bluuebunny/crossref_metadata_2025 huggingface dataset:
An alpha slow-moving high-density-lipoprotein subfraction in serum of a patient with radiation enteritis and peritoneal carcinosis.
Authors: Peynet J , Legrand A , Messing B , Thuillier F , Rousselet F
Published: January 2020

An alpha slow-moving high-density-lipoprotein (HDL) subfraction was seen in a patient presenting with radiation enteritis and peritoneal carcinosis, who was given long-term cyclic parenteral nutrition. This subfraction, observed in addition to normal HDL, was precipitated with low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) by sodium phosphotungstate-magnesium chloride. The patient's serum lipoproteins were analyzed after fractionation by density gradient ultracentrifugation. The alpha slow-moving HDL floated in the ultracentrifugation subfractions with densities ranging from 1.028 to 1.084 kg/L, and their main apolipoproteins included apolipoprotein E in addition to apolipoprotein A-I. These HDL were larger than HDL2. The pathogenesis of this unusual HDL subfraction is hypothesized.
Use of DNA probes in detecting carriers of Duchenne muscular dystrophy: selected case studies.
Authors: Prior T W , Blasco P A , Dove J L , Leshner R T , Gruemer H D
Published: January 2020

Detection of Duchenne muscular dystrophy carriers by genetic analysis is illustrated by four case studies. The technique is most useful in obligate families, in excluding carrier status in isolated cases, and in families in which the affected child demonstrates a molecular deletion. A major limitation of this technique is that the accuracy of carrier status in isolated (i.e., no family history) cases is limited by the probability that the affected child may represent a new mutation. To improve the carrier risk estimate generated by the DNA data, particularly in isolated cases, we suggest that measuring creatine kinase activities in the serum and performing the genetic analysis on the nonaffected males may be helpful.
Modulated adhesion: a proposal for the role of myelin-associated glycoprotein in myelin wrapping.
Authors: Attia J , Tropak M , Johnson P W , Newerly-Abranow W , Pawson T , Roder J C , Dunn R J
Published: January 2020

Myelin-associated glycoprotein (MAG) is a 100-kDa integral membrane glycoprotein expressed by oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. It is found first in loosely wrapped myelin and then periaxonally after compaction. Clinical findings, structural analysis, and cell assays indicate a role for MAG in adhesion. We propose that the phosphorylation state of MAG modulates its adhesion and that a minimum spatial requirement for the separation of the kinase and phosphatase activities postulated by this model may explain the correlation between axon size and myelination state.
Genes encoding neuronal nicotinic acetylcholine receptors.
Authors: Deneris E S , Boulter J , Connolly J , Wada E , Wada K , Goldman D , Swanson L W , Patrick J , Heinemann S
Published: January 2020

Four genes (alpha 2, alpha 3, alpha 4, and beta 2), which encode proteins homologous to the Torpedo electric organ and vertebrate muscle nicotinic acetylcholine receptors, have been identified by cloning rat brain cDNAs. Injection of transcripts derived from these cDNAs into Xenopus laevis oocytes results in the formation of three nicotinic acetylcholine receptors. Two of these receptors, alpha 3/beta 2 and alpha 4/beta 2, have the characteristics of ganglionic nicotinic receptors. The third (alpha 2/beta 2) exhibits a previously undescribed pharmacology and thus represents a novel subtype that may be expressed in the brain. The wide distribution of alpha 2, alpha 3, alpha 4, and beta 2 transcripts in the brain indicates that neuronal nicotinic acetylcholine receptors are a major neurotransmitter receptor system.
Isolation and characterization of myoglobin and its two major isoforms from sheep heart.
Authors: Wu J T , Pieper R K , Wu L H , Peters J L
Published: January 2020

We isolated myoglobin from sheep heart by homogenizing cardiac muscle in 70%-saturated ammonium sulfate, followed by chromatography on a column containing carboxymethyl(CM)-Sephadex gel. Two major isoforms of myoglobin, designated Mb 7.9 and Mb 8.1, were separated by chromatofocusing and were distinguished by their different patterns seen on either isoelectrofocusing or on electrophoresis on polyacrylamide gel. The isoelectric points of the major bands of Mb 7.9 and Mb 8.1 were 7.4 and 7.16, respectively. Both isoforms were identical in size when examined by gel filtration chromatography but differed slightly when analyzed by polyacrylamide gradient gel in the presence of sodium dodecyl sulfate. The Mr of Mb 7.9 (15,900 Da) is slightly smaller than that of Mb 8.1 (18,400 Da). When reacted against rabbit anti-sheep myoglobin, two isoforms also appeared as two nonidentical precipitin lines on agarose gel.
Direct determination of four sulfates and seven glucuronides of 17-oxosteroids in urine by fluorescence "high-performance" liquid chromatography.
Authors: Iwata J , Suga T
Published: January 2020

We describe a direct method for determining four sulfates and seven glucuronides of 17-oxosteroids (17OS) in urine without hydrolysis, by use of "high-performance" liquid chromatography (HPLC) with fluorometric detection. After pretreatment of urine samples with a Sep-Pak C18 cartridge, four 17OS sulfates and seven 17OS glucuronides in the pretreated urine samples were reacted with tetrapentylammonium ions to form ion pairs. Ion-paired 17OS sulfates were extracted with benzene. By adding sodium sulfate to the remaining sample, we could then extract ion-paired 17OS glucuronides with dichloromethane. Each extract was labeled with dansyl-hydrazine in an acetic acid-acetonitrile solution. The labeled steroids were separated by HPLC on a reversed-phase Capcell-Pak C8 (silicon-polymer-coated silica gel modified with octyl groups). We monitored each effluent with a fluorometric detector (330 nmexcitation, 535 nmemission).
Phospholipid surface tension: the diagnostic utility of amniotic fluid and its lipid extract, an analysis of the value of precipitation with cold acetone.
Authors: Ruiz Budría J , Abbad Bara J M , Fabre Gonzalez E , Higueras Sanz M T , Serrano Ostáriz J L
Published: January 2020

In prevention of the respiratory distress syndrome (RDS), measurements of surface tension values (a biophysical property) of amniotic-fluid samples are correlated with their lecithin/sphingomyelin (L/S) ratios (a biochemical property). According to some authors, precipitation of phospholipid with cold acetone is essential for determining the L/S ratio, because it separates surfactant and nonsurfactant phospholipidic fractions. Here we report the first study of the ability of three amniotic-fluid components to decrease surface tension: The complete lipid extract (without precipitation), and the fractions precipitated and (or) remaining soluble after addition of cold acetone. Addition of increasing aliquots of lipid extracts to these three samples showed that: (a) measurement of surface tension rapidly and reliably indicates fetal lung maturity, and (b) both precipitated and soluble phospholipid fractions decrease surface tension similarly, making it unlikely that the precipitation step in fact separates surfactant and nonsurfactant material.
Statistical interpretation of concentrations of magnesium, zinc, calcium, potassium, cholesterols, and creatine kinase isoenzymes in men at different stages of ischemic heart disease.
Authors: Speich M , Auget J L , Arnaud P
Published: January 2020

We present a statistical interpretation of plasma (Pl) and (or) erythrocyte (Erc) concentrations of magnesium, zinc, calcium, potassium, and total high-density lipoprotein (HDL) cholesterol, as well as of the activity of total creatine kinase (CK) and its CK-MB isoenzyme, in 26 men with pre-infarction syndrome (PIS) and 34 men with acute myocardial infarction (MI). Discriminant analysis allowed overall comparison of both groups and determination of the most significant variables: CK and Pl-Zn. By non-hierarchical cluster analysis we defined three homogeneous subgroups among MI men, with CK, CK-MB, and Pl-Zn differing significantly between the groups. In PIS men, Pl-Zn was correlated with Pl-Ca, whereas in MI men Pl-Zn was correlated with Pl-Mg. Stepwise regression indicated that Pl-Zn was the most significant regressor of CK in PIS men and of CK-MB in MI men. All these statistical interpretations support a special role of Pl-Zn in diagnosis and perhaps prognosis. After MI, interleukin-1 release may possibly mediate observed hypozincemia via formation of a heart Zn-metallothionein.
Application of a silver-binding assay to the determination of protein in cerebrospinal fluid.
Authors: Krystal G , Lam V , Schreiber W E
Published: January 2020

We evaluated a silver-binding assay for use in measuring total protein in cerebrospinal fluid. The advantage of this procedure over other methods is that, because of its sensitivity, it requires only a 0.5-microL sample. The procedure, which takes approximately 40 min to complete, involves dilution of 0.5-microL samples to 1 mL with distilled water containing sodium dodecyl sulfate, followed by addition of glutaraldehyde and an ammoniacal silver solution. After color development for 30 min, the reaction is terminated with sodium thiosulfate and the absorbance is measured at 420 nm. This assay displayed within-run and day-to-day precision (CV) of 3.1% to 13% over the range of 210 to 1370 mg/L. It showed substantially less protein-to-protein variation than the Coomassie Blue dye-binding procedure when tested with albumin, globulin, and transferrin. It also yielded an accurate estimation of hemoglobin. Moreover, preliminary studies suggested that it was capable of quantifying immunoglobulin light chains and glycoproteins. In a study of 54 human cerebrospinal fluid samples, results of the silver-binding assay corresponded more closely with those obtained with a rate biuret assay (intraclass correlation coefficient = 0.91) than did either the dye-binding or classical Lowry methods.
Circadian variations and reference intervals for some enzymes in urine of healthy children.
Authors: Feldmann D , Flandrois C , Jardel A , Phan T M , Aymard P
Published: January 2020

Circadian variations of alanine aminopeptidase (EC 3.4.11.2), gamma-glutamyltransferase (EC 2.3.2.2), and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) in urine were studied in 10 healthy children, ages six to 15 years. Urine specimens were collected during 24 h, grouped into four time intervals. Enzymes were measured spectrophotometrically, with automation. These enzymes all showed diurnal variation, with morning (8 a.m.-12 noon) excretion being highest. We also analyzed timed urinary specimens (8 a.m.-12 noon) from 136 healthy children, ages two to 11 years. Reference intervals are presented for these enzymes. High excretion of the three enzymes was observed in children two and three years old.
Concentrations of tumor-associated trypsin inhibitor and C-reactive protein in serum in acute pelvic inflammatory disease.
Authors: Paavonen J , Lehtinen M , Lehto M , Laine S , Aine R , Räsänen L , Stenman U H
Published: January 2020

We measured tumor-associated trypsin inhibitor (TATI) and C-reactive protein (CRP) in serum of 29 patients with proven pelvic inflammatory disease (PID). TATI values were increased in seven (24%), paralleling increases in CRP. TATI was increased by about 3.5-fold in seven of eight patients with CRP concentrations greater than 90 mg/L, but in none of 21 patients with CRP concentrations less than 90 mg/L. TATI concentration and severity of PID as determined by laparoscopy or endometrial biopsy were not correlated. These results suggest that, in severe infections, regulation of TATI synthesis resembles that of acute-phase proteins.
Enzyme-linked immunosorbent assay of apolipoprotein B in blood spotted onto filter paper, suitable for neonatal screening.
Authors: Wang X L , Dudman N P , Wilcken D E
Published: January 2020

This assay was developed to measure apolipoprotein (apo) B in blood samples spotted onto filter paper. The long-term aim is to detect young families with the dominantly inherited familial hypercholesterolemia, during current neonatal screening programs. The interassay CV was 5.6%, and apo B concentrations correlated closely with values measured by radial immunodiffusion. In the present assay, the primary apo B standard selected and the serum samples behaved similarly. Use of Triton X-100 in the extraction of apo B from dried blood prevented the decrease in apo B immunoreactivity that otherwise occurred during storage for 20 days at 4 degrees C. In 57 neonates two to six days postpartum, the mean (and SD) apo B concentration in whole blood was 186 (78) mg/L, and the apo A-I/apo B ratio was higher in female than in male neonates (P less than 0.001), as is also true for adults. The assay is suitable for use in screening programs for newborns, and the observations add to our understanding of lipid metabolism in neonates.
Liquid-chromatographic monitoring of cytosine arabinoside and its metabolite, uracil arabinoside, in serum.
Authors: Wermeling J R , Pruemer J M , Hassan F M , Warner A , Pesce A J
Published: January 2020

This is a "high-performance" liquid-chromatographic method for quantifying the antileukemic drug cytosine arabinoside (cytarabine; 1-beta-D-arabinofuranosylcytosine; Ara-C), with a structural analog, 5-methylcytidine, as the internal standard. We used a C18 reversed-phase column and ammonium acetate (0.5 mol/L, pH 6.5) as the mobile phase, monitoring the column effluent at 280 nm. Tetrahydrouridine was present in the sample-collection tubes to inhibit conversion of cytosine arabinoside to uracil arabinoside. The standard curve is linear to 100 mg/L. Analytical recovery is 98%. Coefficients of variation for within-run and between-run imprecision were 2.0% and 4.3% at 20 mg/L and 2.7% and 2.7% at 80 mg/L, respectively. Assay sensitivity was limited by the amount of endogenous material in each patient's serum, making assay of a pre-infusion sample necessary for accurate calculations. In a trial patient population, the assay was shown to have potential for the detection of toxic concentrations in patients receiving high doses of Ara-C.
Modified magnesium method in the aca III: elimination of interference by bilirubin.
Authors: Rehak N N , Chiang B T
Published: January 2020

Bilirubin interferes with the Du Pont magnesium method in the aca at 510 nm. We determined that bilirubin concentrations in serum samples up to 380 mg/L did not affect the absorbance measured in the aca III at 540 nm, and therefore we modified the Du Pont setting of spectrophotometer for the magnesium method to 540 and 600 nm. Accuracy and precision of the modified method were comparable with the unmodified method and with atomic absorption spectrophotometry. For comparison, we analyzed serum samples with normal (n = 37) and increased (n = 22) bilirubin concentration with the modified method in the aca III (y) and in the atomic absorption spectrophotometer (x). The results (range 0.56-1.25 mmol/L) were in good agreement (x = y = 0.84 mmol/L, D(x - y) = -0.001, SD = 0.004, t = 0.238, P greater than 0.50) and bilirubin did not interfere with the modified method.
Nonenzymic glycation of human immunoglobulins does not impair their immunoreactivity.
Authors: Morin L G , Austin G E , Rodey G E , Johnson J E
Published: January 2020

Diabetic patients have an increased proportion of their immunoglobulins nonenzymically glycated. To investigate the possibility that this may contribute to increased susceptibility to infection, we compared the immunoreactivity of glycated and nonglycated human immunoglobulins against rubella and hepatitis; streptococcal exoenzyme and infectious mononucleosis; human lymphocytotoxic antigens (HLA); and Varicella zoster in terms of antigen-antibody binding, cell agglutination, cytotoxicity, and complement-fixation properties, respectively. We found no evidence to support the supposition that glycated immunoglobulins are functionally impaired.
Do nonesterified fatty acids displace thyroxin from its plasma binding sites in severe nonthyroidal illnesses?
Authors: Nicolson R E , Reilly C P , Pannall P R , Esposito L , Wellby M L
Published: January 2020

Severe nonthyroidal illnesses have been associated with increases in nonesterified fatty acids (NEFA) and the dialyzable fraction of thyroxin (T4) in plasma. We have further investigated their possible relationship in severe nonthyroidal illnesses as well as in induced in vivo and in vitro situations involving increased NEFA. We demonstrate that there is no relationship between NEFA and the dialyzable fraction of T4, either in severe nonthyroidal illnesses or in the other situations, unless plasma NEFA concentrations exceed 5 mmol/L in normal persons or 1.7 mmol/L in nonthyroidal illnesses, and that this concentration was not reached in the patients we studied, with one exception. We conclude that NEFA are unlikely to contribute to an inhibition of the binding of T4 to the binding proteins that might be present in plasma of patients with severe nonthyroidal illnesses unless their NEFA concentrations are very high.
Strategic use of individual and combined enzyme indicators for acute pancreatitis analyzed by receiver-operator characteristics.
Authors: Werner M , Steinberg W M , Pauley C
Published: January 2020

The optimal strategy for the diagnosis of acute pancreatitis with enzyme assay results as indicators was evaluated in 67 emergency cases in whom this condition was suspected. We measured urine amylase expressed as activity concentration (U/L), timed excretion (U/h), and amylase/creatinine clearance ratio, and also serum amylase, elastase, lipase, and trypsinogen, at admission and repeatedly during hospitalization. The receiver-operator characteristic function was used to evaluate the diagnostic discrimination of each variable among initial findings and among the highest individual findings established retrospectively. We applied the same treatment to multiple univariate discrimination, using the six possible pairs and the four possible triplets of serum indicators. The results suggest that such urine assays should be abandoned, that all individual serum assays combine about 0.9 sensitivity with 0.9 specificity, that pairing of two assays does not clearly enhance discrimination, and that triplets of tests may degrade discrimination. The trade-off between sensitivity and specificity is a function not only of the chosen decision threshold but also of the sampling strategy (initial vs highest values) and of the interpretation rule (Boolean "and" vs "or" strategy).
Methods for determining "reference changes" from serial measurements: plasma lipid-bound sialic acid.
Authors: Shahangian S , Fritsche H A , Hughes J I , Johnston D A
Published: January 2020

Lipid-bound sialic (neuraminic) acid (LSA) was measured in EDTA-treated plasma of 26 healthy subjects at three-month intervals for up to one year. The change in LSA concentration for consecutive measurements ranged from -54 to 42 mg/L (mean, -2.1 mg/L; SD, 19.6 mg/L; n = 56). The "reference change" for plasma LSA (+/- 2 SD), calculated from distribution of the differences, was +/- 39 mg/L. The 88th percentile of the intra-individual variance was 338 mg2/L2 and the mean variance was 159 mg2/L2. Using the homeostatic, autoregressive time-series model, a reference change of +/- 51 mg/L between two consecutive measurements was determined to be statistically significant (i.e., expected by chance no more than 5% of the time) in 88% of the healthy subjects. Only 73% of the healthy subjects would have had intra-individual variances corresponding to the reference change of +/- 39 mg/L according to the autoregressive model. The concentration of LSA in plasma was significantly decreased upon surgery in five of 10 patients with colorectal adenocarcinomas of Dukes stages A-C when we used +/- 39 mg/L as the reference change, but in only two of the 10 when we used +/- 51 mg/L as the reference change.
Immunoaffinity purification of creatine kinase-MB from human, dog, and rabbit heart with use of a monoclonal antibody specific for CK-MB.
Authors: Landt Y , Vaidya H C , Porter S E , Dietzler D N , Ladenson J H
Published: January 2020

We describe a simple, rapid immunoaffinity procedure for purifying the MB isoenzyme of creatine kinase. Immunoaffinity gel is prepared by linking a monoclonal antibody ("Conan-MB"), specific for this isoenzyme, to Sepharose 4B. Heart tissue is homogenized and fractionated with 40-70% saturated ammonium sulfate before it is applied to the immunoaffinity gel. CK-MB activity, retained on the gel, is then eluted with a high-pH diethylamine buffer (0.1 mol/L, pH 10.5). The purified CK-MB isoenzyme is stabilized by collection directly into tubes containing glycerol (to prevent dissociation of the enzyme subunits) and pH-neutralizing buffer. This procedure compares favorably in yield, specific activity, and technical ease with a multi-column purification method previously used in our laboratory. We have used the immunoaffinity procedure to purify to homogeneity CK-MB from human, dog, and rabbit heart, with yields of 50.0%, 53.1%, and 49.3% and specific activities of 540, 477, and 377 kU/g, respectively. The preparations are pure as judged by protein staining with silver nitrate after electrophoresis on sodium dodecyl sulfate/polyacrylamide gel.
Supercritical fluid chromatography and microbore liquid chromatography for drug analysis.
Authors: Wong S H
Published: January 2020

Supercritical fluid and microbore liquid chromatography offer potential applications for drug analysis. In supercritical fluid chromatography (SFC), the mobile phase is a gas (e.g., carbon dioxide) maintained at its supercritical state--that is, above its critical temperature and pressure, above which it cannot be liquefied even with further increases in applied pressure. The SFC mobile phase has low viscosity, approximating that of a gas, and high diffusivity, between those of a gas and a liquid. These properties yield favorable column efficiency, between that of capillary gas chromatography (GC) and liquid chromatography (LC). SFC analysis may be performed by either packed or open tubular capillary columns and with GC and LC detectors. SFC, interfaced with mass spectrometry, may become a viable alternative to GC/MS for drug identification in clinical and forensic toxicology. Advantages of microbore liquid chromatography include enhanced mass sensitivity, reduced solvent consumption, and others. Microbore columns (internal diameters 1 to 2 mm) may be packed with 3-, 5-, or 10-micron particles. Potential applications include micro-sample analysis (5-200 microL) for neonatal and pediatric drug monitoring, and drug confirmation analysis for toxicology.
Antipyrine interferes with chemical determination of bromide in simultaneous estimation of total body water and extracellular water.
Authors: Brans Y W , Shaffer S G , Bell E F , Schwartz C A , Martin L A
Published: January 2020

Antipyrine and bromide dilution are frequently used to estimate total body water and extracellular water volumes, respectively. We assessed a potential error with the simultaneous use of these methods. In vitro the bromide concentration of solutions measured by the gold chloride or rosaniline methods decreased linearly with increasing concentrations of antipyrine. With the gold chloride method, the "inhibitory" effect of antipyrine was more pronounced for solutions in plasma than in those in water. With the rosaniline method, the "inhibitory" effect was similar for water and plasma. Antipyrine had no effect on bromide concentration measured by fluorescein or fluorescence excitation methods. We conclude that antipyrine potentially affects the measurement of bromide space by certain colorimetric methods. The mean values of previously published data do not appear to have been affected by the technical problems described in this study, although these problems may account, at least in part, for the large variability of the results.
Creatine kinase and lactate dehydrogenase isoenzymes in serum and tissues of patients with stomach adenocarcinoma.
Authors: Hirata R D , Hirata M H , Strufaldi B , Possik R A , Asai M
Published: January 2020

Total activities of creatine kinase (EC 2.7.3.2; CK) and lactate dehydrogenase (EC 1.1.1.27; LD) and their isoenzymes were estimated in serum and tissue samples from patients with stomach adenocarcinomas who were to undergo gastric resection. Total CK activity (U/g protein) appeared to be markedly decreased in neoplastic stomach tissue. CK-BB was the predominant isoenzyme in both neoplastic and normal stomach tissues; however, the CK-BB/total CK ratio was increased in adenocarcinoma tissue. Macro CK type 1 was found in two neoplastic tissues and macro CK type 2 in 11. LD4 and LD5 isoenzymes were predominant in gastric tissues, but LD5 and the LD5/LD1 ratio were higher in adenocarcinoma tissue. At 24 h before surgery, CK-BB was demonstrated in sera of all patients and CK-MB in 69%. The CK-BB probably originated from neoplastic stomach tissue, which contains high CK activity, with BB isoenzyme predominating. After gastrectomy, CK and LD isoenzymes in sera were markedly increased by 24 h postsurgery. These alterations were attributed to release from damaged tissue during gastric resection.
Determination of magnesium in granulocytes.
Authors: Hosseini J M , Yang X Y , Elin R J
Published: January 2020

Determination of magnesium in mononuclear blood cells may provide useful information about intracellular magnesium. Mononuclear blood cell preparations are frequently contaminated with granulocytes. We determined the normal reference interval for magnesium content and concentration of granulocytes and compared it with magnesium in mononuclear blood cells. The mean granulocyte magnesium concentration and content were 6.95 (SD 1.25) mmol/L and 3.11 (SD 0.54) fmol per cell, respectively, vs 10.95 (SD 1.70) mmol/L and 3.27 (SD 0.46) fmol per cell in mononuclear cells. The concentration of magnesium in granulocytes was significantly less (P less than 0.001) than in mononuclear blood cells, but the magnesium contents were not significantly different.
Sensitive, rapid assay of subforms of creatine kinase MB in plasma.
Authors: Puleo P R , Guadagno P A , Roberts R , Perryman M B
Published: January 2020

The subforms of creatine kinase (CK; EC 2.7.3.2) in plasma have received recent attention as potential markers for the early diagnosis of acute myocardial infarction. Because changes in CK-MM subforms are not specific for myocardial injury, we developed an assay, based on high-voltage electrophoresis, that is sufficiently sensitive to detect the CK-MB subforms at concentrations substantially below the upper limit of normal (14 U/L). The assay can detect 1.25 U of either MB subform per liter with a precision of 0.20 U/L and gives responses that vary linearly with activity concentration from 0.0 through 30.0 U/L, with an identical signal response for both subforms. When both subforms are present in a serum sample, the assay accurately measures both the relative percentage and the absolute quantity of each: assay activity/known activity was 1.03 for each subform at a total MB subform activity of 5.0 U/L (r = 0.98). Assay time is 25 min, and there is no loss of CK during electrophoresis. Thus, this system can be used to rapidly, sensitively, and precisely quantify the two CK-MB subforms at activities well within the normal reference interval.
Influence of blood pressure, heart rate, age, and sex on concentrations of atrial natriuretic factor and cyclic GMP in 124 volunteers.
Authors: Wencker M , Hauptlorenz S , Moll W , Puschendorf B
Published: January 2020

The significance of increased atrial natriuretic factor (ANF) in relation to blood pressure and age is still controversial. We investigated the influence of blood pressure, age, and some other variables on ANF and its putative second messenger, cGMP. Samples for ANF and cGMP detection were taken from 124 ostensibly healthy individuals who were donating blood. Samples were also collected from 27 volunteers before and after blood donation, to study the influence of bleeding. During blood donation, ANF increased from 78.9 to 87.4 ng/L (P = 0.0035), whereas cGMP remained unchanged. ANF concentrations in 124 healthy individuals, corrected for the influence of bleeding, were 61.5 (SD 26.1) ng/L, with a 95% confidence interval of 10.0 to 112.1 ng/L. Mean cGMP concentrations in plasma were 2.9 (SD 1.45) nmol/L, with a 95% confidence interval of 0.4 to 5.75 nmol/L. Multivariance analysis revealed no significant influence of blood pressure, age, heart rate, or sex on concentrations of either ANF or cGMP in plasma.
Effect of lyophilization on results of five enzymatic methods for cholesterol.
Authors: Kroll M H , Chesler R , Elin R J
Published: January 2020

We studied the effect of lyophilization of serum pools on the determination of cholesterol with the enzymatic methods used in five automated analyzers: SMAC (Technicon), the RA-1000 (Technicon), the aca (Du Pont), the TDx (Abbott), and the Ektachem 700P (Kodak). We prepared two serum pools: pool A (2.2 g/L) and pool B (1.9 g/L). We separated each pool into three groups for treatment by lyophilizing or freezing at -20 and -70 degrees C. We determined cholesterol by the above methods at regular intervals during the next 270 days. For all methods, the measured concentration of cholesterol was less for the lyophilized serum than for frozen (P less than 0.007 by the paired-sample t-test). After adjusting for the dilutional effects of reconstitution, the decrease in original value was 0.7% for the SMAC, 1.7% for the RA-1000, 5.7% for the Ektachem, 9.4% for the TDx, and 14.3% for the aca. Lyophilization of cholesterol standards or serum samples may hamper the effective standardization of enzymatic methods and may be a source of apparent inaccuracy (bias) among enzymatic methods.
Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.
Authors: Urdea M S , Kolberg J , Clyne J , Running J A , Besemer D , Warner B , Sanchez-Pescador R
Published: January 2020

We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.
Investigation of protoporphyrin IX standard materials used in acid-extraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX.
Authors: Gunter E W , Turner W E , Huff D L
Published: January 2020

Erythrocyte protoporphyrin (EP) has been used for more than 30 years as an indicator of lead intoxication, iron deficiency, and porphyrias. Recently, numerous analytical problems associated with various EP methods have been reported, including a lack of consensus among investigators regarding the best calibration material or analytical procedure. We investigated commercially available protoporphyrin IX (PPIX) standard materials and measured the millimolar absorptivity (m epsilon) of these materials, focusing on variables affecting the determination of their absorptivities. Among the five forms of PPIX available, PPIX dimethyl ester, when hydrolyzed to PPIX free acid, gave the most consistent and reproducible results. This work confirmed our earlier observations, made on more than 600 separate occasions during 12 years, that the m epsilon of PPIX free acid in 1.5 mol/L HCl at the Soret maximum is 297 +/- 1.3 L.mmol-1.cm-1, 19% higher than the arbitrary value of 241 L.mmol-1.cm-1 generally accepted by most investigators but based on unpublished data. We propose that the m epsilon of 297 L.mmol-1.cm-1 for PPIX be adopted and that PPIX dimethyl ester be used for the calibration of acid-extraction methods. A detailed protocol for the preparation and verification of PPIX from the dimethyl ester is available upon request.
Estimation of true values in radioimmunoassays.
Authors: Riesco O F , Hajri-Mezghani S , Cekan S Z
Published: January 2020

We tested the accuracy of radioimmunoassays for two hormones, progesterone and estradiol, in relation to the use of two different antisera in each assay and to the degree of purification from plasma before the assay was done. Each analyte was assayed either in a diethyl ether extract, in a zone eluted from a Sephadex LH-20 chromatographic column, or in fractions of a chromatographic zone (from Sephadex LH-20 or Celite) that passed a test of "radiochemical purity." Statistically indistinguishable results were obtained in the assay of radiochemically pure fractions of both analytes, irrespective of the antiserum used. In addition, one of the antisera from each hormone gave equivalent results in the radioimmunoassay of ether extracts, even with no preceding chromatography. We demonstrated in this way that results obtained with use of highly specific antisera may, after a single chromatography, but even without any chromatographic purification, be as nearly accurate and as closely reflect the true value as those obtained in the assay of radiochemically pure hormones, an assay that has a character of a reference method (as defined by the IFCC).
Simple, sensitive assay of polyamines by high-performance liquid chromatography with electrochemical detection after post-column reaction with immobilized polyamine oxidase.
Authors: Maruta K , Teradaira R , Watanabe N , Nagatsu T , Asano M , Yamamoto K , Matsumoto T , Shionoya Y , Fujita K
Published: January 2020

This simple, rapid liquid-chromatographic assay of urinary polyamines (putrescine, spermidine, spermine, and cadaverine) involves electrochemical detection with a post-column immobilized enzyme, polyamine oxidase (EC 1.4.3.6) from soybean seedlings. Polyamines are separated by isocratic ion-pairing reversed-phase chromatography, then enzymatically converted, with release of hydrogen peroxide, via the post-column reactor with immobilized polyamine oxidase; the hydrogen peroxide is detected by electrochemical oxidation on a platinum electrode. The detection limits for injected putrescine, spermidine, and spermine were 0.3, 0.5, 0.6, and 4 pmol, respectively, with linear ranges of two to three orders of magnitude. Reproducibility was also good, with CV values less than 7%. The efficiency of the immobilized enzyme column was not decreased after analysis of 300 urine samples. Putrescine and spermidine excretion in urine from patients with blood cancers and solid cancers was significantly increased.
A 125I-radioimmunoassay for measuring androstenedione in serum and in blood-spot samples from neonates.
Authors: Thomson S , Wallace A M , Cook B
Published: January 2020

We developed a radioimmunoassay with a gamma-emitting radioligand to measure androstenedione in human serum and in dried blood-spot samples from newborns. Antisera were raised in rabbits against androstenedione linked to bovine serum albumin at positions 3, 6, or 11 on the steroid nucleus. Radioligands were prepared by linking [125I]iodohistamine at positions 3, 6, or 11. Linkages were through either carboxymethyloxime or hemisuccinate bridges. All label and antibody combinations were examined, and the most sensitive and specific combination (antiserum raised against androstenedione-3-carboxymethyloxime-bovine serum albumin with an androstenedione-carboxymethyloxime-[125I]iodohistamine label) was selected for full evaluation. We report the performance of these selected reagents in an immunoassay for androstenedione in both serum and dried blood-spot samples from neonates. We measured concentrations of androstenedione in serum under normal and pathological conditions such as congenital adrenal hyperplasia and polycystic ovarian disease. Diurnal variation in normal men was observed. Androstenedione was measured in blood spots from neonates born at term or prematurely, with respiratory distress syndrome, or with congenital adrenal hyperplasia.
Accuracy and precision of analyses for total cholesterol as measured with the Reflotron cholesterol method.
Authors: Bachorik P S , Bradford R H , Cole T , Frantz I , Gotto A M , Roberts K , Warnick G R , Williams O D
Published: January 2020

We compared plasma cholesterol measurements made with the Boehringer Mannheim Reflotron reflectance photometric analyzer in 1298 capillary blood samples with measurements made in venous blood samples collected at the same time and analyzed in four standardized Lipid Research Clinics laboratories. The Reflotron measurements averaged 0.8% to 7.8% lower than the laboratory values. Correlations (r) between the two sets of measurements ranged from 0.92 to 0.96. In some samples, however, the Reflotron values differed from the laboratory values by greater than or equal to 12%; the cholesterol concentrations in these samples tended to be higher than in those for which better agreement was observed. The smaller negative biases were observed when test strips were used that were calibrated with reference to the Centers for Disease Control Reference Method for cholesterol. The agreement between sequential Reflotron values averaged less than or equal to 4.3%. There was an average difference of less than or equal to 1.0% between Reflotron measurements made in each of two sequential capillary blood samples taken from a single finger puncture.
Murine monoclonal antibody adsorbed onto vinylidene fluoride floccules used to eliminate antibody interference in "sandwich"-type immunoassays.
Authors: Newman E S , Moskie L A , Duggal R N , Goldenberg D M , Hansen H J
Published: January 2020

We have previously reported that human anti-mouse IgG antibody (HAMA) can cause false-positive and false-negative results in "sandwich"-type monoclonal antibody (MAb) assays. To eliminate HAMA interference in "sandwich"-type MAb assays, we investigated the use of MAb on solid-phase, vinylidene fluoride floccules, which we have previously used as a solid-phase second antibody for RIA. The simple procedure effectively removes greater than 95% of HAMA from the most positive serum we have obtained from patients hyperimmunized to murine MAb, and it allows for accurate quantification of carcinoembryonic antigen. The solid-phase complex, added to blood, effectively removes HAMA and (or) "HAMA-type" heterophilic antibody from the sera or plasma.
Characterization of sulfasalazine's interference in the measurement of conjugated bilirubin by the Ektachem slide method.
Authors: Franquemont D W , Sutphen J L , Herold D A , Bruns D E
Published: January 2020

We describe the cases of four patients who were taking sulfasalazine for inflammatory bowel disease, whose conjugated bilirubin concentrations in serum exceeded their corresponding total bilirubin concentrations as measured with a multilayer film analyzer, the Ektachem 400. Sulfasalazine added to pooled human serum at therapeutic concentrations increased the apparent conjugated bilirubin, as measured with the Ektachem, in a linear and dose-related fashion. Measured unconjugated bilirubin was simultaneously decreased to values less than -3 mg/L. The same interference occurred on the Ektachem 700, but an algorithm prevented the instrument from reporting the results. The major metabolites of sulfasalazine in blood did not interfere with analysis for those fractions of bilirubin. Sulfasalazine's strong absorbance at 400 nm explains its interference with determination of conjugated bilirubin in this instrument.
Estimation of reference intervals for total protein in cerebrospinal fluid.
Authors: Lott J A , Warren P
Published: January 2020

Protein in cerebrospinal fluid (CSF) was measured by a modified biuret procedure and in two automated instruments, the Du Pont aca and the Kodak Ektachem. The latter's dry-slide reagent was also evaluated for precision, linearity, and effect of potential interferents. In vitro, ampicillin and vancomycin increase the apparent value for CSF protein as measured with the Ektachem slides. We excluded patients with disorders of the central nervous system, and we estimated the central 95% percentile reference intervals for CSF protein for each of the three methods. We found no age or gender dependence of values. By the biuret procedure, the reference interval is 140 to 620 mg/L.
Enzyme-linked immunosorbent assay for free thyroxin in human serum.
Authors: Chui S H , Wan K C , Lam C W , Lewis W H
Published: January 2020

We describe a simple enzyme-linked immunosorbent assay (ELISA) in which microtiter plates are used for determining the free thyroxin concentration (FT4) in serum. Only commercially available chemicals and reagents are needed, including the antiserum. The working range is 1 to 60 ng/L. Turnaround time is about 4 h. Within-run coefficients of variation (CV) for FT4 concentrations of 9.0, 19.0, and 35.0 ng/L were less than 5%; between-run CVs were 10% to 11%. Results by a radioimmunoassay method for 150 sera correlated well (r = 0.922).
Normal biochemical and hematological values in New Zealand white rabbits.
Authors: Hewitt C D , Innes D J , Savory J , Wills M R
Published: January 2020

Animal model systems are used extensively for experimental in vivo studies. When biochemical and hematological measurements are made, reference intervals for the animal species must be determined. Here, the subjects for our study were young adult male New Zealand White rabbits. Blood was sampled from 110 normal healthy rabbits. Biochemical and hematological blood variables were measured by methods available in our routine service laboratory, and we report the ranges.
An interlaboratory study of blood-gas analysis: dependence of pO2 and pCO2 results on atmospheric pressure.
Authors: Burnett R W , Itano M
Published: January 2020

Blood-gas data from a large interlaboratory survey were analyzed to determine whether pO2 and pCO2 results are affected by the atmospheric pressure at the measuring location. A small but statistically significant dependence was found, averaging about 2.3% per 100 mmHg (1.7% per 10 kPa) for pO2 and 1.0% per 100 mmHg (0.75% per 10 kPa) for pCO2.
Cost-effective assays for use in monitoring carbamazepine, phenobarbital, and phenytoin in serum.
Authors: Moore F M , Simpson D
Published: January 2020

Using Syva "EMIT" reagents and the Cobas Bio centrifugal analyzer, we have developed cost-effective assays for therapeutic drug monitoring of carbamazepine, phenobarbital, and phenytoin. With these optimized methods one can assay up to 2470 samples with a single 100-test EMIT kit with good precision and accuracy. Between-batch CVs, based on results of repeat analysis of routine patients' samples, were 4.6%, 7.0%, and 5.3%, respectively. Results correlated well with those by methods previously used [gas chromatography and fluorescence polarization (Abbott TDx)], but there was a negative bias (-10.9%) of our method for phenobarbital results in patients' samples, as compared with those obtained with the TDx.
Amiodarone and the thyroid.
Authors: Kennedy R L , Griffiths H , Gray T A
Published: January 2020

An antiarrhythmic drug, amiodarone, contains 37% iodine by weight and is structurally similar to the thyroid hormones. The drug inhibits hepatic 5'-deiodinase, resulting in increases in serum thyroxin and "reverse" triiodothyronine, whereas the concentration of triiodothyronine in serum is decreased. There is a significant incidence of either hypothyroidism or hyperthyroidism in patients who are being treated with the drug. This is largely the effect of iodine released from the drug during chronic therapy, but in susceptible individuals amiodarone may unmask autoimmune thyroid disease. Some effects of the drug suggest that it may interfere with the action of thyroid hormones at the cellular level, inducing a state of localized hypothyroidism.
Quantification of strontium in plasma and urine with flameless atomic absorption spectrometry.
Authors: Leeuwenkamp O R , van der Vijgh W J , Hüsken B C , Lips P , Netelenbos J C
Published: January 2020

This analytical method for determination of Sr in plasma and urine involves flameless atomic absorption spectrophotometry (FAAS). Drying, charring, and atomization were optimized with respect to temperature, temperature ramp, and duration for Sr in dilute HNO3 and Sr in plasma diluted 20-fold with dilute HNO3. Calibration curves (r greater than 0.995) were linear in the concentration range 5-250 micrograms/L for Sr in various media, with intercepts negligibly small except for the calibration curves in 1:1-diluted plasma and undiluted urine. The estimated detection limits for Sr in 20-fold-diluted plasma and 50-fold-diluted urine were 2 and 3 micrograms/L, respectively. Endogenous Sr in plasma and urine was estimated at 16 (SD 8) micrograms/L and 158 (SD 26) micrograms/L (n = 6), respectively. Intra- and interassay CVs were 9.1% and 5.3% for 20-fold-diluted plasma at a Sr concentration of 25 micrograms/L, and 6.9% and 4.8% at a concentration of 250 micrograms/L. The respective CVs were 8.2% and 1.2% for 50-fold-diluted urine at the low concentration, and 4.0% and 4.6% at the high concentration. In a pharmacokinetic pilot study of 2.5 mmol of Sr orally administered to a healthy volunteer, the peak plasma concentration of Sr, 4.4 mg/L, decayed bi-exponentially [t1/2, alpha = 24 h, t1/2, beta = 77 h]; the estimated first-order absorption rate constant was 0.005 min-1; and the observed decay (day 0-6) of the urinary Sr/creatinine ratio closely paralleled the plasma decay [t1/2 = 70 h].
Purification and characterization of intact lactoferrin found in the urine of human milk-fed preterm infants.
Authors: Hutchens T W , Henry J F , Yip T T
Published: January 2020

Little is known about the metabolic fate of ingested lactoferrin in human-milk-fed term or preterm infants. Enzyme-linked immunosorbent assays and sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis (SDS-PAGGE) with immunoblots have demonstrated that the urinary excretion of lactoferrin by preterm infants fed exclusively human milk exceeded that by formula-fed infants. The origin and molecular integrity of the excreted lactoferrin, however, are unclear. We have developed extraction and separation procedures involving a stationary phase of immobilized single-stranded DNA (ssDNA) that allows the efficient (greater than 80%) and rapid isolation of pure, intact lactoferrin from infants' urine. We purified lactoferrin to apparent homogeneity from infants' urine in one step, using the immobilized ssDNA with mobile phases containing up to 6 mol of urea per liter. The purified lactoferrin was evaluated by SDS-PAGGE; silver-staining revealed one protein band at 78 kDa; immunoblots confirmed the presence of intact lactoferrin. High-performance affinity chromatography with use of immobilized metal ion (Cu2+) suggested the presence of intact, iron-saturated lactoferrin. Subsequent chromatography on high-performance reversed-phase (C18) columns independently verified sample identity and purity.