Icteric Serum Research Articles

Exploring the Relationship between Canine Paraoxonase-1 (PON-1) Serum Activity and Liver Disease Classified by Clinico-Pathological Evaluation.

Paraoxonase-1 (PON-1), a liver-synthesized enzyme, acts as a negative acute-phase reactant during systemic inflammation in dogs. Given the hepatic synthesis of this enzyme, the presence of liver diseases may influence PON-1, thus affecting its reliability as a biomarker for inflammatory/oxidative systemic diseases. The aim of this study is to investigate PON-1 activity variations among dogs suspected of liver injury or failure, evaluating the influence of hepatic diseases on PON-1 activity. A total of one-hundred-sixty dogs were retrospectively enrolled and categorized into three groups based on clinical presentation and laboratory results: control (C = 20), suspected liver injury (INJ = 114), and suspected liver failure (FAIL = 26). The INJ group was further divided into subgroups based on the severity of the alanine aminotransferase (ALT) increase. Both the INJ and FAIL groups were further divided based on serum macroscopic appearance. The PON-1 activity was quantified using a paraoxon-based method, which is already validated in dogs. No significant difference in PON-1 activity was observed between the C and INJ groups, despite a significant increase in the subgroups with moderate and severe elevations of ALT. The dogs with icteric serum exhibited decreased PON-1 activity, while lipemic serum was associated with an increased PON-1 activity. A significant reduction in PON-1 activity was noted in the FAIL group, compared to both C and INJ groups (p < 0.0001), regardless of serum appearance. Given the retrospective nature of this study, additional evaluations (e.g., histopathology, imaging) were not performed. The results obtained here suggest the importance of interpreting PON-1 activity cautiously in dogs with suspected liver disease.

When results matter: reliable creatinine concentrations in hyperbilirubinemia patients.

Background Failure to report a creatinine concentration, especially in icteric patients who are eligible for a liver transplant, can result in a life-threatening situation. We assessed the influence of bilirubin interference on several creatinine assays and investigated ways to circumvent icteric interference without interfering with our normal automated sample logistics. Methods Using icteric patient sera (total bilirubin >255 μmol/L) we determined creatinine concentrations using an enzymatic and Jaffé assay (Roche Diagnostics) in both normal (i.e. undiluted) and decreased mode (i.e. diluted) as well as an enzyme-coupled amperometric assay on a Radiometer ABL837 FLEX analyzer. Creatinine concentrations from the five methods were compared with an in-house developed LC-MS/MS method. Passing and Bablok (proportional and constant bias) as well as difference plot parameters (bias and 95% limits of agreement [LoA]) were calculated. Interferograph-based regression analysis of the enzymatic and Jaffé results was used to investigate if such an approach could be used to report corrected creatinine concentrations in icteric patient sera. Results In icteric patient sera the enzyme-coupled amperometric assay was hardly influenced by icteric interference as shown by a difference plot bias of -1.5% (95% LoA -11.6 to +8.5%). The undiluted Jaffé method had a bias of -1.4% with a very broad 95% LoA (-35.1 to +32.2%) emphasizing the poor specificity of this method. The undiluted enzymatic method had the largest bias (-13.4%, 95% LoA -35.8 to +9.0%). Diluting sera in the enzymatic method did not improve the bias (-10.5%, 95% LoA -25.4 to 4.4%), while diluting the Jaffé method resulted in a bias increase (+11.4%, 95% LoA -14.7 to 37.5%). Using interferograph-based regression analysis we were able to reliably correct enzymatic creatinine concentrations in 97 out of 100 icteric patient sera. Conclusions Analytically, quantifying creatinine in icteric sera using the Radiometer ABL837 FLEX analyzer is the method of choice within our laboratory. However, not all laboratories are equipped with this method and even if available, the limited number of highly icteric patient sera makes this method costly. For those laboratories using the Roche enzymatic method, mathematically correcting an icteric creatinine concentration using an interferograph based on an LC-MS/MS reference method is a suitable alternative to report reliable creatinine results in icteric patients.

BILIRUBIN CONCENTRATIONS IN CLINICALLY HEALTHY AND DISEASED CAPTIVE WATERBUCK (KOBUS ELLIPSIPRYMNUS) AT THE SAN DIEGO ZOO SAFARI PARK.

Captive waterbuck ( Kobus ellipsiprymnus ) that appear clinically healthy have been noted to have high serum bilirubin concentrations compared with other ruminants; however, questions remain about the physiologic factors affecting bilirubin concentration and its potential association with underlying disease and icteric serum or mucous membranes. Serum bilirubin concentrations of healthy and diseased waterbuck housed at the San Diego Zoo Safari Park from 1989 to 2012 were retrospectively analyzed to determine any link between icteric serum, total bilirubin concentration (tBili), and disease entities in this species. Total bilirubin and direct (dBili) bilirubin concentrations and the prevalence of icteric serum were compared by subspecies, age group, and health status; associations with complete blood count and biochemical results and clinical diagnosis were assessed. No significant differences were found in tBili or dBili between Ellipsen (n = 32) and Defassa (n = 29) subspecies or in juveniles (n = 22) versus adults (n = 39). Clinically healthy waterbuck (n = 40) had significantly higher tBili (mean ± 2SD, 7.9 ± 1.2 mg/dl; P < 0.001) and dBili (3.7 ± 1.0 mg/dl; P < 0.001) than did diseased waterbuck (n = 21; tBili: 4.9 ± 2.56 mg/dl; dBili: 2.2 ± 0.8 mg/dl). No waterbuck had icteric tissues on physical examination. Twelve (19.7%) waterbuck (six healthy, six diseased) had icteric serum. Few minor correlations were seen between tBili or dBili and clinical, laboratory, or necropsy evidence of disease, though an inverse correlation between dBili and blood glucose was noted. Of the 40 healthy animals, reference intervals were calculated for tBili (5.5-10.3 mg/dl), dBili (1.7-5.7 mg/dl), and indirect bilirubin (2.2-6.2 mg/dl). These results suggest healthy waterbuck have relatively high tBili and dBili compared with related species. Icteric serum may be seen in up to 15% of healthy animals in the absence of icteric tissues.

Refractometric total protein concentrations in icteric serum from dogs

To determine whether high serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among refractometer measurements. Evaluation study. Sera from 2 healthy Greyhounds. Bilirubin was dissolved in 0.1M NaOH, and the resulting solution was mixed with sera from 2 dogs from which food had been withheld to achieve various bilirubin concentrations up to 40 mg/dL. Refractometric total protein concentrations were estimated with 3 clinical refractometers. A biochemical analyzer was used to measure biuret assay-based total protein and bilirubin concentrations with spectrophotometric assays. No interference with refractometric measurement of total protein concentrations was detected with bilirubin concentrations up to 41.5 mg/dL. Biases in refractometric total protein concentrations were detected and were related to the conversion of refractive index values to total protein concentrations. Hyperbilirubinemia did not interfere with the refractometric estimation of serum total protein concentration. The agreement among total protein concentrations estimated by 3 refractometers was dependent on the method of conversion of refractive index to total protein concentration and was independent of hyperbilirubinemia.

Does bilirubin interfere with capillary electrophoresis of serum proteins?

Capillary electrophoresis of serum proteins is a fast, reliable and simple technique, but many interference exist. The objective of our work is to study the interference of bilirubin on this technique; 70 icteric sera were analysed on Capillarys ™ (Sebia). A second electrophoresis was performed on 40 samples after bilirubin photodegradation. The bilirubin and serum proteins were determinated respectively by Jendrassik and Grof and biuret methods on Konélab 20i ™ (Thermo Electron Corporation). We found abnormal spreading of the albumin fraction of the anode side wich constitute sometimes an isolated fraction in the traditional area of pre-albumin migration. This fraction varies from 2.0 ± 2.0% (0.0 to 7.3%) or 0.98 ± 1.53 g/L (0 to 5.3 g/L) and it seems to be related to the direct bilirubin since, following overloading sera with a solution of bilirubin, no further fraction was recovered. An average decrease of bilirubin after photodegradation of 58 ± 17% (26-89%) is followed by a decrease in the same order 64 ± 38% (10-100%) of the additional fraction. Acetate cellulose electrophoresis of the same samples showed no variation. The high bilirubin levels seem modify slightly the electrophoretic profile. However the impact of the interference on the interpretation of electrophoretic trace is negligible.

Role of training activities for the reduction of pre-analytical errors in laboratory samples from primary care

BackgroundThe presence of pre-analytical errors (PE) is a usual contingency in laboratories. The incidence may increase where it is difficult to control that period, as it is the case with samples sent from primary care (PC) to clinical reference laboratory. Detection of a large number of PE in PC samples in our Institution led to the development and implementation of preventive strategies. The first of these has been the realization of a cycle of educational sessions for PC nurses, followed by the evaluation of their impact on PE number. MethodsThe incidence of PE was assessed in two periods, before (October–November 2007) and after (October–November, 2009) the implementation of educational sessions. Eleven PC centers in the urban area and 17 in the rural area participated. In the urban area, samples were withdrawn by any PC nurse; in the rural area, samples were obtained by the patient's reference nurse. The types of analyzed PE included missed sample (MS), hemolyzed sample (HS), coagulated sample (CS), incorrect sample (ISV) and others (OPE), such as lipemic or icteric serum or plasma. ResultsIn the former period, we received 52,669 blood samples and 18,852 urine samples, detecting 3885 (7.5%) and 1567 (8.3%) PEs, respectively. After the educational intervention, there were 52,659 and 19,048 samples with 5057 (9.6%) and 1.256 (6.5%) PEs, respectively (p<0.001). According to the type of PE, the incidents compared before and after compared incidences were: MS, 4.8% vs. 3.8%, p<0.001; HS, 1.97% vs. 3.9%, p<0.001; CS, 0.54% vs. 0.25%, p<0.001; ISV, 0.15% vs. 0.19% p=0.08; and OPE, 0.3% vs. 0.42%, p<0.001. ConclusionsSurprisingly the PE incidence increased after the educational intervention, although it should be noted that it was primarily due to the increase of HS, as the other EP incidence decreased (MS and CS) or remained unchanged (ISV). This seems to indicate the need for a comprehensive approach to reduce the incidence of errors in the pre-analytical period, as one stage interventions do not seem to be effective enough.

Albumin-based or albumin-linked calibrators cause a positive bias in serum proteins assayed by the biuret method

Assay of total serum protein by the biuret method calibrated with albumin standards according to the reference method provides results with a positive bias approximately 3%-5% exceeding the total error of 3.4% allowable for total protein in serum analysis made by analysers using two-part reagents and short-term procedures. We used two types of two-part biuret reagents utilised in a short-term measurement in analysers with albumin or serum calibrators, in which protein was attested by the Kjeldahl method. Tests with potentially interfering substances proved that serum blanking used in a short-term biuret procedure is not capable of sufficiently eliminating effects of serum interferents. A short-term blanking is evidently capable of suppressing only an absorbance caused by serum-present coloured and turbid interferents, but its capacity to transform them (oxidise, hydrolyse, saponify, etc.) to some other not-interfering substances is very low compared with a long-term blanking. Lipids and bilirubin are responsible for significant positive bias of total protein in normal serum samples (approximately 3%) and even a greater positive offset in lipaemic and icteric sera (approximately 5%). We verified that interference tests based on a normal serum spiked with endogenous lipids and bilirubin give quite false and misleading results in the biuret reaction. A pure albumin, not depending on its bovine/human origin, gives absorbance responding only to its copper complexes with protein with a biuret regent, while its absorbance with a serum also includes the absorbance of interferents present in serum. The simplest way to improve current short-term biuret procedures is the use of a human serum calibrator with total protein attested by the Kjeldahl method. A serum calibrator, behaving analogously to serum samples, compensates for a positive bias in most normal sera. Reagents with a greater concentration of active biuret components (copper and alkali, reference method included) seem to be unnecessarily aggressive to proteins and are responsible for a lower accuracy when used in short-term measurements. Standard Reference Material 927c based on pure bovine albumin is still recommended and used as the primary standard for assays of total protein by colourimetric methods. The albumin calibrator is responsible for a positive bias of approximately 3%-5% in serum total protein assayed by the biuret reaction both in the reference and in current methods. Its substitution by a serum calibrator attested by the Kjeldahl method could solve this drawback. Clin Chem Lab Med 2009;47:91-101.

Development of an ELISA method for detecting immune complexes between tissue‐nonspecific alkaline phosphatase and immunoglobulin G

A convenient method for measuring immune complexes between tissue-nonspecific alkaline phosphatase (TNSALP) and immunoglobulin G (IgG) (i.e., TNSALP-IgG) would be highly useful for routine analyses. Here, we identified a surface-active agent that would dissolve membrane but not dissociate TNSALP-IgG complexes. Next, we developed an enzyme-linked immunosorbent assay (ELISA) method for detecting TNSALP-IgG complexes with two monoclonal antibodies (MoAbs): 3-29-3R was coated on assay plates and captured TNSALP-IgG from a specimen; an horseradish peroxidase (HRP)-conjugated anti-human IgG1 then reacted with captured TNSALP-IgG to form an "immunocomplex sandwich." The immunocomplex was detected via the absorbance of an HRP substrate, resulting in a semiquantitative assay. The mean absorbance of 0.195 (n=5) measured in sera from healthy donors was designated as an arbitrary unit (AU/mL) of TNSALP-IgG concentration. The ELISA values of patient sera known to contain TNSALP-IgG complexes were greater than those of normal sera (normal, 1.86 plusmn; 0.61; patient, 9.30 plusmn; 5.44), and these data were confirmed by electrophoresis. Thus, the ELISA could detect TNSALP-IgG complexes. The intraassay coefficient of variation (CV) was within 7.4% and analytical recovery was excellent. There was no significant interference from hemolytic, lipemic, or icteric serum. In summary, an ELISA using 3-29-3R MoAb and HRP-conjugated anti-human IgG1 constitutes a reliable and convenient method for the semiquantitative detection of TNSALP-IgG complexes in human serum.

805. Fatality in Mice Due to Oversaturation of Cellular Micro/Short Hairpin RNA Pathways

Induction of RNA interference (RNAi) by expression of short hairpin RNA (shRNA) holds significant promise for functional genomics and human gene therapy. Therefore, numerous non-viral and viral shRNA expression vectors are currently being developed and evaluated in pre-clinical models of human disease. Here we report efficient expression of shRNAs in entire murine livers from intravenously delivered Adeno-associated virus (AAV) serotype 8, carrying a novel stabilized double-stranded (sds) vector genome with the shRNA cassette flanked by an intact AAV-2 and a truncated AAV-4 terminal repeat (ITR). As opposed to conventional dsAAV with two AAV-2 ITRs, the use of heterologous repeats prevented recombination of the genome ends and thus markedly enhanced vector stability and yield. We engineered a panel of 49 distinct sdsAAV-8 vectors to express shRNAs unique in stem length (19 to 29 nucleotides), sequence, loop structure, and target (human alpha-1-antitrypsin or luciferase gene, or human Hepatitis B or C virus genome). Strikingly, their in vivo evaluation in various wildtype mice, or in animals transgenic for the targets, showed that 36 shRNAs resulted in dose-dependent and often massive liver injury. Typical observations were rises in liver enzymes, emergence of icteric sera, drops in liver-derived albumin, and as a result of the latter, frequent occurrence of ascites. Moreover, histological analyses revealed the presence of megakaryocytes, indicative of liver damage and hepatocyte repopulation. In fact, shRNA-induced liver injury was often associated with a transient RNAi effect, peaking within weeks after vector delivery and completely lost thereafter. Immunohistochemical and Southern blot analyses confirmed that shRNA expression caused individual cell death and liver regeneration with hepatocytes lacking the AAV/shRNA, explaining the transient phenotype. Most strikingly, 23 of the tested shRNA vectors ultimately caused death of the treated animals within a month after the injection. Northern blot analysis of total liver RNA showed that morbidity correlated with high shRNA expression levels, which were in turn frequently associated with down-regulation of liver-derived micro-RNAs. The latter remained stable in alternative models of liver injury, implying a global competition mechanism specifically from shRNA overexpression. In fact, subsequent in vitro and in vivo studies hinted at the karyopherin Exportin-5 as a limiting factor in the common pathway. At this point, we hypothesize that this carrier is used by shRNAs and miRNAs for nuclear export, and that its saturation with over-abundant shRNAs is a main cause for the observed liver toxicity and animal morbidity. Our findings thus strongly imply that control over intracellular shRNA expression levels will be mandatory for the clinical RNAi use, and generally underscore the inherent risks of this technology. These can likely be minimized by optimizing shRNA dose and sequence, as shown by our findings of persistent and therapeutic RNAi against human Hepatitis B virus in vivo.

Determination of monoethylglycinexylidide by fluorescence polarization immunoassay in highly icteric serum samples: modified precipitation procedure and HPLC compared

Hyperbilirubinemia, which frequently occurs in severe liver disease, interferes with the fluorescence polarization immunoassay (FPIA) monoethylglycinexylidide (MEGX) assay manufactured by Abbott Diagnostics. Because the MEGX test is particularly helpful in this clinical situation, strategies have been developed to overcome this problem. Precipitation of serum with the Abbott Digoxin II precipitation reagent eliminates bilirubin. Therefore, we compared FPIA results after precipitation of 81 icteric samples from 27 MEGX tests to results obtained using a validated HPLC method. The precipitation did not substantially alter the performance characteristics of FPIA: detection limit, 8 microg/L; between-days imprecision, 5.3-6.2%; recovery, 102-104% (50-200 microg/L). This pretreatment of serum did not eliminate all interference, and only a poor correlation was observed between serum MEGX concentrations measured with HPLC or modified FPIA (r2 = 0.46; S(y/x) = 20.0 microg/L). In contrast, MEGX formation values calculated by subtraction of the prelidocaine MEGX concentration were in close agreement (r2 = 0.98; S(y/x) = 2.3 microg/L). Because only MEGX formation is clinically relevant, this modified FPIA procedure offers a simple and rapid alternative to HPLC.

A new enzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase

A new enzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase (BOD) has been developed. At pH 5.5 BOD can oxidize only conjugated bilirubin in the presence of reagents such as sodium fluoride and N-acetylcysteine which can decrease BOD reactivity to unconjugated bilirubin and bilirubin covalently bound to albumin (delta bilirubin). The resulting decrease in absorbance at 450 nm is linearly related to the concentration of conjugated bilirubin in serum. The BOD in this new assay was confirmed to oxidize conjugated bilirubin, and neither unconjugated nor delta bilirubin, based on both its reactivity to unconjugated bilirubin and HPLC results. This assay was found to give satisfactory results, such as in terms of the range of measurement, the reproducibility of the results, the lack of interference with coexisting substances in serum and the stability of the reagent solutions, in practical applications. The serum conjugated bilirubin concentrations determined using this assay correlate well with those determined by the HPLC analysis. This assay can be used for accurate monitoring of changes in the conjugated bilirubin concentration in patient sera. These findings suggest that the conjugated bilirubin assay is useful for fractional determination of bilirubin in icteric sera.

Capillary Electrophoretic Enzyme Immunoassay for Digoxin in Human Serum

The combined use of capillary electrophoretic (CE) separation and homogeneous enzyme immunoassay for analyzing drugs in hemolyzed, lipemic, or icteric serum samples was investigated. An FDA-approved EMIT assay kit for digoxin in human serum was used. After the enzyme immunoassay, the enzymatic reaction product (NADH) and remaining substrate (NAD+), together with internal standard (p-nitrophenol, NP), were electrokinetically injected into a polyacrylamide-coated electrophoresis capillary and separated under applied potential. Detection was made by monitoring the UV adsorption at wavelength of 260 nm. The digoxin level in human serum was determined by comparing the peak area ratio of NADH and NP to the ratios established by the known digoxin standards. In this study, the factors that influence the CE separation were also investigated. Under the optimum conditions, NADH, NAD+, and NP were separated at electric field strength of 438 V/cm in the coated capillary (100 microns x 57 cm) with 200 mM Tris-borate buffer (pH 7.9) containing 0.2% hydroxypropyl methylcellulose. CE analyses of serum samples spiked with NADH standards at concentrations of 100 and 400 microM resulted in detection variabilities of less than 2% and analytical recoveries of 98-102%. Both an internal calibration plot for NADH and a dose-response curve for digoxin in serum were constructed. Calibrator serum, patients' sera with hemolyzed, lipemic, and icteric interference factors, and other pigmented blood components (e.g., serum albumin, bilirubin, hemoglobin, uric acid, coproporphyrin, melanin, protoporphyrin IX, and uroprophyrin) demonstrated no interference in this method. The authors believed that this method is useful for analyzing digoxin in hemolyzed, lipemic, and icteric blood samples that are known to create problems in conventional EMIT assays and may be applicable to other EMIT-based assays for monitoring drugs in complex biological matrices.

Discrepancies between lipoprotein(a) concentrations in icteric sera measured by immunonephelometry and electroimmunodiffusion

We compared the lipoprotein(a) [Lp(a)] levels in 32 icteric sera determined both ba an electroimmunodiffusion assay (EIA), using the Hydragel Lp(a) kit (Sebia, France) and by two immunonephelometric assays, one on a Behring Nephelometer Analyzer (BNA), using antiserum from Immunofrance, and the other on a Beckman analyzer (Array), using antiserum from, Dako (Denmark). With the EIA assay, the Lp(a) level was 0.09 ± 0.09 (mean ± SD in g/L), with the BNA assay, 1.01 ± 1.51 and with the Array assay, 0.05 ± 0.05. Sample blanks values (0.76 ± 1.28 g/L) demonstrated that the high Lp(a) levels obtained in the BNa assay are caused by nonspecific precipitation. Analysis of the precipitate indicated the presence of Lipoprotein X, and abnormal lipoprotein that appears in the serum of patients with obstructive jaundice or with lecithin-cholesterol acyltransferase deficiency. The precipitant seems to be polyethyleneglycol (PEG) that was added to the reaction medium in both the BNA and the Array assays to stabilize the Lp(a)-anti Lp(a) immune complex. In the Array assay, interference by this nonspecific precipitation is eliminated by preliminary centrifugation of the diluted sample. However, coprecipitation of Lp(a) could occur during this step. Consequently, the results of Lp(a) measurement in serum from patients with hepatobiliary diseases should be interpreted with caution when immunonephelometric assays are used with a medium containing PEG.

Simple sensitive solid-phase extraction of paraquat from plasma using cyanopropyl columns.

We report an inexpensive, sensitive paraquat quantitation method which is simple to perform. First, 5 mL of blanks, standards, or patient plasma are applied to 1-mL cyanopropyl extraction columns equipped with 15-mL reservoirs. The samples are drawn through the columns under vacuum, followed by a rinse with 15-20 mL of 0.1M NH4OH. Paraquat is eluted with 0.8 mL 0.1M HCl, which is then neutralized with 25 microL concentrated NH4OH. Sodium dithionite reagent (0.23M in 4M NaOH) is added (100 microL) and the color produced is measured by absorbance difference (A395-A460). The assay is linear up to at least 4.35 microM paraquat. The lower limit of quantitation is 0.23 microM. Lipemic and icteric sera do not affect the method, but easily visible hemolysis elevates the concentrations measured by up to 0.7 microM, independent of paraquat concentration. Equimolar amounts of diquat with paraquat, at paraquat concentrations from 0.4 to 4.0 microM, elevate apparent paraquat concentrations by 0.08-0.28 microM. At 0.632, 1.92, and 4.06 microM paraquat, within-run coefficients of variation (CVs) were 6.27, 7.23, and 2.14%, and between-run CVs were 6.82, 8.42, and 4.43%, respectively.

Effects of serum-isolated vs synthetic bilirubin-albumin complexes on dye-binding methods for estimating serum albumin

Bush and Reed reported (Clin Chem 1987;33:821-3) that the reaction of albumin with bromcresol purple but not with bromcresol green underestimated the concentration of albumin in synthetically obtained bilirubin-albumin (Bd) by 29%. Their unproven assumption was that chemically synthesized Bd behaved in a manner indistinguishable from the natural Bd in icteric serum. Here we verify that Bd, whether synthetically obtained or isolated from serum, causes an underestimation of albumin in the bromcresol purple but not in the bromcresol green method. The molar ratio of Bd from either source to underestimated albumin approximates 1.0, suggesting that one molecule of Bd would react equivalently to a molecule of albumin in the bromcresol purple method. This underestimation might falsely suggest hypoalbuminemia in patients with increased serum Bd.

A simple method for determination of serum catalase activity and revision of reference range

A rapid, cost-efficient, spectro-photometric assay for serum catalase activity was developed. It was a combination of optimized enzymatic conditions and the spectrophotometric assay of hydrogen peroxide based on formation of its stable complex with ammonium molybdate. Lipemic and icteric sera increased the absorbance without influencing the catalase assay. Due to the high catalase activity in erythrocytes artificial hemolysis increased serum catalase activity. The imprecision of the method was CV < 5.8% within run as well and day-to-day. The catalase assay performed using polarographic and spectrophotometric determination of hydrogen peroxide yielded a good correlation ( r = 0.9602, b = 1.011, a = −0.648, n = 440). In 742 healthy individuals the mean and SD values of serum catalase were 50.5 ± 18.1 kU/l with 17.7% higher activity in males than in females. Between 14–60 yr the serum catalase increased with age.

Bilirubin diglucuronide as the main source for in vitro formation of delta bilirubin

To clarify which of the bilirubin moieties is responsible for the formation of bilirubin bonded to albumin (delta bilirubin) in icteric serum, the in vitro formation of delta bilirubin from bile acid-free bilirubin glucuronides and unconjugated bilirubin was examined by high-performance liquid chromatography. Bovine serum albumin (150 mumol/liter) was mixed with equimolar bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG), or unconjugated bilirubin (UCB) and incubated in the dark at 37 degrees C under argon gas saturation. Although no delta bilirubin was formed immediately, formation eventually occurred and increased with time. A similar amount of delta bilirubin was formed when human serum albumin was used instead of bovine serum albumin. Of the three types of bilirubin, BDG was found to be the greatest source of delta bilirubin, whereas UCB produced the least. On the other hand, photoirradiation of a mixture of bovine serum albumin and UCB at a molar ratio of 1:1 resulted 6 hr later in the formation of three times as much delta bilirubin as in nonirradiated specimens. This photoinduced delta bilirubin formation increased further when the UCB/albumin molar ratio was increased to 2:1.

Icteric Serum as an Indicator of Fetal Vascular Access During Intravascular Transfusion for Hemolytic Disease

Correct needle placement during intravascular transfusion of the fetus may be difficult with an anterior placentation. Elevated bilirubin levels due to red cell hemolysis in the fetus of an alloimmunized pregnancy will result in icteric serum. The findings of such serum in centrifuged capillary tubes can be used to confirm fetal vascular access.

A rapid turbidimetric immunoassay for serum antistreptolysin-o2

We designed a rapid, quantitative turbidimetric immunoassay for serum antistreptolysin-O (ASO), based on the immunoprecipitating velocity with purified streptolysin-O (SLO), which can be measured turbidimetrically at 340 nm. The assay system involves polyoxyethylene mono-p-nonylphenyl ether to accelerate and enhance the immunoprecipitation reaction. No antibody excess phenomenon is observed, up to 1,700 U/ml. Grossly icteric sera can be assayed. Patient sera gave acceptable results. Free cholesterol offers no interference. The standard curve is linear for values up to 800 U/ml. Results measured by the proposed method and those by the conventional microtitration method correlated well. Analytical recovery averaged 90.1% (80.3%-102.5%). Within-run and day-to-day CVs ranged from 2.1% to 3.5% and from 2.5% to 6.9%, respectively. Working SLO is stable for at least 15 days at 4 degrees C. The proposed method has proven to be superior to other available methods for measuring serum ASO.

A sensitive, direct colorimetric assay of serum copper using 5-Br-PSAA

A direct colorimetric assay of serum copper in 0.2-ml sized samples, using a new, water-soluble reagent, 2-(5-bromo-2-pyridylazo)-5-( N-propyl- N-sulfopropylamino) aniline Na salt (5-Br-PSAA ε = 6.5 × 10 4 1/mol per cm at 580 nm), is presented. Protein-bound serum copper is dissociated with guanidine HCl which acts also as a protein denaturant. Metal ions, except for cobalt and palladium, do not affect the determination even when present at levels exceeding those normally found in serum. Although bilirubin does interfere with the Cu-5-Br-PSAA complex formation, the copper values in the icteric sera with total bilirubin concentrations of < 137/gmmol/l (80 mg/1) can be corrected by a correction factor which varies with the bilirubin content. Within-run and between-run precisions were in the ranges of 0.8–2.3% and 1.1–3.0% coefficient of variation (CV). Good correlation was obtained between this method and atomic absorption spectrometry.