of the observers, and 0 to 3+, 1 to 3+, and 2 or 3+ by one each of the observers. This study shows that (1) in vitro fluorometric LSI correlates well with IL concentrations; (2) in vivo correlations of LSI with FFA, cholesterol, and TG are poor; and (3) personnel are unable to reliably grade turbidity by visual examination of hematocrit tubes. Infants on IL should be monitored by TG and FFA levels.

New continuous-flow analysis for simultaneous determination of creatinine and uric acid in 200 microliters of serum without use of a dialyzer.

We present a new method for direct continuous-flow (AutoAnalyzer II) measurement of serum creatinine and uric acid. The manifold is simple, inexpensive, and can be constructed in the laboratory. Only 200 microliters of serum is needed; analysis rate is 60 samples per hour. The incorporation of sodium dodecyl sulfate and the simultaneous provision of blank subtraction make it possible to omit the dialysis step. Our method does not require the linearizer, since instrument response and concentration of creatinine and uric acid are linearly related to 200 and 120 mg/liter, respectively. The percentages of steady state, interaction, and recovery are acceptable, Precision is excellent and the results obtained from the new method correlate well with those obtained by the comparison methods. Interferences are few and, when encountered, are generally smaller than in the modified Technicon method. Marked hemolysis interferes only with the uric acid assay; marked turbidity has no effect on results for creatinine. Icteric serum with total bilirubin of 50 and 100 mg/liter interferes significantly with results for creatinine and uric acid, respectively, by the new method.

Spectrophotometric evaluation of interferences in three iron reactions for the determination of serum total cholesterol

A spectrophotometric and chemical evaluation of reported interferences for three iron reactions for the determinations of serum cholesterol has been presented. It has been shown that all three reactions are affected by various interfering substances, such as 2-thiouracil, nitrate, azide, bromide, diethylstilbesterol, and steroids. Spectral differences between the reactions are probably due to solvent and anion effects. The incorporation of uranyl acetate as a precipitating agent into the ferric acetate-uranyl acetate procedure did not make the results obtained comparable with the Abell-type extract of a very icteric serum. Incorporation of ferrous sulfate does not noticeably affect the intensity or stability of color development with standards. As proposed, the ferric acetate-uranyl acetate procedure for the determination of cholesterol represents a modified iron reagent, but the reaction mechanism and the procedure described for it is neither new nor direct. The use of the ferric acetate reagent for serum cholesterol as opposed to ferric chloride, ferric ammonium chloride, or ferric perchlorate appears to have no real advantages as a color reaction. In fact the reaction is less sensitive while reagent preparation is more tedious, time consuming, and expensive than the ferric chloride procedure. Although no quantitative studies were performed, the only effects that chloride had on the reactions were in the region of 400 nm, a wavelength sufficiently far enough away from the 560-nm peak that it did not affect determinations. The concept that the ferric acetate-uranyl acetate reagent contains only acetate and sulfate anions is nullified as soon as one adds serum to the reagents.

467 EVALUATION OF METHODS TO MONITOR INTRALIPID

Nephelometric measurement of light scattering index (LSI) and visual estimates of turbidity have been advocated to monitor serum Intralipid (IL) levels. Ten percent IL was diluted with either saline or serum to various concentrations (0-250 mg/dl). The LSI showed an excellent correlation with known IL standard solutions in saline or serum (R=.99) and triglyceride (TG) concentration (R=.98). One hundred fifteen blood samples were obtained from 35 patients (28 neonates) receiving continuous IL. The LSI was plotted against free fatty acids (FFA) (R=.60), cholesterol (Chol) (R=.22) and TG (R=.18). The ability of clinical personnel to grade visually the degree of turbidity was evaluated by having them assign a turbidity score of 0−4+ to 39 hematocrit tubes which contained clear, hemolyzed, or icteric serum, each of which had IL concentrations varying from 0-291 mg/dl. The 15 tubes of identical IL concentration of 99 mg/dl were graded between 0−4+ by 2 of the observers, between 1−4+ by 3 of the observers and between 0-3+, 1-3+ and 2-3+ by 1 each of the observers. This study shows that: 1) in vitro nephelometric LSI correlates well with IL concentrations. However, 2) correlations of nephelometric LSI with FFA, Chol and TG are poor; and 3) personnel are unable to grade turbidity by visual examination of hematocrit tubes. Therefore, infants on IL should be monitored by TG and FFA levels.

Microdetermination of unbound bilirubin in icteric newborn sera: An enzymatic method employing peroxidase and glucose oxidase

An enzymatic assay method for the microdetermination of unbound bilirubin in newborn icteric sera is described. Unbound bilirubin is oxidized to colorless compounds by peroxidase in the presence of hydrogen peroxide derived from glucose by the mediation of glucose oxidase. In this method, the bilirubin is not significantly degraded before the addition of peroxidase, in contrast to the method using hydrogen peroxide. The oxidation rate is determined by spectrophotometry and chloroform extraction is eliminated. The unbound bilirubin concentration can be determined fromthe initial oxidation velocity of total bilirubin. The Michaelis constant, K M was approximately 20 μM. The coefficient of variation for icteric serum determination was 4.4–6.5%. The concentration of unbound bilirubin was reduced after five days of storage at −20° C. The bilirubin-albumin binding affinity was studied with purified albumin and adult serum. The dissociation constants were 2 × 10 −8 M and 5 × 10 −9 M, respectively, at bilirubin/albumin molar ratios below 1.0. Clinically, serum samples from 75 icteric newborn infants were analysed, and the sera of premature infants were found to have remarkably high levels of unbound bilirubin compared to those of fullterm infants. The sera of a Rhesus immunization infant and an ABO incompatibility infant were remarkably higher than that of the nonhemolytic icteric sera. The unbound bilirubin concentration was also affected, in an in vitro study, by the addition of hemolysate.

Rapid Analysis for Theophylline in Serum by Use of High-Pressure Cation-Exchange Chromatography

We have developed a quantitative assay for serum theophylline by high-pressure cation-exchange chromatography. As little as 0.1-ml aliquots of serum were prepared for analysis simply by diluting them with a solution of the internal standard (8-chlorotheophylline). Theophylline and the internal standard were eluted in 17 and 27.5 min, respectively, and the peaks for them were distinct from those of other xanthines and uric acids. We encountered no interference when we used: (a) plasma obtained from blood anticoagulated with citrate; (b) hemolyzed, lipemic, or icteric serum; or (c) 52 samples from patients who were receiving various other medications. An analysis of calibration suggested that errors greater than plus or minus 2.6 mg/liter, a clinically acceptable range, were highly unlikely. Sensitivity was sufficient to identify less than 2.5 mg/liter. This assay was compared with the usual method involving solvent extraction and absorbance measurement in the ultraviolet and found to have similar accuracy, although it is easier, faster, requires less sample, and potentially is more specific.

Fluorescent dye method for determination of the bilirubin-binding capacity of serum albumin

A simple, rapid microfluorometric method utilizing a fluorescent dye, Direct Yellow 7, for quantitative measurement of serum albumin-binding capacity for bilirubin has been developed. The dye binds exclusively to serum albumin with enhancement of fluorescence. The dye shares bilirubin-binding sites on albumin with up to 2 mols of bilirubin per mol of albumin with differing affinities to the first and second sites. Lowered pH reduces albumin-binding capacity and exposure to light increases binding capacity of icteric sera. The binding capacity of adult human sera is greater than that of human cord sera or purified human serum albumin compared on albumin molar basis.

Radioimmunoassay of Serum Triiodothyronine on Small, Reusable Sephadex Columns

Abstract We have developed a radioimmunoassay method for serum triiodothyronine on small (9 x 23 mm), reusable Sephadex G-25 columns. Recoveries of triiodothyronine added to sera are 97-105%, and the coefficients of variation for within-assay and between-assay determinations are 5.8 and 9.0% , respectively. Maximum sensitivity is in the hypothyroid range (0-100 ng of T3 per dl). Triiodothyronine concentrations (ng/dl) in human serum are (mean ± SD): euthyroid 96 ± 26, euthyroid females receiving oral contraceptives 132 ± 17, hypothyroid 24 ± 22, and hyperthyroid 263 ± 58. Mild hemolysis and lipemia do not interfere, but 10-20% artifactual elevation is found with icteric sera. Interference by thyroxine is negligible. An improved, reproducible carbodiimide coupling procedure for triiodothyronine—albumin (19:1) antigen is described. A parenthetic observation of potential importance is that immunized rabbits accumulate large concentrations (30 000-90 000 ng/dl) of serum bound triiodothyronine. Our procedure offers the advantages that, on a single column, triiodothyronine is quantitatively extracted from serum that is premixed with 125l-labeled triiodothyronine, serum proteins are removed, binding with antiserum is accomplished in 1 h, and the free and bound triiodothyronine are separated. The columns are regenerated for repetitive use by eluting residual iodothyronines with excess thyroxine-binding globulin (dilute plasma).

Evaluation of the Adaptation of the Glucose Oxidase/Peroxidase-3-Methyl-2-benzothiazolinone hydrazone-N, N-Dimethylaniline Procedure to the Technicon "SMA 12/60," and Comparison with Other Automated Methods for Glucose

Abstract We have evaluated the Technicon SMA 12/60 modification of a glucose oxidase/peroxidase-3-methyl-2-benzothiazolinone hydrazone-N, N-dimethylaniline method, and find it acceptable. Added bilirubin, creatinine, dextrans, and uric acid did not interfere. We compared values for patients’ sera by this method to those by the glucose oxidase/peroxidase-2,2'-azinodiethylbenzothiazoline-6-sulfonic acid, neocuproine, o-toluidine, ferricyanide, and hexokinase methods. For comparisons to the hexokinase method (which we used as a reference method), n = 371, slope = 1.00, y-intercept = 1.57 mg/dl, and bias = 2.05 mg/dl. For the method being evaluated, the run-to-run average monthly standard deviation was 2.9 mg/dl for a control product for which the mean was 85 mg of glucose per deciliter, and 4.3 mg/dl for a product for which the mean was 240 mg/dl. All six methods were compared for effects of uremic sera, icteric sera, and sera from patients receiving ascorbic acid therapy or hypoglycemic drugs.

FLUORESCENT DYE METHOD FOR DETERMINATION OF BILIRUBIN-BINDING CAPACITY OF SERUM ALBUMIN

A simple, rapid microfluorometric method for quantitative measurement of serum albumin binding capacity for unconjugated bilirubin (UB), utilizing a fluorescent dye, Direct Yellow 7, has been developed. Sephadex G-200 column chromatographic study revealed exclusive association of serum albumin fraction with the dye. Spectrofluorometric study demonstrated enhanced fluorescence (ΔF) of the dye when bound to serum albumin. The addition of increasing concentrations of UB (5 to 70 mg%) to adult human sera resulted in a progressive inhibition of ΔF with complete inhibition at 55 mg%. Multiple regression analysis yielded two significantly different slopes (p<.001), first, from 0 to 27 mg% and second slope, 27 to 55 mg%. UB concentration of 27 mg% corresponded to UB: albumin molar ratio of 0.97, while 55 mg% to 1.97. Enhanced fluorescence of the dye resulted entirely from binding of the dye to two bilirubin-binding sites on albumin, since ΔF was completely inhibited by addition of two moles of UB per mole of albumin. Lowered pH reduced albumin binding capacity and exposure to light increased binding capacity of icteric sera. The binding capacity of purified human serum albumin was 7.1±0.8 ΔF/gm albumin (SD) (n=4), approximately one-half that of the adult human sera, 13.4±0.5 ΔF/gm (SD) (n=4), while human cord sera gave 9.3±1.1 ΔF/gm (SD) (n=41), 30% less than that of the adult sera on albumin molar basis.

Factors Affecting the Results of Serum Cholesterol Determinations: An Interlaboratory Evaluation

Abstract We present results of an interlaboratory survey of cholesterol assays in which methodology, standardization technique, type of standard, reagent source, and control sera used are considered. Each of these factors is evaluated, on both clear and icteric sera, with respect to accuracy, interlaboratory precision, and associated random error. We find that most participants use direct techniques, that these laboratories do not use correction procedures where these are needed, and that not all extractants effectively prevent bilirubin interference. Our data illustrate that precision on clear sera is best by direct procedures, but not in the presence of bilirubin. Participants uniformly exhibit better intralaboratory precision than interlaboratory agreement, regardless of the variable being studied. It is appropriate to consider the interactions of variables if a technique is to be properly evaluated.

Progress in Liver Disease

Whether or not the Australia antigen represents a virus remains to be established; nonetheless, identification of this antigen in the serum of hepatitis patients constitutes a fascinating development that may, hopefully, presage eradication of transfusion-induced hepatitis. Other recent developments discussed by the 51 contributors to Popper and Schaffner's book range from hepatic culture and transplantation to umbilical vein catheterization and phototherapy. Intense blue light, according to Hsia and Porto, decomposes the bilirubin in icteric serum and sometimes controls neonatal jaundice. They caution, however, against an ill effect from the degradation products. Reporting on another development, Chiandussi tells how to catheterize the umbilical vein. Umbilico-portal catheterization, he notes, permits selective splenic and portal phlebography, measurement of direct portal venous pressure, and decompression of the portal bed during hemorrhage from esophageal varices. He also notes that a physician can use the transumbilical catheter to infuse chemotherapeutic agents directly into a cancerous liver.

Some factors regulating duration of incubation for serum hepatitis.

The duration of incubation for icteric viral hepatitis appears to be shorter for fresh blood, serum, and plasma than for stored serum or plasma, pooled or unpooled. More than half of the cases of serum hepatitis from blood occur prior to the 60th day and three fourths before the 90th day. It has become evident also that one may extend the incubation period for infectious serum and plasma almost at will, especially by the use of ultraviolet light or by storage of liquid plasma or serum at temperatures between 20 and 35 C for three months or longer. Eventually, these extensions of icteric serum hepatitis reach infinity, in that no cases of serum hepatitis are observed. It is the purpose of this paper to present data from several sources in support of these statements and to discuss their possible relationship to the Australia antigen. In general, the longer the duration

An improved method for the colorimetric determination of free fatty acids

An improved method is proposed for the colorimetric microdetermination of free fatty acids in biological fluids, especially in icteric sera. The previous method could not be applied to icteric sera, because some pigment which had passed into the chloroform phase interfered with colorimetry at 440 mμ. Therefore, the following two improvements were introduced. 1. 1. Omission of the pigment from the final colored phase which is to be measured. 2. 2. Alteration of the wavelength of colorimetry from 440 mμ to 600 mμ by using bicyclohexanoneoxalyldihydrazone instead of diethyldithiocarbamate for the color-developing reagent. The sensitivity was intensified by more than 20% compared with that of the previous method.

Comparison of atomic absorption spectrophotometry and fluorometry for the measurement of serum calcium.

An atomic absorption spectrophotometric (AAS) and a fluorometric method for the measurement of serum calcium were found to be comparable in acceptable reproducibility. The recovery figures obtained by the fluorometric procedure were considerably lower than those given by AAS in the 2-4 mEq./L. range, although recoveries at higher levels (6 mEq./L.) were acceptable. The average recovery for the fluorometric method throughout the range 2-6 mEq./L. was 83.8%; for AAS, 98.9%. Marked interference was observed in the fluorometric method when hemolyzed or icteric serum was assayed. In four groups of hospital patients—premature infants, newborn infants, older children, and adults—AAS gave ranges for serum calcium values which were higher than those obtained by fluorometry.

Observations on diagnostic kits for the determination of total cholesterol

Summary Seventeen diagnostic kits or reagent sets from 15 manufacturers for the determination of total cholesterol were assessed. The kits were assessed for adequacy of literature supplied, the repro- ducibility of the procedures, the accuracy of results compared with those ob- tained by the reference method of Abell et al. (1952), the recovery of added cholesterol, the interference caused by icteric sera and the stability of the reagents throughout the test period. Eight kits failed to show sufficient precision to ensure results within accept- able limits of error of ±10%. Six kits yielded values significantly higher than those of the reference method and one kit gave lower results. A number of kits gave higher values in the presence of icteric sera. Only some of the manufacturers cautioned the users in their enclosed literature to be aware of this source of interference with their method. In general, the literature provided by the manufacturer is inadequate.