bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparative chromatographic purification of cyclosporine metabolites
Authors: Brooks C A , Cramer S M , Rosano T G
Published: January 2020

Polar and primary metabolites of cyclosporin A (CsA) have successfully been isolated by a novel separation protocol. An efficient, easy-to-scale-up chromatographic adsorption/desorption operation recovers polar and primary CsA metabolite pools from large volumes of urine; purified CsA metabolites are subsequently obtained by high-resolution preparative elution chromatography of the semipurified metabolite pools. Separations performed on a semipreparative scale [with a 250 x 9.4 mm (i.d.) reversed-phase HPLC column] yielded microgram quantities of CsA metabolites at > 97% purity, as determined by fast atom bombardment mass spectrometry. These separations also yielded two previously unreported CsA metabolites, similar to AM1A but with an additional hydroxylation. The yield of metabolites was increased to several milligrams by performing the separations with a preparative-scale [250 x 21.2 mm (i.d.)] reversed-phase column. The production rate of purified primary CsA metabolites was greatly increased by performing the separation with the preparative-scale column under conditions of severe mass overloading. In a single chromatographic run, we successfully isolated 11.0 and 5.0 mg of AM1 and AM1c, respectively, at a purity of > 97%. As expected, this increase in the yield of purified metabolites was accompanied by a decrease in the overall recovery. This separation scheme enables the rapid processing of large volumes of urine for isolation of the milligram quantities of CsA metabolites necessary to assess their biological activity. The procedure is applicable to small- or large-scale metabolite isolation and provides a ready source of purified metabolites for in vitro and whole-animal studies.
Characteristics and clinical application of a radiometric Escherichia coli-based phospholipase A2 assay modified for serum analysis
Authors: Aufenanger J , Zimmer W , Kattermann R
Published: January 2020

Determination of activities of phospholipase A2 (PLA2) in human sera was based on the hydrolysis of phospholipids from [1-14C]oleic acid-labeled Escherichia coli biomembranes. The E. coli membranes served as substrate specifically for the PLA2 of human serum and were essentially resistant to other lipases in human sera, i.e., lipoprotein lipases, hepatic triacylglycerolipase, or pancreatic lipase in acute pancreatitis. Exchange of phospholipids between the serum and the biomembrane compartment aggravates the determination of PLA2 activity in human serum, which is naturally rich in phospholipids. In our modified E. coli assay, which overcomes these difficulties, the main substrate components phosphatidylethanolamine (70%) and cardiolipin (25%) were > 90% labeled in the sn-2 position. Fatty acids released by PLA2 activity were eluted from an aminopropyl solid-phase column directly into scintillation vials, where the radioactivity was counted. The ratio of [1-14C]oleic acid to released total fatty acids was used to calculate true enzymatic activity. The linear assay range extended from 0 to 3.6 U/L (0-60 nkat/L), with a detection limit of < 0.03 U/L (< 0.5 nkat/L). Within-assay imprecision (CV) was < 6% and between-assay is < 10% over the whole activity range. The normal range for men was 0-0.44 U/L (0-7.33 nkat/L) and for women 0.044-1.11 U/L (0.73-18.4 nkat/L). Patients with septicemia, pancreatitis, acute respiratory distress syndrome, or other severe diseases had PLA2 values up to 540 U/L (9000 nkat/L).
One-step competitive immunochromatographic assay for semiquantitative determination of lipoprotein(a) in plasma
Authors: Lou S C , Patel C , Ching S , Gordon J
Published: January 2020

Numerous studies have associated high concentrations of lipoprotein(a) [Lp(a)] with atherosclerosis. We developed a rapid, one-step competitive immunochromatographic assay to measure Lp(a) in plasma. The assay is performed on a nitrocellulose membrane strip and the result is determined by a visual readout of rust-colored colloidal selenium. The assay is based on the principle that Lp(a) in the sample will compete with Lp(a)-coated colloidal selenium for binding to the anti-Lp(a) monoclonal antibody immobilized on the assay strip in the format of four ladder bars. The number of capture bars that appear as a result of the formation of colloidal selenium color is proportional to the concentration of the Lp(a) protein in the samples. The strip assay semiquantitatively measures Lp(a) concentrations ranging from 0 to 180 mg/L of Lp(a) protein in serum, plasma, or fingerstick whole-blood samples. This assay appears very useful for quick identification of individuals with above-normal concentrations of plasma Lp(a) protein (> 70 mg/L), and has potential for monitoring a patient's response to treatment with Lp(a)-lowering drugs.
Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation
Authors: Risteli J , Elomaa I , Niemi S , Novamo A , Risteli L
Published: January 2020

We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.
Lipid peroxidation and trace element status in diabetic ketotic patients: influence of insulin therapy
Authors: Faure P , Corticelli P , Richard M J , Arnaud J , Coudray C , Halimi S , Favier A , Roussel A M
Published: January 2020

Lipid peroxidation is known to accelerate aging and microvascular lesions in diabetic patients. We studied the acute influence of improved glycemic control on the concentrations of plasma lipid peroxidation intermediates [malondialdehydes (MDA), organic hydroperoxides (OHP)] in ketotic insulin-dependent diabetic patients, as well as the interplay of enzymes such as glutathione peroxidase (GPX) and CuZn superoxide dismutase (CuZn-SOD), and trace elements (Zn, Se, Cu) postulated to be involved in free radical protection. These plasma components were measured on the first day of hospitalization (T0) and when the patient had attained a stable glycemic state after continuous insulin treatment (T1). Plasma MDA and OHP concentrations were high at the beginning of the study but approached reference values after glycemic equilibration. Plasma zinc concentrations were significantly (P < 0.05) decreased during the ketotic state, but also approached reference values with glycemic equilibration. Plasma selenium concentrations and GPX activity were relatively unchanged between T0 and T1. Erythrocyte GPX activity measured at T1 in six patients was below the reference values, whereas CuZn-SOD activity was not affected. Our results show that enhanced lipid peroxidation is associated with decreased zinc plasma concentrations in ketotic patients and underline the beneficial effects of continuous insulin infusion. The mechanisms involved are still unclear but may indicate the importance of extracellular zinc transfer secondary to glycemic disorders.
Fast HPLC determination of serum free fatty acids in the picomole range
Authors: Püttmann M , Krug H , von Ochsenstein E , Kattermann R
Published: January 2020

We developed a method for determining individual free fatty acids in serum by using a modified one-step Dole extraction, derivatization, and a new high-performance liquid chromatographic (HPLC) separation. Sample handling is minimized to a single transfer of the fatty acids (upper layer of the Dole extract), which are readily derivatized at 85 degrees C with p-bromophenacyl bromide without significant hydrolysis of esterified fatty acids. The derivatization mixture is directly injected into the HPLC apparatus. The new method, which uses C6 (3-microns particle) column material and an isocratic acetonitrile-water eluent, separates nearly to baseline 12 of the physiologically most abundant long-chain fatty acids (C12-C22) in < 20 min with a detection limit of approximately 2 pmol. It is therefore suitable for routine analysis even with basic HPLC equipment and can easily analyze a series of 10-20 samples in about 2 h including extraction until first results are available. The method is also applicable to other matrices than serum, e.g., for determination of precursor fatty acids such as arachidonic acid in platelets or of fatty acid patterns liberated by lipases or phospholipases A1/A2 in test systems.
Increased fetal hemoglobin in insulin-treated diabetes mellitus contributes to the imprecision of glycohemoglobin measurements
Authors: Kilpatrick E S , Small M , Rumley A , Dominiczak M H
Published: January 2020

An increased prevalence of fetal hemoglobin (HbF) has been described in pediatric insulin-dependent patients. The popular electroendosmotic method for glycohemoglobin includes HbF. In an adult population comprising 50 insulin-treated and 57 non-insulin-treated diabetic patients and 57 control subjects, we measured HbF by HPLC and measured glycohemoglobin by both HPLC and an electroendosmotic method. Of the insulin-treated patients, 46% had concentrations of HbF > or = 0.5%, compared with 25% of non-insulin-treated patients and 23% of controls (P < 0.02). In the insulin-treated patients, the two glycohemoglobin methods correlated best when the HPLC measurements included HbF (r = 0.92 vs r = 0.84). Fructosamine concentrations correlated best with glycohemoglobin concentrations determined by methods that accounted for HbF. The true between-batch CV of the electroendosmotic assay increased (from 4.33% to 8.33%) when variable interpatient HbF concentrations were included. Thus, HbF must be considered when interpreting glycohemoglobin measured by an electroendosmotic method and when comparing it with other measures of glycemic control.
International cooperation in analytical chemistry: experience of antidoping control at the XI Pan American Games
Authors: Segura J , Pascual J A , Ventura R , Ustaran J I , Cuevas A , Gonzalez R
Published: January 2020

We describe the experience of the international cooperation carried out for antidoping control at the XI Pan American Games. A temporary accreditation was granted by the International Olympic Committee to the accredited laboratory of Barcelona (Spain) to set up a Doping Control Laboratory in Havana. Two other laboratories from Mexico and Cuba contributed personnel, materials, and instrumentation. The main issues associated with the preparation and organization of the project are described. During 16 days, 741 urine samples were tested for stimulants, narcotics, anabolic steroids, beta-blockers, diuretics, cannabinoids, local anesthetics, and human chorionic gonadotropin by gas-liquid chromatography, gas chromatography/mass spectrometry, high-performance liquid chromatography, and immunoassay techniques. Analytical methodologies, quality-control strategies, and the main results are reported.
Interference of lysophosphatidylcholine in hormone radioimmunoassays
Authors: Lepage N , Roberts K D , Langlais J
Published: January 2020

The interference of synthetic and naturally occurring detergents in immunoassays is well documented. In the present study, we evaluated the effect of lysophosphatidylcholine (LPC) and found that the lysophospholipid interfered with formation of the antigen-antibody complex in hormone immunoassays. In the presence of LPC (100 mumol/L), progesterone was overestimated by 29%. Furthermore, physiological concentrations of LPC (140 mumol/L) interfered with the assays of cortisol, progesterone, and aldosterone, resulting in overestimations of 35%, 30%, and 27%, respectively. The addition of albumin decreased the interference by LPC to 7% in the assay of cortisol and progesterone when the LPC:albumin ratio was unity. Adding cholesterol (100 mumol/L) also reduced by 50% the interference induced by LPC. Finally, treating plasma to increase the endogenous LPC concentration also resulted in interference in the cortisol assay. Thus, interpretation of the results of these assays should take into consideration the endogenous serum albumin:LPC ratio.
Role of antibody valency in hapten-heterologous immunoassays
Authors: Piran U , Silbert-Shostek D , Barlow E H
Published: January 2020

We studied the effects of hapten heterology on immunoassays of triiodothyronine (T3), digoxin, and cortisol, in a format involving labeled monoclonal antibodies and immobilized, protein-conjugated ligands. Replacing the homologous conjugated ligands T3, digoxin, and cortisol with their respective analogs diiodothyronine, digitoxin, and corticosterone led in each case to a decrease in the midpoint of displacement (ED50) for the same zero-dose signal. The mechanism of this phenomenon was studied by converting the bivalent anti-T3 to a monovalent whole antibody (bispecific monoclonal anti-T3 x anti-glucose-6-phosphate dehydrogenase) by cell fusion. The monovalent antibody was effective as a tracer in the homologous T3 assay, but generated a very low zero-dose signal with the heterologous solid phase, thus precluding sensitivity enhancement. On the basis of these results and additional kinetic and double-labeling experiments, we propose that the use of hapten heterology relies on bivalent binding of the antibody to the solid phase to compensate for a lower intrinsic affinity. This binding mechanism leads to lower assay concentrations of the ternary complex analyte-labeled antibody-immobilized hapten, thereby providing enhanced sensitivity.
Medically significant concentrations of prostate-specific antigen in serum assessed
Authors: Bernstein L H , Rudolph R A , Pinto M M , Viner N , Zuckerman H
Published: January 2020

Vol. 36

p. 517. In the article by L.H. Bernstein, R.A. Rudolph, M.M. Pinto, N. Viner, and H. Zuckerman entitled "Medically significant concentrations of prostate-specific antigen in serum assessed," 1990;36:515-8, the text describing Table 4 reports reversed values for sensitivity and positive predictive value and likewise for specificity and negative predictive value. The text should read, "Table 4, a reorganized Table 3, defines the occurrence of binary class patterns with respect to carcinoma of the prostate by stage compared with nondisease. The table allows the estimation of 44.4% sensitivity, 97.1% specificity, a negative predictive value of 79%, and a positive predictive value of 87.8% for PSA >22.8 ng/mL."

Vol. 38

p. 2557. In the latter to the editor by J.D. Mitchell, B.J. Perrigo, and V.A. Mason-Daniel entitled "Falsely negative urine drug assay results due to filtration," 1992;38:2556-7, the units in the first paragraph at the top of p. 2557 should have been micrograms per liter (µg/L), not pg/L.
Biological variability in concentrations of serum lipids: sources of variation among results from published studies and composite predicted values
Authors: Smith S J , Cooper G R , Myers G L , Sampson E J
Published: January 2020

To obtain the best estimates of the average intraindividual biological variability (CVb) in the concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), and triglyceride serum lipids in a person's blood, we evaluated results from 30 studies published from 1970 to 1992. The usually more applicable random-effects model estimated an average CVb of 6.1% for TC, 7.4% for HDLC, 9.5% for LDLC, and 22.6% for triglyceride. Composite estimates of the average CVb from all evaluated published studies by different models of estimation ranged from 6.0% to 6.4% for TC, from 6.2% to 7.5% for HDLC, from 7.0% to 9.6% for LDLC, and from 22.4% to 22.9% for triglyceride. Two important factors influenced the reported biological variation of the study subjects: (a) the magnitude of the variability of the analytical method used and (b) the design characteristics of the study--primarily the number of subjects, the sampling interval, and the number of measurements per subject. For TC, we found a statistically significant positive correlation between the reported mean CVb and both the number of study subjects and the analytical variation. For TC and LDLC we estimate CVb as a function of the study design features. The number of patient specimens required to obtain reliable estimates for serum lipid concentrations are determined from the CVb and the current analytical variation.
Evaluation of colorectal cancer-associated mucin CA M43 assay in serum
Authors: van Kamp G J , von Mensdorff-Pouilly S , Kenemans P , Verstraeten R , Yedema C A , Wobbes T , Bon G G , Kok A , Brown J P , Hilgers J
Published: January 2020

The technical performance of a newly developed assay for CA M43, a serum marker for colorectal cancer, was evaluated and its preliminary clinical potential assessed. The heterologous double-determinant enzyme immunoassay for the detection of the tumor-associated mucin CA M43 utilizes two monoclonal antibodies (CT 43 and CT 66) selected for their binding capacity to two distinct epitopes present on mucins in the sera of patients with colorectal cancer. CT 66 recognizes both Lewis(a) and sialylated Lewis(a) antigen; CT 43 is directed toward a mucin epitope of an as-yet uncharacterized structure. Precision experiments revealed interassay CVs of 11.8%, 5.9%, and 4.9% at 9.3, 11.9, and 78.9 units/mL, respectively; intraassay precision was 2.0% at 95.1 units/mL. The upper normal value was set at 7.5 units/mL, which included 99% of the values found in healthy controls. In colorectal cancer patients, CA M43 showed a positivity rate equivalent to that of carcinoembryonic antigen (CEA) and superior to that of CA 19.9, with only one CA 19.9-positive serum being negative for CA M43. Interestingly, CA M43 appeared to be complementary to CEA, with CA M43 and CEA together reaching 87% positivity in metastatic disease.
Analytical evaluation of i-STAT Portable Clinical Analyzer and use by nonlaboratory health-care professionals
Authors: Jacobs E , Vadasdi E , Sarkozi L , Colman N
Published: January 2020

We evaluated the performance of the i-STAT Portable Clinical Analyzer, a hand-held instrument that, with its current cartridge, analyzes for electrolytes, urea nitrogen, glucose, and hematocrit in approximately 60 microL of whole blood in approximately 90 s. Accuracy, imprecision, and linearity studies were performed with aqueous controls and standards and by split-sample analysis. Intrarun imprecision (CV) ranged from 0.34% to 3.97%. Total imprecision over a 2-month period ranged from 0.42% to 4.83%, with urea nitrogen and glucose analyses generating the higher values. Patients' results from the Portable Clinical Analyzer correlated well with those obtained for whole blood or plasma by the Nova Stat Profile 5, the Beckman Synchron CX3, or the Technicon H1 Hematology Analyzer, with Sylx values < 0.2 mmol/L for potassium; < 1.5 mmol/L for sodium, glucose, and urea nitrogen; < 2.4 mmol/L for chloride, and < 2.4% for hematocrit. We also ascertained imprecision and accuracy of the system placed in a cardiothoracic intensive-care unit and operated by nurses. There were no significant differences in either the imprecision or accuracy of the system in this setting. We conclude that operator technique is not a factor in the analytical performance of the system and that it can be used by nonlaboratorians with a high degree of confidence that reliable results will be obtained.
Reported effects of long-term freezer storage on concentrations of retinol, beta-carotene, and alpha-tocopherol in serum or plasma summarized
Authors: Comstock G W , Alberg A J , Helzlsouer K J
Published: January 2020

A literature review of the effects of long-term freezer storage on concentrations of antioxidant micronutrients in serum or plasma showed that a high proportion of the studies that compared the concentrations of retinol, beta-carotene, or alpha-tocopherol in fresh and stored serum were deficient because of small numbers of observations, imprecise descriptions of procedures, and short periods of storage. Data from nested case-control studies of the associations of these micronutrients with cancer are confounded by differences in the study populations. Nevertheless, the overall impression is strong that retinol is stable at storage temperatures as warm as -20 degrees C for at least 15 years, that moderate losses of alpha-tocopherol occur at temperatures above -40 degrees C during that period, and that only a small proportion of beta-carotene persists at storage temperatures above -40 degrees C. At -70 degrees C or colder, all three micronutrients appear to be stable for at least 15 years.
Commercial kits for 1,25-dihydroxyvitamin D compared with a liquid-chromatographic assay
Authors: Bertelloni S , Baroncelli G I , Benedetti U , Franchi G , Saggese G
Published: January 2020

Three commercially available kits for 1,25-dihydroxyvitamin D [1,25(OH)2D] determination were compared with a liquid-chromatographic method. Serum samples were analyzed for 1,25(OH)2D from 70 healthy subjects (age 2.2-17.5 years; 35 males, 35 females), some (n = 5) given orally high vitamin D3 doses. In addition, 1,25(OH)2D was measured in 28 patients with untreated diseases associated with low (n = 16) or high (n = 12) serum concentrations of the hormone. The results showed that the commercial kits were sufficiently accurate with respect to the comparison method and suitable for routine clinical work.
Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles
Authors: Brousseau T , Clavey V , Bard J M , Fruchart J C
Published: January 2020

We describe a fast sequential separation of very-low-density, low-density, and high-density lipoproteins from 400 microL of serum, using the Beckman TL100 ultracentrifuge. The cumulative centrifugation time is 9.5 h. The purity of lipoprotein fractions was verified by a gel-filtration procedure. The major contaminant is the serum albumin, which can be eliminated by a second centrifugation at the same density. Enzymatic measurement of lipids shows good recovery (> 91%) and weak within-sample variation (< 7%). In comparison with a density-gradient procedure, the deleterious effects on the lipoprotein structure appear to be limited, as shown by the low concentrations of apolipoprotein (apo) E and apo A-I in the fraction > 1.21 kg/L. Furthermore, the micro-ultracentrifugation also gives a better recovery rate. Finally, we have studied the distribution of lipids, apolipoproteins, and lipoprotein particles (LpA-I:A-II, LpB:C-III, LpB:E) in each fraction separated from 10 serum samples from healthy subjects.
Redox status and protein binding of plasma aminothiols during the transient hyperhomocysteinemia that follows homocysteine administration
Authors: Mansoor M A , Guttormsen A B , Fiskerstrand T , Refsum H , Ueland P M , Svardal A M
Published: January 2020

We administered reduced L-homocysteine perorally (67 mumol/kg of body wt) to 12 healthy subjects and injected the same dose into one person, and determined the kinetics of the alterations in reduced, oxidized, and protein-bound concentrations of homocysteine, cysteine, and cysteinylglycine. After oral intake, reduced homocysteine increased rapidly (tmax < or = 15 min), reaching concentrations [3.97 (SD 2.99) mumol/L] 20-fold above fasting values, and then declined towards the normal concentration within 2 h. There was a similar increase in reduced cysteine and a moderate increase in reduced cysteinylglycine. During this response, we observed a positive correlation between the reduced/total ratio for homocysteine and cysteine. When homocysteine was injected, the increase in reduced homocysteine preceded the increase in reduced cysteine by about 3 min. After oral loading, oxidized homocysteine showed a transient increase (tmax = 30 min) that lagged behind the increase of reduced homocysteine. Oxidized cysteine and cysteinylglycine were stable or decreased slightly. Protein-bound homocysteine increased the least rapidly after homocysteine administration (tmax = 1-2 h), and returned to normal values slowly. Changes in protein-bound homocysteine essentially mirrored a concurrent decrease in protein-bound cysteine, suggesting displacement of bound cysteine. These data show that plasma homocysteine has a pronounced, direct effect on the redox status and protein binding of other plasma thiol components. Such effects should be recognized when studying the mechanisms behind the atherogenic effect of increased plasma homocysteine.
Quantification of lipoprotein(a) particles containing various apolipoprotein(a) isoforms by a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apolipoprotein B detection antibody sandwich enzyme immunoassay
Authors: Taddei-Peters W C , Butman B T , Jones G R , Venetta T M , Macomber P F , Ransom J H
Published: January 2020

A quantitative sandwich ELISA for lipoprotein(a) [Lp(a)], utilizing a monoclonal capture antibody that recognizes human and rhesus monkey apolipoprotein(a) [apo(a)] isoforms in combination with a polyclonal anti-apolipoprotein B-peroxidase conjugate was developed. This assay generates a linear calibration curve from 31.2 to 1000 mg/L, is highly reproducible (intra- and interassay CV of < 5% and < or = 12%, respectively), and shows no interference from plasminogen (1 g/L), low-density lipoprotein (6.00 g/L), triglycerides (27.00 g/L from chylomicrons and 10.00 g/L from very-low-density lipoprotein), hemoglobin (5 g/L), or bilirubin (30 mg/L). This assay format quantifies the concentration of Lp(a) on an equal molar basis regardless of apo(a) isoform. In contrast, a commercially available ELISA [Macra Lp(a)] method with a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apo(a) conjugate was found to underestimate the Lp(a) concentrations of individuals with lower-M(r) apo(a) isoforms--whether quantifying the Lp(a) in plasma or the purified lipoprotein. This demonstrates the importance of assay format selection in quantifying Lp(a).
Simple method for extracting RNA from cultured cells and tissue with guanidine salts
Authors: Zolfaghari R , Chen X , Fisher E A
Published: January 2020

We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.
Enzyme immunoassay of beta-hexosaminidase A and B in serum: carrier detection of GM2-gangliosidoses, and equivalence of enzyme activity and enzyme protein reactivity
Authors: Isaksson A , Hultberg B , Masson P , Landels E , Fensom A
Published: January 2020

beta-Hexosaminidase (Hex; EC 3.2.1.52) isoenzymes A and B were analyzed in sera from a control group of 22 apparently healthy subjects, 13 obligate carriers of Tay-Sachs disease (TSD), 10 obligate carriers of Sandhoff disease (SHD), and 4 affected TSD patients by enzyme immunoassay methods based on enzyme activity. No Hex A activity was detected in the sera of patients with TSD. The activities of Hex A in the obligate carriers of TSD and SHD tended to be lower (nonsignificantly) than in the control group. Hex B activities tended to be higher in TSD patients as well as in carriers of TSD, although the mean activities did not significantly differ from the corresponding mean for the control group. However, Hex B activities were decreased in the carriers of SHD in comparison with the other groups. Sera from 900 postmenopausal women, all of age 55 years, were also analyzed for Hex isoenzymes; the results indicated a carrier frequency of about 1 in 200 for both TSD and SHD. We also compared the enzyme immunoassay method based on enzyme activity with one based on the antigenic (enzyme protein) reactivity alone. Because both methods yielded similar information, we conclude that no significant amounts of inactive enzyme protein are present in the circulation.
Governmental perspectives on evaluating laboratory performance
Authors: Boone D J
Published: January 2020

The quality of laboratory analytical performance required to support medical decision-making has been defined in four major ways: (a) by the analytical variance of the state of the practice; (b) by the total variance, including analytical and biological variability; (c) by the loss of diagnostic efficiency attributable to analytical error; and (d) by medical-usefulness criteria. From the federal government's perspective, the answer to the question "How good must a laboratory test result be to be medically relevant?" must take into account the clinical context of the test, with accompanying concerns about access, timeliness, and cost, as well as limits for precision and accuracy in the analytical process and the frequency and potential patient-care impact of error in the pre- and postanalytical steps of the total testing process. Therefore, medically relevant goals should encompass not only analytical precision and accuracy but also goals to provide access to clinically effective tests and to reduce errors in the total testing process that can lead to medically misleading information. Development of more appropriate regulatory requirements for laboratories, as well as any needed improvements in instrumentation and methodology, should focus on ensuring that goals for medically relevant results are met by appropriate design and management of the entire process of laboratory testing.
Application of gene transfer technologies to the production of enzyme reference materials: example of gamma-glutamyltransferase
Authors: Siest G , Oster T , Visvikis A , Thioudellet C , Angèle C , Schiele F , Wellman M
Published: January 2020

Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.
Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization
Authors: Andersson A , Isaksson A , Brattström L , Hultberg B
Published: January 2020

We describe a versatile high-performance liquid-chromatographic method for determining homocysteine and other plasma sulfhydryls. Using three different procedures for preparation of plasma, we determined total, free (non-protein-bound), and reduced forms of homocysteine, cysteine, glutathione, cysteinylglycine, and gamma-glutamylcysteine in human plasma. Sample preparation involves disulfide reduction with dithiothreitol and protein precipitation with sulfosalicylic acid. The assay utilizes isocratic reversed-phase ion-pair liquid chromatography at pH 2.4, postcolumn derivatization with 4,4'-dithiodipyridine, and colorimetric detection at 324 nm. The intra-assay precision (CV) of the method for total homocysteine is 1.5%; the interassay precision over 2.5 months is 2.5%. The detection limit for homocysteine is < 50 nmol/L plasma.
Determination of clozapine, norclozapine, and clozapine-N-oxide in serum by liquid chromatography
Authors: Volpicelli S A , Centorrino F , Puopolo P R , Kando J , Frankenburg F R , Baldessarini R J , Flood J G
Published: January 2020

We report a new assay to measure the serum concentrations of the atypical antipsychotic drug clozapine and two major metabolites, norclozapine and clozapine-N-oxide. The analytes and an internal standard (triprolidine) were extracted from alkalinized samples into ethyl acetate and back-extracted into 0.1 mol/L HCl. The acid extracts were chromatographed on a reversed-phase liquid chromatographic column with photodiode array detection (210-340 nm). With the 254-nm signal, between-run imprecision (CV) was < 2% for clozapine and norclozapine at 400 micrograms/L, and 4.1% for clozapine-N-oxide at 100 micrograms/L. Absolute recovery exceeded 65%, and the detection limit was approximately 3-4 micrograms/L. In 25 patients at steady state at a mean daily clozapine dosage of 269 mg (3.09 mg/kg), clozapine averaged 231 +/- 144 micrograms/L (mean +/- SD); norclozapine and clozapine-N-oxide concentrations averaged 84% and 23% that of clozapine. Analyte concentrations were significantly correlated with daily dose. The method's ability to quantify clozapine and two major metabolites simultaneously with precision and sensitivity makes it useful in pharmacokinetic studies and therapeutic monitoring.
Free thyroxine assessed with three assays in sera of patients with nonthyroidal illness and of subjects with abnormal concentrations of thyroxine-binding proteins
Authors: Docter R , van Toor H , Krenning E P , de Jong M , Hennemann G
Published: January 2020

Three methods for estimating free thyroxine (FT4) in serum were studied: equilibrium dialysis, the SPAC-ET FT4 radioimmunoassay kit, and the Amerlite MAB FT4 luminometric assay. Serum samples from 10 subjects with above-normal thyroxine-binding globulin (TBG), 6 with low TBG, 30 with familial dysalbuminemic hyperthyroxinemia (FDH), 13 with nonesterified fatty acids (NEFA) concentrations in serum > 1.0 mmol/L, and 178 patients with various degrees of nonthyroidal illness (NTI) were measured and compared with samples from 42 euthyroid blood donors. The Amerlite MAB FT4 assay compared well with equilibrium dialysis, whereas the SPAC-ET assay averaged 40% lower. All three assays were not influenced by changes in TBG and showed no or only little changes in the presence of NEFA. Mean FT4 values in the FDH samples were somewhat higher than in controls when measured with the SPAC-ET assay, about equal with equilibrium dialysis, and somewhat below the mean control value with the Amerlite MAB FT4 assay, although individual results were within the control reference range. In NTI patients, no FT4 values were below the control reference range by the Amerlite MAB FT4 assay, 4 of 178 were below this range by equilibrium dialysis, and 1 of 178 was below this range by the SPAC-ET assay. In all assays a large proportion of the NTI samples showed FT4 values above the control reference range, a result that will interfere with the efficacy of these assays for assessing thyroid function in NTI patients.
Fast, manual, nonradioactive method for DNA sequencing
Authors: Debuire B , Chabli A , Frenoy N
Published: January 2020

We describe a protocol that allows nonradioactive detection of sequencing products after manual, direct, solid-phase sequencing of polymerase chain reaction-amplified DNA. The amplified DNA fragment to be studied is biotinylated at the 5' end of one of the two oligonucleotide primers used for amplification, allowing coupling to streptavidin-coated magnetic beads. The immobilized double-stranded DNA is then separated into single strands by alkaline treatment. A 5'-biotinylated sequencing primer is used after saturating with a biotin solution any possible remaining affinity sites on the streptavidin-coated magnetic beads. Sequencing is performed by using T7 DNA polymerase, and the sequencing products are electrophoresed in denaturing polyacrylamide sequencing gel. After transfer of the products to a nylon membrane, the sequencing pattern is revealed by chemiluminescence. Biotinylated alkaline phosphatase is bound to the 5' end of the sequencing primer via a streptavidin bridge and catalyzes the reaction by cleaving a phosphate group from a chemiluminescent substrate. The emitted photons are detected by exposing the membrane to x-ray film. This method is simple, rapid, and consistently successful and reproducible.
A method to quantify deviations from assay linearity
Authors: Krouwer J S , Schlain B
Published: January 2020

We present a statistical method to quantify deviations from linearity for assays that veer from linear assay responses. Our procedure handles the common case of unequally spaced analyte levels and nonconstant variance and provides a least-squares estimate with a confidence interval for the amount of deviation from assay linearity at a specified analyte concentration. This estimate of assay bias due to nonlinearity goes beyond the NCCLS EP6 lack-of-fit test, which tests for only the presence of nonlinearity. Knowing that nonlinearity is present is insufficient; users need to know the magnitude of the bias caused by nonlinearity. Our method can also be used with multifactor designs that estimate other systematic assay effects such as drift and carryover, thus obviating the need for a separate protocol to assess linearity. The procedure is carried out by adding extra columns to the design matrix corresponding to the concentration level(s) of interest. The extra columns, which replace the quadratic column, are orthogonal to all other columns. We describe a general method of constructing the new columns, and illustrate the procedure with a manual ammonia assay example dataset from EP6.
Primary aldosteronism in a patient with an aldosterone-producing adenoma
Authors: Pekarske S L , Herold D A
Published: January 2020

We describe an unusual patient presenting with a history of refractory hypertension and hypokalemia. Initial screening tests for adrenal hypertension were consistent with primary aldosteronism and an abdominal computed tomography scan showed an 8-mm left adrenal mass. However, adrenal venous sampling revealed markedly suppressed plasma aldosterone in the left adrenal vein but increased plasma aldosterone in the right adrenal vein. Therefore, on the basis of the clinical, radiographic, and laboratory findings, we concluded that the patient had a nonfunctioning adrenocortical adenoma on the left and an aldosterone-producing adrenocortical adenoma on the right, with the aldosteronoma resulting in hypertension and hypokalemia. Right adrenalectomy decreased the hypertension and corrected the hypokalemia. The right adrenal contained a 7-mm nodule microscopically consistent with the diagnosis of a cortical adenoma. The case highlights key steps and potential pitfalls in the evaluation of adrenal hypertension.
Development of third-generation immunochemiluminometric assays of follitropin and lutropin and clinical application in determining pediatric reference ranges
Authors: Pandian M R , Odell W D , Carlton E , Fisher D A
Published: January 2020

We developed dioxatane-based immunochemiluminometric assays (ICMAs) for lutropin (LH) and follitropin (FSH), using monoclonal antibodies. These ICMAs have a minimal detectable dose (analytical sensitivity) of 0.01 IU/L, extending the lower limit of sensitivity 10-fold (from 0.10 IU/L) when compared with immunoradiometric assays (IRMA) (second generation), and thus provide a true third-generation assay. Daytime FSH and LH concentrations were measured in 236 boys and 195 girls. Unlike the previous assays, all the samples had detectable concentrations of LH and FSH. In agreement with results from earlier methods, the present results indicate that for both sexes mean FSH and LH concentrations are relatively high during the early months of life, fall to baseline prepubertal concentrations by 12-18 months, and remain low until the onset of puberty. During puberty, the mean concentrations of FSH and LH increase significantly in both girls and boys with each stage of puberty, but there is considerable overlap between stages. These third-generation FSH and LH ICMAs reliably separate daytime plasma FSH and LH concentrations of prepubertal children from those of sexually mature children, and therefore can more reliably distinguish between the major causes of precocious puberty (e.g., gonadotropin dependent and independent). Our LH assay is also useful in monitoring the gonadotropin-releasing hormone therapy of patients with gonadotropin-dependent precocious puberty.
New reagent for colorimetric assay of calcium in serum
Authors: Chapoteau E , Czech B P , Zazulak W , Kumar A
Published: January 2020

We report a new single-reagent colorimetric assay for the determination of calcium in serum. The assay is based on a novel chromogenic tetracarboxylic acid chelating agent. The method has several advantages over the o-cresolphthalein complexone (CPC) method, including improved linearity, better accuracy, increased stability, and lower working pH (9.0). The calibration curve for the assay is linear up to 4 mmol/L. The results for 47 patients' samples correlated well with results obtained with an Instrumentation Laboratory atomic absorption spectrophotometer. The mean (+/- SD) calcium values for the new method were 2.27 +/- 0.06 mmol/L and 2.26 +/- 0.05 mmol/L for the comparison method. The on-system stability of the reagents is 1 week vs 8 h for the CPC method.
Microaffinity chromatographic separation and characterization of lipoprotein fractions in rat and mongolian gerbil serum
Authors: Tschantz J C , Sunahara G I
Published: January 2020

A rapid, routine microaffinity chromatographic procedure with spectrometric detection was evaluated to measure the concentration of total and free cholesterol, triglycerides, and phospholipids linked to the alpha- and beta-/pre-beta-lipoproteins in very small serum samples from small experimental animals and from humans. This procedure overcomes many of the limitations presented by methods involving ultracentrifugation, electrophoresis, or precipitation. Using this method, we report that in 35 adult male Mongolian gerbils (Meriones unguiculatus) fed a basal commercial diet with no cholesterol supplementation, the mean (+/- SD) concentration of total and free cholesterol linked to the alpha-lipoprotein fraction (in mmol/L) is 2.85 +/- 0.81 and 0.33 +/- 0.14, respectively; phospholipids were 1.99 +/- 0.41, and triglycerides were 0.64 +/- 0.42. The concentration (mmol/L) of total and free cholesterol associated with the beta-/pre-beta-lipoprotein fraction is 0.92 +/- 0.26 and 0.22 +/- 0.06, respectively; phospholipids were 0.28 +/- 0.17, and triglycerides were 0.49 +/- 0.31.
Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction
Authors: Findlay J B , Atwood S M , Bergmeyer L , Chemelli J , Christy K , Cummins T , Donish W , Ekeze T , Falvo J , Patterson D
Published: January 2020

An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.
Rare earth cryptates and homogeneous fluoroimmunoassays with human sera
Authors: Mathis G
Published: January 2020

The rare earth cryptates are used as long-lived fluorescent labels. They are formed by inclusion of a europium ion in the intramolecular cavity of a macropolycyclic ligand containing bipyridine groups as light absorbers. The use of fluoride ions in the measuring medium allows a total shielding of the label fluorescence. Here, I have established the conditions under which the signal of these cryptates can be amplified through the use of nonradiative energy transfer, and applied these conditions to homogeneous immunoassays by using allophycocyanin as the acceptor. Because only a low proportion of the cryptate label is involved in the transfer amplification, and because allophycocyanin emission occurs between the europium emission lines, time-resolved measurement of europium and allophycocyanin emission allows real-time correction of the optical properties of the assay medium. These features have allowed the development of a rapid homogeneous immunoassay of prolactin that can detect as little as 0.3 microgram/L (3rd International Standard 84/500).
Diagnostic accuracy of pancreatic enzymes evaluated by use of multivariate data analysis
Authors: Kazmierczak S C , Catrou P G , Van Lente F
Published: January 2020

We analyzed pancreatic enzyme data from 508 patients with suspected pancreatitis by neural network analysis, by an Expert multirule generation protocol, and by receiver-operator characteristic (ROC) curve analysis of a single test result. Neural network analysis showed that use of lipase provided the best means for diagnosing pancreatitis. Diagnostic accuracies achieved by using amylase only, lipase only, and amylase and lipase in combination were 76%, 82%, and 84%, respectively. Use of the Expert rule generation protocol provided a diagnostic accuracy of 92% when rules for single and multiple samplings were combined. ROC curve analysis for initial enzyme activities showed the maximal diagnostic accuracy to be 82% and 85% for amylase and lipase, respectively; use of peak enzyme activities yielded accuracies of 81% and 88%, respectively. The evaluation of laboratory test data should include analysis of the diagnostic accuracy of laboratory tests by multivariate techniques such as neural network analysis or an Expert systems approach. Multivariate analysis should allow for a more realistic assessment of the diagnosis accuracy of laboratory tests because all the available data are included in the evaluation.