bluuebunny/crossref_metadata_2025 huggingface dataset:
Drug screening with EMIT reagents: a quantitative approach to quality control
Authors: Standefer J C , Backer R C
Published: January 2020

We investigated the precision, linearity, accuracy, and stability of quantitative results for five drugs of abuse [amphetamines, benzoylecgonine, opiates, phencyclidine, and the cannabinoid-tetrahydrocannabinol (THC)-9-acid metabolite], analyzed in control specimens by using EMIT d.a.u. reagents (Syva Co.) with a Monarch 2000 analyzer with a nonlinear interpolation curve-fitting algorithm. The within-day and between-days coefficients of variation (CVs) were less than 5% for all drugs except THC-9-acid, which had a CV between 10% and 20%. The drift of control values during a 30-day stability study was less than 10% from target values for three weeks after a single calibration, except for THC-9-acid control values, which were stable for only two to three days. Daily calibration reduced the drift away from target values during the 30-day stability study and produced optimum precision of all drug assays. Mean control values near the National Institute on Drug Abuse cutoff limits were within 10% of their target values.
kappa/lambda index for confirming urinary free light chain in amyloidosis AL and other plasma cell dyscrasias
Authors: Levinson Stanley S
Published: January 2020

Primary systemic amyloidosis (AL), a disease involving the deposition of immunoglobulin light chains in tissue, is caused by a plasma cell dyscrasia. In the case of amyloidosis reported here, no monoclonal component was seen upon routine protein electrophoresis of serum or urine nor was a bone marrow analysis positive for AL. Immunofixation electrophoresis did not show a typical paraprotein band but did show, in the gamma region, two large diffuse bands and a lower concentration of oligoclonal-type bands, all of which stained for free lambda but not for free kappa chain. The ratio of kappa to lambda chains in urine was 0.178, much less than the ratio in serum (1.3). Six other urine samples from a group of patients with documented Bence Jones proteinuria also exhibited kappa/lambda ratios that differed manyfold from the ratios in their corresponding serum samples. On the other hand, the kappa/lambda ratios from seven controls (seven patients with generalized proteinuria unrelated to plasma cell dyscrasia) were similar in serum and urine. This difference between the kappa/lambda ratios from serum and urine can be expressed as a kappa/lambda index. The index was significantly different (P less than 0.01) between the two patient groups compared here, and was useful in confirming the presence of Bence Jones protein in this case with a difficult-to-interpret electrophoretic pattern. Although the kappa/lambda ratio has been widely used for confirmation and identification of monoclonal components in serum, its use in clinical laboratories has not been widely extended to urine. Comparison of serum and urine kappa/lambda ratios as a kappa/lambda index may help reduce the need for more complex immunoelectrophoresis techniques in identifying free light chains in urine.
Bone protein and peptide assays in the diagnosis and management of skeletal disease
Authors: Deftos Leonard J
Published: January 2020

The measurement in blood of bone proteins is an important adjunct to imaging procedures for clinical assessment of the skeleton. The discovery of new bone proteins and their structural characterization has led to immunochemical procedures of improved sensitivity and specificity for their quantification. An appreciation of the advantages and disadvantages of these evolving methodologies will assist in their clinical application.
Cholesterol distribution between high-density-lipoprotein subfractions HDL2 and HDL3 determined in serum by discontinuous gradient gel electrophoresis
Authors: Atger Véronique , Malon Denise , Bertiere Marie Claude , N'Diaye Françoise , Girard-Globa Anik
Published: January 2020

We used discontinuous gradients of polyacrylamide gel to determine the high-density-lipoprotein (HDL) subfractions HDL2 and HDL3 of serum lipoproteins. Serum (40 microL) prestained with Sudan Black was electrophoresed in cylindrical tubes over successive layers of 3.5%, 6%, 13%, and 17.5% acrylamide gels in a Tris.glycine buffer (3-4 h, 300 V). Very-low- (VLDL) and low-density lipoprotein (LDL) were retained by the 3.5% and 6% gels. HDL2 was concentrated at the interface between the 13% and 17.5% gels, and HDL3 migrated into the 17.5% gel. The distribution between HDL2 and HDL3 was obtained by densitometric scanning. Application of the respective percentages to HDL cholesterol assayed after phosphotung-state-Mg2+ precipitation of VLDL and LDL gave calculated concentrations of HDL2 and HDL3 cholesterol. The calculated values for HDL2 cholesterol were in excellent agreement with those for HDL2 isolated by ultracentrifugation (r = 0.920 for n = 120 sera; differences nonsignificant by Student's paired t-test). Besides being highly discriminating, the method is rapid, easily performed, and economical.
Toxicological screening of drugs by microbore high-performance liquid chromatography with photodiode-array detection and ultraviolet spectral library searches
Authors: Turcant A , Premel-Cabic A , Cailleux A , Allain P
Published: January 2020

We use ultraviolet data, acquired with a photodiode-array detector coupled to a reversed-phase liquid-chromatographic system, to identify unknown drugs in plasma samples of acutely poisoned patients. Both retention time and spectra of the peaks obtained with a microbore Hypersil ODS column under gradient elution are compared with a library of approximately 350 compounds. We present our three-year experience with this system, which identifies drugs in less than 1 h, with a high degree of confidence.
Liquid-chromatographic determination of nicotine and cotinine in urine from passive smokers: comparison with gas chromatography with a nitrogen-specific detector
Authors: Hariharan M , VanNoord T
Published: January 2020

We describe a simple, sensitive, and specific high-performance liquid-chromatographic method with ultraviolet detection (256 nm) for the simultaneous analysis of nicotine and cotinine in urine of passive smokers. The analytes are extracted and purified from the complex and impure matrix in two stages; first, by liquid-liquid extraction and followed by solid-phase extraction (C2 column). We used a "DB" C8 5-microns-particle column (25 x 0.46 cm) and a mobile phase of phosphate-citrate buffer and acetonitrile (91:9 by vol) containing 5 mL of triethylamine and 600 mg of heptanesulfonate per liter, adjusted to pH 4.4, to separate the compounds. Two internal standards (2-phenylimidazole and N-ethylnorcotinine) were used. The detection limit of the HPLC assay was less than 1 microgram/L for both analytes. The average interassay CV for nicotine was 7.6%, for cotinine 6.5%, in the concentration range 0-60 micrograms/L. The mean analytical recovery of nicotine with respect to the internal standard N-ethylnorcotinine was 102% and that for cotinine was 99%; the mean absolute recoveries of the compounds were as follows: nicotine 85%, cotinine 87%, and N-ethylnorcotinine 87%. To compare the HPLC assay results of 20 samples with the more-sensitive in-laboratory gas-chromatographic method with a nitrogen-phosphorus detector, we used both N-ethylnornicotine and N-ethylnorcotinine as internal standards. The mean correlation coefficient for nicotine values between the two methods was 0.934; for cotinine, 0.987.
Angiotensin-converting enzyme determination in plasma during therapy with converting enzyme inhibitor: two methods compared
Authors: Gorski T P , Campbell D J
Published: January 2020

For normal and above-normal concentrations of angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity in plasma, results of a manual fluorometric method [with hippuryl-histidyl-leucine (HHL), 5 mmol/L, as substrate] correlated well with those of an automated spectrophotometric method [with 3-(2-furylacryloyl)-L-phenylalanyl-glycyl-glycine (FAPGG), 2 mmol/L, as substrate]. However, for patients receiving converting enzyme inhibitor (CEI) therapy, the spectrophotometric method showed much greater suppression of plasma ACE activity than did the fluorometric method. To determine which of the two methods provided a more reliable indication of ACE inhibition in vivo, we measured plasma ACE, angiotensin I (ANG I), and angiotensin II (ANG II) in patients receiving the CEI perindopril. During perindopril therapy, changes in the ratio of ANG II:ANG I, an index of ACE activity in vivo, showed a close agreement with changes in plasma ACE activity measured with FAPGG as substrate, but not with HHL as substrate. We conclude that measurement of ACE activity in vitro with FAPGG as substrate provides a reliable measure of changes in conversion of ANG I to ANG II in vivo during CEI therapy.
Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin
Authors: Obzansky D M , Rabin B R , Simons D M , Tseng S Y , Severino D M , Eggelte H , Fisher M , Harbron S , Stout R W , Di Paolo M J
Published: January 2020

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.
Novel solid-phase assay of ketone bodies in urine
Authors: Kundu S K , Judilla A M
Published: January 2020

This simple dip-and-read color-matching assay measures ketone bodies in urine. The result is matched with any of the seven colors provided in a color chart. The intensity and hue of the color formed depends on the concentration of acetoacetic acid and follows a gradation starting from light purple (0.2 mol/L acetoacetate) to dark purple and finally to dark violet (9 mol/L). The color developed is independent of pH in the range pH 4-9.5, and is stable for at least 1 h. 3-Hydroxybutyric acid produced no color at the concentrations tested (less than or equal to 100 mmol/L), and acetone was detectable only when greater than or equal to 15 mmol/L. The test was compared with the Ames Ketostix assay in subjects who were on restricted-calorie weight-loss programs. The two methods showed good agreement, except that 20% (21/103) of samples that were negative by Ketostix were read as positive by the new method, which may be more sensitive (detection limit = 0.2 mmol/L). Measurement of urine ketones was unreliable (in comparison with measurement of blood or breath acetone) for monitoring the rate of fat loss during a negative calorie balance.
Inclusion of proteins into calcium oxalate crystals precipitated from human urine: a highly selective phenomenon
Authors: Doyle I R , Ryall R L , Marshall V R
Published: January 2020

The abundance of protein in the matrix of calcium oxalate uroliths has fueled speculation regarding its role in stone genesis. In this study, we wanted to characterize the composition of the proteins associated with early stages of calcium oxalate crystallization in urine. Calcium oxalate crystallization was induced in urine from healthy men and women by the addition of an oxalate load. The crystals were harvested and demineralized, and the proteins remaining were separated and characterized by polyacrylamide gel electrophoresis and Western blotting. Most urinary proteins were not detected in the crystals or were present in only small quantities. The most abundant urinary macromolecule, Tamm-Horsfall glycoprotein, was notably absent from the crystal extracts. The predominant protein associated with the crystals, a previously unknown urinary constituent that we call crystal matrix protein (CMP; molecular mass, 30,000 Da), was more prevalent in the crystals derived from female urine. We conclude that most urinary proteins play no direct role in calcium oxalate crystal formation. However, the protein CMP exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis.
Enzymatic digestion, solid-phase extraction, and gas chromatography/mass spectrometry of derivatized intact oxazepam in urine
Authors: Langner J G , Gan B K , Liu R H , Baugh L D , Chand P , Weng J L , Edwards C , Walia A S
Published: January 2020

Enzymatic digestion with beta-glucuronidase (EC 3.2.1.31) was used to release intact oxazepam from urine samples containing the d5-analog internal standard. The resulting specimens were extracted with Du Pont PREP Type W cartridge (processed by a PREP Automated Sample Processor), Bond Elut Certify, and J.T. Baker "spe" columns for comparison of the columns' extraction recovery and overall effectiveness. Methyl iodide/tetrahexylammonium hydrogen sulfate and N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (10 g/L) were used for the methylation and trimethylsilylation studies. We used a Hewlett-Packard HP 5790 mass-selective detector equipped with a 13-m J & W DB-5 column (5% phenyl polysiloxane phase) for gas chromatography/mass spectroscopy (GC/MS) analysis and the Thru-Put Target software package for data processing. After several exploratory experiments, we adopted the Du Pont PREP system methylation procedure because of its effective recovery, the superior stability of the derivatization product, the possibility of incorporating a clean-up step, and the potential for high throughput. The extraction recovery from a set of control samples was 87%. Coefficients of variation obtained for six replicates of GC/MS analysis and for the overall procedure were 1% and 3%, respectively. Excellent linearity was established in the 50-8000 micrograms/L concentration range studied. With the use of 3-mL samples, a 20-microL final reconstitution volume, oxazepam at 50 micrograms/L was easily detected under the adopted operation conditions.
Ektachem Slides for Direct Potentiometric Determination of Sodium in Plasma: Effect of Natremia, Blood pH, and Type of Electrolyte Reference Fluid on Concordance with Flame Photometry and Other Potentiometric Methods
Authors: Boeyckens Ann , Schots Jan , Vandenplas Hugo , Senesael Freedy , Goedhuys Wim , Gorus Frans K
Published: January 2020

With electrolyte reference fluid (ERF)00, results from Kodak Ektachem slides for the direct potentiometric assay of sodium in plasma were significantly correlated with results from flame photometry, but also appeared to be systematically higher, especially in hypernatremic patients. Indirect potentiometry with the Technicon RA-1000 yielded intermediate values. In 23 hypernatremic patients with greater than or equal to 6 mmol/L difference in sodium between Ektachem ERF00 and flame photometry, a clinical survey disclosed the frequent association of large between-method differences with renal failure, diabetes mellitus, and gastrointestinal disease. However, there was no correlation between differences in sodium on the one hand and anion gaps or (lipo)protein concentrations on the other, nor did in vitro addition studies implicate metabolites that often accumulate in the above-mentioned disorders. Unlike indirect methods, sodium measurements by direct potentiometry on Ektachem and Corning were influenced by in vitro changes of pH between 7.0 and 7.9. However, in a group of patients that included many acidotic individuals, between-method differences in sodium appeared not significantly correlated with in vivo blood pH. Use of the equitransferant ERF04 on Ektachem strongly diminishes the systematic differences with flame photometry, reduces the pH-dependency of the results to that of the direct Corning method, and brings the mean analytical recovery of sodium to below 95% (instead of 115% previously) without affecting the ability of Ektachem slides to avoid spuriously low results in the presence of increased (monoclonal) protein concentrations.
Simple Quantitative Measurement of Serum Choriogonadotropin Compared with Immunoradiometric, Immunoenzymometric, and Chemiluminescent Assays
Authors: Norman R J , Gilmore T A , McLoughlin J W
Published: January 2020

We evaluated a new simplified quantitative method (Tandem Icon QSR; Hybritech) for choriogonadotropin (hCG), which could theoretically be performed in a ward or with outpatients. The method was compared with immunoradiometric (Bioclone Australia), chemiluminescent (Amerlite; Amersham), and immunoenzymometric assays (Stratus; Dade). We analyzed by each of the methods 104 serum samples from pregnant and nonpregnant patients. For quantitative hCG values indicative of pregnancy (greater than 25 int. units/L), excellent correlation was observed between the Tandem Icon method and the other standard laboratory assays (r = 0.995, 0.990, and 0.992, respectively). Occasional problems arose because of the instability of Tandem Icon QSR reagents at room temperature but this was resolved by storing the reagents at 4 degrees C. We conclude that this simplified quantitative method for hCG is reliable and suitable for use outside of the routine immunoassay laboratory.
Avidin-Biotin Enzyme Immunoassay of Osteocalcin in Serum or Plasma
Authors: Jaouhari J , Schiele F , Dragacci S , Tarallo P , Siest J P , Henny J , Siest G
Published: January 2020

We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).
Analysis for Cerebrospinal Fluid Proteins by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Authors: Mashige F , Shimizu T , Iijima S , Ohkubo A
Published: January 2020

Cerebrospinal fluid (CSF) proteins with molecular masses of < 150,000 Da were identified by immunoblotting after two kinds of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With PAGE 1 (17-27% gradient gel), CSF proteins were clearly separated into seven to nine bands with molecular masses of 3000-67,000 Da; seven bands were identified as beta 2-microglobulin, lysozyme, prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, and albumin by immunoblotting. With PAGE 2 (10-20% gradient gel), proteins were clearly separated into 11-16 bands with molecular masses of 15,000-150,000 Da; 11 were identified as prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, albumin, alpha 1-antitrypsin, transferrin (separated into two bands), immunoglobulin fragments, haptoglobin, and IgG. We analyzed CSF samples collected from 81 patients with cerebrospinal signs by these SDS-PAGE methods and observed prominent bands in some cases.
Automated Assay of Vitamin B-12 by the Abbott IMx® Analyzer
Authors: Kuemmerle S C , Boltinghouse G L , Delby S M , Lane T L , Simondsen R P
Published: January 2020

A nonisotopic assay of vitamin B-12 in human serum or plasma is described, performed with the Abbott IMx analyzer. The sample is first treated at pH > 12.5 to release bound vitamin B-12 and to convert all forms to cyanocobalamin. Next, the analyte is bound, at lower pH, by vitamin B-12-specific binding protein, immobilized to a solid phase of polymeric microspheres. Detection involves monitoring the activity of the tracer enzyme (alkaline phosphatase) coupled to a derivative of cyanocobalamin. Total assay precision is 7.9% for vitamin B-12 at 200 ng/L, 6.6% at 400 ng/L, and 6.7% at 800 ng/L. Assay sensitivity, calculated as 2 SD from the zero calibrator, is 37 (+/- 9) ng/L. The dynamic range extends to 2000 ng/L. Analytical recovery of 300 and 600 ng/L additions of vitamin B-12 to sera with basal concentrations of 30-400 ng/L was 102.5%. Results of the assay correlated well with those of commercially available radioisotope assays. No interference was observed in specimens from patients with pernicious anemia, chronic or acute myelogenous leukemia, or renal failure. Cross-reactivity with cobinamide (1 g/L) was < 0.00003%. Vitamin B-12 measurements for blood specimens drawn into serum, EDTA, or heparinized plasma-collection tubes agreed within 3%.
In Vitro Effects of Light on Serum Bilirubin Subfractions Measured by High-Performance Liquid Chromatography: Comparison with Four Routine Methods
Authors: Ihara H , Nakamura H , Aoki Y , Aoki T , Yoshida M
Published: January 2020

The effects of light on serum bilirubin subfractions in vitro were investigated by HPLC and four routine methods for bilirubin analysis. By HPLC, the rate of photodegradation of unconjugated bilirubin (Bu) was nearly twice that of monoconjugated bilirubin (mBc) and threefold that of diconjugated bilirubin (dBc); delta bilirubin (Bd) was most stable against photoirradiation. In the diazo method, the rate of photodegradation of direct bilirubin was almost the same as that of the sum of mBc, dBc, and Bd determined by the HPLC method. However, the rate of photodegradation of indirect bilirubin was significantly lower (P < 0.001) than that obtained by HPLC, because approximately 30% of the bilirubin photoproducts reacted with the diazo reagent as indirect bilirubin. The rate of photodegradation of total bilirubin determined by the direct spectrometric method was lower than that determined by the diazo method, but equal to that of the total peak areas of HPLC. In the Ektachem method, bilirubin photoproducts affected total bilirubin negligibly, and Bc and Bu positively, so that the value of Bd decreased. In the bilirubin oxidase method, bilirubin photoproducts were oxidized enzymatically by both the total and direct bilirubin reagents. We re-emphasize the importance of shielding serum from light to avoid generating bilirubin photoproducts that interfere with the accurate determination of serum bilirubin subfractions. We also recommend HPLC analysis as a standard method for bilirubin measurement.
Superiority of Dynamic Over Static Reference Intervals for Intact, Midmolecule, and C-Terminal Parathyrin in Evaluating Calcemic Disorders
Authors: Lepage R , Whittom S , Bertrand S , Bahsali G , D'Amour P
Published: January 2020

We compared the clinical performance of assays of intact, C-terminal, and midmolecule parathyrin (PTH), when used with either a dynamic reference interval (based on the range of serum PTH concentrations observed in 35 healthy individuals during acute modifications of their blood calcium concentrations) or a gaussian (mean +/- 2 SD) interval derived from normocalcemic individuals. Dynamic intervals were substantially different from gaussian intervals, with half of the area delimited by the gaussian limits for calcium and intact PTH concentrations, and one-third for both C-terminal and mid-PTH assays, being outside the range of values observed during the dynamic tests. Use of the dynamic intervals increased the average clinical sensitivity of the three assays for detecting primary hyper- and hypoparathyroidism from 68% to 97% (and up to 100% for the intact and C-PTH assays). Even though only the intact PTH assay allowed complete separation between primary hyperparathyroid and nonparathyroidal hypercalcemic patients, the average proportion of patients correctly classified in this latter category was increased from 40% to 70% by the use of dynamic intervals. We conclude that gaussian reference intervals are largely responsible for the poor clinical sensitivity of many types of PTH immunoassays and that they should be replaced by dynamic reference intervals when evaluating calcemic disorders.
Parathyroid Hormone-Related Protein: Structure, Function, and Measurement
Authors: Burtis W J
Published: January 2020

Parathyroid hormone-related protein (PTHRP) is a 139- to 173-amino-acid protein with N-terminal homology to parathyroid hormone (PTH). Initially isolated from tumors from patients with humoral hypercalcemia of malignancy, PTHRP appears to be of far more widespread physiological importance. Its complex gene is expressed in a surprising diversity of tissues. The primary amino acid sequences of both the PTH-like and non-PTH-like regions of the protein are highly conserved across species. In addition to classical and nonclassical PTH-like activities of the N-terminal region, other biological functions have been ascribed, including augmentation of calcium transport by midregion PTHRP and potent inhibition of bone resorption by a C-terminal peptide. There is emerging evidence that the protein undergoes extensive processing, including glycosylation and defined proteolytic cleavages. Several region-specific immunoassays are now capable of measuring circulating concentrations of PTHRP in patients with humoral hypercalcemia of malignancy. Molar concentrations of different regions may differ by more than an order of magnitude, reflecting diversity in processing and (or) metabolism. By analyzing the results of these assays, taking into account the specificities of known endoproteases, one can hypothesize about some of the endoproteolytic cleavages that occur in PTHRP metabolism. Clinically, selected PTHRP assays can be very helpful in diagnosing PTHRP-mediated hypercalcemia, but are not yet sufficiently sensitive to accurately measure normal concentrations of the protein.
Simple Method for Correcting Total Protein in Plasma for Actual Fibrinogen Content
Authors: Bakker A J , Gorgels J P , Draaisma J , Jongendijk M , Altena L , Hamersma A , Weiland A
Published: January 2020

Using plasma instead of serum for routine chemistry analyses has many advantages. To overcome the disadvantage of inclusively measuring fibrinogen in the plasma total protein assay without changing the clinical significance of the total protein assay, we investigated the possibility of subtracting the actual amount of fibrinogen from the plasma total protein. The correlation between serum and plasma total protein was excellent (plasma total protein = 0.989 x serum total protein + 6.7 g/L; r = 0.969; n = 131; mean difference = 5.55 g/L; P < 0.001). When the plasma total protein was corrected for the actual amount of fibrinogen, the correlation with serum total protein was equally good but the intercept was practically eliminated (corrected plasma total protein = 1.009 x serum total protein + 0.25 g/L; r = 0.985; n = 131; mean difference = 0.78 g/L; P = 0.47). The mean concentration of fibrinogen was 2.5 g/L (range: 1.38-3.62 g/L; n = 404) for blood donors, 3.6 g/L (n = 2707) for patients from the outpatient department, 4.6 g/L (n = 2023) for patients admitted to the hospital, and 6.6 g/L (n = 219) for patients whose concentration of C-reactive protein was > 50 mg/L. We conclude that the plasma total protein result should be corrected for the actual amount of fibrinogen.
Two-Site Assays of Bone Gla Protein (Osteocalcin) Demonstrate Immunochemical Heterogeneity of the Intact Molecule
Authors: Deftos L J , Wolfert R L , Hill C S , Burton D W
Published: January 2020

We developed a panel of monoclonal antibodies to human bone gla protein (BGP; osteocalcin) peptides that span the linear sequence of the molecule, specifically BGP 1-12 (N-terminal), BGP 15-30 (midregion), and BGP 38-49 (C-terminal). These antibodies were evaluated in various combinations of two-site formats in studies of serum BGP concentrations. For clinical studies, we selected from a panel of antibodies the two most sensitive antibody pairs for the intact molecule (N-C); we also used a polyclonal RIA based on BGP-C. For the two-site format, we used two N-terminal antibodies, 029 and 052, adsorbed to polystyrene beads, and radioiodinated a C-terminal antibody, 663. The standard for each of the assays was purified human BGP. The following BGP serum concentrations (microgram/L, mean +/- SE) were measured with the various assays: by the 029-663 assay, results for normal subjects were 7 +/- 3, for patients with renal failure 25 +/- 8, and for patients with Paget disease 12 +/-4; by the 052-663 assay, the respective results were 22 +/- 4, 44 +/- 12, and 31 +/- 7; by the polyclonal assay, the results were 3 +/- 0.2, 13 +/- 2, and 5 +/- 1. The two intact (N-C) assays were significantly (P < 0.01) correlated (r = 0.94), but their serum values differed by more than twofold in terms of the same BGP standard. The polyclonal assay significantly correlated with each of the intact assays (r = 0.83, 0.77), but it, too, gave different serum values for BGP. These studies demonstrate the immunochemical heterogeneity of circulating BGP, heterogeneity that is manifest even in immunoassays specific for the same region of the molecule.
Placental Alkaline Phosphatase: A Model for Studying COOH-Terminal Processing of Phosphatidylinositol-Glycan-Anchored Membrane Proteins
Authors: Amthauer R , Kodukula K , Udenfriend S
Published: January 2020

Placental alkaline phosphatase (PLAP) has been used as a model for studying the biosynthesis of the phosphatidylinositol-glycan (PI-G)-protein linkage in intact cells and in cell-free systems. However, for the study of processing in cell-free systems, a small protein devoid of glycosylation sites is preferable. A PLAP-derived cDNA was engineered that codes for a nascent protein (mini-PLAP) of 28 kDa in which the NH2- and COOH-termini are retained but most of the interior of PLAP is deleted. In vitro translation of mini-PLAP mRNA in the presence of rough microsomal membranes yields mature PI-G-tailed mini-PLAP. Processing of nascent mutant proteins occurs only when a small amino acid is located at the site of cleavage and PI-G attachment (omega site). Mutations adjacent and COOH-terminal to the omega site have revealed that the omega + 1 site is promiscuous in its requirements but that only glycine and alanine are effective at the omega + 2 site. Rough microsomal membranes from T cells deficient in PI-G biosynthesis do not support processing of mini-PLAP; addition of exogenous PI-G restores activity. Translocation of the proprotein, most likely requiring ATP and GTP, precedes COOH-terminal processing.
Decreased Abundance of Alkaline Phosphatase and Calbindin-D9K mRNAs in the Intestine of the Spontaneously Hypertensive Rat
Authors: Leetun D T , Bruns M E , Bruns D E
Published: January 2020

Alkaline phosphatase activity and calbindin-D9K immunoreactivity are decreased in the intestines of spontaneously hypertensive rats (SHRs). To investigate the potential role of altered gene expression in these decreases, we measured, by Northern blot analyses, the abundances of alkaline phosphatase and calbindin-D9K mRNAs in the proximal regions of the small intestines of 14-week-old SHR and control Wistar-Kyoto (WKY) rats. Alternate 4-cm segments of intestine were used for measurements of the proteins (0-4 cm, 8-12 cm, and 16-20 cm from pylorus, segments A1, B1, and C1, respectively) and mRNAs (4-8 cm, 12-16 cm, and 20-24 cm, segments A2, B2, and C2). Calbindin-D9K (immunoassay) was decreased in SHR vs WKY rats by 27%, 64%, and 67% in segments A1, B1, and C1, respectively (P < 0.01); its mRNA was decreased to a similar extent (69%, 82%, and 80%, respectively; P < 0.002 by analysis of variance). Alkaline phosphatase activity was decreased in SHRs by 58%, 54%, and 51% in segments A1, B1, and C1, respectively (P < 0.01); the abundance of its 3.0-kb mRNA was decreased to a similar extent: 57%, 80%, and 69% in segments A2, B2, and C2, respectively (P < 0.02). The mean decreases of the 2.7-kb mRNA of alkaline phosphatase were statistically significant (P < 0.02) but smaller (38%, 40%, and 35%). The mean abundance of vitamin D receptor mRNA in the same animals was decreased slightly in SHR vs WKY rats (3%, 36%, and 20% in segments A2, B2, and C2, respectively), but the difference in the values was not statistically significant. Decreases in alkaline phosphatase activity and calbindin-D9K immunoreactivity may reflect decreased mRNA abundance and not decreased enzyme-specific activity or increased protein degradation.
Dextran Sulfate Complexes with Potassium Phosphate to Interfere in Determinations of High-Density Lipoprotein Cholesterol
Authors: Olmos J M , Lasunción M A , Herrera E
Published: January 2020

The interference of dextran sulfate with the colorimetric determination of free cholesterol causes an increase in the absorbance that is proportional to the dextran sulfate concentration in the sample. This makes estimation of the free cholesterol concentration unreliable in high-density lipoprotein-containing supernates obtained after plasma precipitation with dextran sulfate/MgCl2. Analysis of the absorption spectrum demonstrated that the absorbance increase was due to turbidity. We observed this effect with colorimetric reagents based on potassium phosphate buffer but not with those based on sodium phosphate or Tris buffers. This effect is ascribed to dextran sulfate because neither the omission of Mg2+ ions nor their chelation with EDTA prevented turbidity, whereas precipitation of dextran sulfate with Ba2+ counteracted this effect. Therefore, potassium phosphate-buffered reagents appear unsuitable for colorimetric lipid determinations in samples containing dextran sulfate.
Ratio of Serum Tartrate-Inhibitable Acid Phosphatase to Total Serum Protein in Benign Prostatic Hypertrophy and Prostatic Carcinoma
Authors: Petrovich George , Ursich-Jankovich Jasna , Prijovich Zeijko
Published: January 2020

The activity concentration and the specific activity (the ratio of enzyme activity to total serum protein) of the tartrate-inhibitable fraction of acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2; TIAP] were related to benign prostatic hypertrophy and to prostatic carcinoma. As expected, the TIAP activity concentrations assayed in the sera of patients with benign prostatic hypertrophy were within the range of those assayed in normal human sera. In contrast, the specific activities of TIAP determined in the sera of patients with benign prostatic hypertrophy were significantly higher than those determined in the control group. In the sera of prostatic carcinoma patients, both the TIAP activity concentrations and the TIAP specific activities differed significantly (F = 730) from the normal values.
Decreases in Apolipoprotein(a) after Renal Transplantation: Implications for Lipoprotein(a) Metabolism
Authors: Black I W , Wilcken D E
Published: January 2020

Serum concentrations of apolipoprotein(a) [apo(a)], the unique glycoprotein of lipoprotein(a), are increased in patients with end-stage renal failure. We prospectively studied serum apo(a) and other lipoproteins in 20 consecutive patients, ages 46 +/- 11 years, before and for six months after successful renal transplantation. All patients received cyclosporine, and no patient was treated for hyperlipidemia. The mean creatinine clearance increased from 7.5 mL/min before transplant surgery to 40.9 mL/min six months afterwards (P less than 0.001). Apo(a) decreased from a median of 403 units/L before transplantation to 184 units/L at one week (P less than 0.001) and was 170 units/L (P less than 0.001) at six months. For the assay used, 1 unit of apo(a) is equivalent to 1 mg of lipoprotein(a). In contrast, from baseline to six months, increases were found for low-density lipoprotein (LDL) cholesterol (P = 0.03), high-density lipoprotein cholesterol (P = 0.06), apo B (P = 0.07), and apo A-I (P = 0.01). The decrease in apo(a) in individual patients was significantly correlated with the increase in creatinine clearance (r = -0.48, P less than 0.001). The single patient who developed nephrotic syndrome after renal transplantation had marked increases in apo(a) (693-1595 units/L), apo B, and LDL cholesterol, which paralleled the degree of proteinuria. These findings suggest that abnormal renal function affects the regulation of lipoprotein(a) metabolism.
Prototype Application of a Robot in the Clinical Laboratory Enabling Fully Automated Quantification of Fecal Porphyrins
Authors: Minder E I , Vuilleumier J P , Vonderschmitt D J
Published: January 2020

Unpleasant specimens, sensitive analytes, and a lengthy chromatographic procedure were the main reasons we implemented fecal porphyrin analysis with a laboratory robot. We describe the system in detail and compare it with the same technique performed manually. The day-to-day variation of assays of standards was lower with the robot than with the manual operation: 8% (CV) for coproporphyrin I and 11% for protoporphyrin IX. We repeatedly analyzed a specimen from a healthy volunteer and determined that the specimen contained (in nmol/g dry wt) 7.1 (SD 0.7) for coproporphyrin I, 3.0 (SD 0.4) for coproporphyrin III, and 44.4 (SD 4.3) for protoporphyrin IX. Upper reference limits as measured in 20 healthy volunteers were 20 nmol/g dry wt for coproporphyrin I, 12 nmol/g for coproporphyrin III, and 80 nmol/g for protoporphyrin IX. We also present characteristic chromatograms for samples from various different porphyrias that exhibit abnormal fecal porphyrin excretion. Calculations of return on investment show that the robot, working at full capacity, is a profitable tool.
Effect of Sample Storage on the Assay of Lipoprotein(a) by Commercially Available Radial Immunodiffusion and Enzyme-Linked Immunosorbent Assay Kits
Authors: Craig W Y , Poulin S E , Forster N R , Neveux L M , Wald N J , Ledue T B
Published: January 2020

Lipoprotein(a) [Lp(a)] was measured by both a radial immunodiffusion (RID) kit from Immuno AG (Zurich, Switzerland) and a Tint Elize enzyme-linked immunosorbent assay (ELISA) kit from CytRx Biopool Ltd. (Umeå, Sweden) in serum samples that had been stored at -20 and -70 degrees C for six months. Storage temperature had no significant effect on the Lp(a) concentrations obtained by either method. After six months, mean Lp(a) degradation was 46% (95% confidence interval, 34-58%) with the RID kit; the ELISA data could not be compared between time points. In fresh sera, Lp(a) concentrations obtained by RID were 41% higher than by ELISA (because of differences in assay calibration materials), but in paired measurements of a set of 215 samples stored at -40 degrees C for an average of 10 years, Lp(a) concentrations were 62% lower by RID. This suggests that RID is more sensitive to the effects of long-term storage than is ELISA.
Performance of Proficiency Survey Samples in Two Immunoradiometric Assays of Human Growth Hormone and Comparison with Patients' Samples
Authors: Pringle P J , Jones J , Hindmarsh P C , Preece M A , Brook C G
Published: January 2020

The immunoradiometric assay (IRMA) used in our laboratory for the measurement of growth hormone (hGH; somatotropin) performed badly in the national proficiency survey program, the U.K. External Quality Assessment Scheme (EQAS). We compared our assay with another IRMA, which gave similar results for patients' samples and performed adequately in EQAS. The samples from EQAS are collected from patients with polycythemia and fall into two categories: those containing endogenous hGH and those supplemented with pituitary-derived hGH. Analysis of the two groups separately showed that the differences between the two IRMAS were in the measurement of the endogenous hormone. The reason for this appears to be a matrix effect related to the fact that the EQAS serum samples are collected from polycythemic patients.
Optimized Spectrophotometric Determination of Aldehyde Dehydrogenase Activity in Erythrocytes
Authors: Johnson R D , Bahnisch J , Stewart B , Shearman D J , Edwards J B
Published: January 2020

We describe a reliable and sensitive semiautomated spectrophotometric assay of aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity in erythrocytes. The hemolysate can be stabilized with sucrose, and the technique involves only microliters of hemolysate on a centrifugal analyzer. The use of microcolumns to remove interfering hemoglobin is avoided, and reproducibility of the assay has been improved by manipulating the inherent lactate dehydrogenase activity of erythrocytes by adding lactate and oxalate to the reaction mixture. These modifications have decreased the analytical imprecision of the assay, allowing a better appraisal of aldehyde dehydrogenase activity in erythrocytes as a biological marker of excess alcohol consumption. Erythrocytic ALDH activity was significantly less in 40 alcoholics than in 145 teetotallers (median activity 128 vs 219 mU/g of hemoglobin, respectively; P = 0.0001), indicating the potential of this assay as a useful marker of excess alcohol consumption.
Triple-Helical Capture Assay for Quantification of Polymerase Chain Reaction Products
Authors: Vary C P
Published: January 2020

This method for rapid, automated analysis of polymerase chain reaction (PCR) products makes use of PCR primers containing 5'-polypyrimidine sequences. Polypyrimidine-"headed" primers confer to the PCR product the ability to form triple helical complexes with a third polypyrimidine oligonucleotide. Third-strand oligonucleotides are modified to serve as either capture reagents or detection reagents for PCR products. Automated quantitative measurement of the PCR product is achieved by using latex bead-based fluorescence analysis. The use of triple-instead of double-helical interactions avoids the usual requirements of complex blocking reagents, time- and labor-intensive washing steps, and long times for color development. The method also provides rapid, sequence-specific capture and detection of PCR products without the need to denature the double-stranded PCR product. The assay is demonstrated with use of both PCR primer-derived and endogenous triple-helix-forming sequences resulting from PCR of several bacterial and viral target nucleic acids.
Measuring Ethanol in Blood and Breath for Legal Purposes: Variability between Laboratories and between Breath-Test Instruments
Authors: Jones A W , Beylich K M , Bjørneboe A , Ingum J , Mørland J
Published: January 2020

We determined the concentrations of ethanol in nearly simultaneous specimens of venous blood (BAC) and end-expired breath (BrAC) after healthy volunteers drank moderate amounts of alcohol. BAC was measured at two laboratories and BrAC was analyzed with two instruments (Intoxilyzer 5000) from the same manufacturer. The mean difference in BAC between laboratories was 0.0105 mg/g (SD 0.0219); 95% of the differences ranged from -0.0333 to 0.0543 mg/g. The mean difference in BrAC between instruments was 0.0153 mg/L (SD 0.0136), and 95% of the differences ranged from -0.0119 to 0.0425 mg/L. The coefficient of variation (CV) between laboratories was 2.9% compared with 4.5% between breath-test instruments. Venous BAC (y) and BrAC (x) were highly correlated (r = 0.978). However, when the Intoxilyzer instruments indicated that BrAC had reached zero, the actual BAC was 0.135 mg/g, according to the average forensic laboratory reports. The Intoxilyzer 5000 breath analyzers used in this study seem to have a constant analytical bias.
Acquired Hypolipoproteinemia
Authors: De Buyzere M , Delanghe J , Labeur C , Noens L , Benoit Y , Baert J , Rosseneu M
Published: January 2020

We present a six-year follow-up of a boy with a novel type of hypolipoproteinemia, with clinical and biochemical features distinct from classical hypoalphalipoproteinemias. There were abnormally low concentrations of total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) B, apo A-I, and apo A-II, and the phospholipids were decreased. The most striking abnormality was an extra fraction containing mainly phospholipids and apo A-I in the HDL3 subfraction. This fraction is reminiscent of concentric 20- to 50-nm-diameter lamellar phospholipid liposomes. Plasma lecithin:cholesterol acyltransferase activity was strongly decreased. We noted a persisting polyclonal hypergammaglobulinemia, hematological abnormalities (hemolytic anemia and thrombocytopenia), and a progressive splenomegaly. After the five-year follow-up, the patient had recurrent severe infections; moderate hematuria and proteinuria developed gradually. Treatment with corticosteroids and immunoglobulins improved thrombocytopenia and hypolipoproteinemia. These clinical and biochemical findings differ from those in the known primary and secondary hypo-alpha-lipoproteinemia syndromes. Although investigation of the relatives suggests a familial predisposition for hypo-alpha-lipoproteinemia, the subject's condition can be regarded as acquired.
Two-Site Monoclonal Antibody-Based Immunoradiometric Assay for Measuring Prostate Secretory Protein in Serum
Authors: Huang C L , Liang H M , Brassil D , Schellhammer P F , Rozzell M , Newhall K , Beckett M L , Wright G L
Published: January 2020

We developed a double-determinant immunoradiometric assay for measuring serum prostate secretory protein (PSP), using monoclonal antibodies (MAb) against two different epitopes: MAb PSP-19 was the capture antibody and MAb PSP-6 was the tracer antibody. Assay sensitivity was 0.1 microgram/L. Analytical recovery of PSP was 93.5-104.6%, whereas the intra- and interassay mean CVs were 4.2% and 6.9%, respectively. In 92 normal men, ages greater than 50 years, the mean PSP concentration was 5.7 micrograms/L, with 10 (10.9%) men having concentrations greater than 10 micrograms/L. In contrast, 20 of 49 (40.8%) patients with benign prostate hyperplasia (BPH; mean PSP concentration 9.4 micrograms/L) and 46 of 100 (46%) patients with prostate cancer (mean PSP concentration 22.2 micrograms/L) had PSP concentrations greater than 10 micrograms/L. Mean serum PSP concentrations of the BPH (P less than 0.05) and prostate cancer (P less than 0.01) groups were significantly different from those of age-matched normal men. In a small group of patients, serial PSP concentrations correlated with the clinical course during therapy. Thus, PSP may be a useful marker for evaluating patients with prostate cancer.