bluuebunny/crossref_metadata_2025 huggingface dataset:
ILIS--An integrated laboratory information system. I. Biochemistry and hematology.
Authors: Deshpande S U
Published: January 2020

IBM System 34 (central processing unit, 128 kilobytes; fixed disks, 128.4 megabytes) with seven cathode-ray tubes has been used by our clinical laboratories for the last 30 months. All data-entry programs are in a conversational mode, for on-line corrections of possible errors in patient identification and results. Daily reports are removed from the medical records after temporary and permanent cumulative weekly reports are received, which keep a three-month track of the results. The main advantages of the system are: (a) the increasing laboratory work load can be handled with the same staff; (b) the volume of the medical record files on the patients is decreased; (c) an easily retrievable large data base of results is formed for research purposes; (d) faster billing; and (e) the computer system is run without engaging any additional staff.
Creatine kinase isoenzymes in serum from cord blood and the blood of healthy full-term infants during the first three postnatal days.
Authors: Jedeikin R , Makela S K , Shennan A T , Rowe R D , Ellis G
Published: January 2020

Isoenzymes of creatine kinase (ATP:creatine phosphotransferase; EC 2.7.3.2; CK) were measured by electrophoresis in serum from cord blood and skin-puncture blood taken from 45 healthy full-term infants during the first three postnatal days. Mean total CK activities (in U/L at 30 degrees C) were 185 in cord samples, 536 in samples taken between 5--8 h postnatally, 494 between 24--33 h, and 288 in the 72-100 h samples. Values for all three isoenzymes increased to a peak over this period, with the highest values generally being found in the samples taken 5--33 h after birth; the subsequent decline was most rapid for CK-BB. Serum CK isoenzymes in cord samples and those taken at 72--100 h in the 11 babies delivered by cesarian section did not differ significantly from those of babies delivered vaginally. However the postnatal increases in total CK, CK-MM, and CK-MB (but not in CK-BB) were significantly greater in those patients born by vaginal delivery. The reasons for the increases in CK isoenzymes after birth are not clear, but our results and reported studies on the ontogeny of CK suggest that CK-MB cannot be regarded as a "cardiac-specific" isoenzyme in the neonatal period.
Interactions among enzyme, antibody, drug, and time in enzyme immunoassays for drugs of abuse.
Authors: Osterloh J , Butrimovitz G P
Published: January 2020

Published information on the major interactive variables of enzymic immunoassays for drugs, whether in urine or serum, is scanty. Using the Syva EMIT-dau Barbiturates assay as an example, we examined the following interactive variables with respect to test response: concentrations of enzyme, antibody, and drug; urine matrix; and time. The results suggest a process for optimizing this type of enzyme immunoassay. Transformation of the data gives indirect evidence of heterogeneous antibody reactions that are drug-concentration dependent.
Laboratory differentiation of hypercalcemic patients.
Authors: Johnson K R , Howarth A T , Hamilton M , Mascall G C
Published: January 2020

We studied the biochemical discrimination of primary hyperparathyroidism from other causes of hypercalcemia. This potentially serious disorder is readily remedied, and because recent work has suggested that even very mild cases in elderly people derive substantial benefit from surgery, the correct diagnosis is crucial. A discriminant function is derived that maximizes the use of data that are readily available from general hospital laboratory; application of the Hamilton "T score" system simplifies its interpretation. We conclude that the best use of this formula is as a negative discriminant, to identify some non-hyperparathyroid cause of hypercalcemia, and also as a partial positive discriminant, to identify some cases of primary hyperparathyroidism. We review previous such discriminants and consider the reasons why they have been less successful.
Improved procedure for lecithin/sphingomyelin ratio in amniotic fluid reduces false predictions of lung immaturity.
Authors: Brown L M , Duck-Chong C G , Hensley W J
Published: January 2020

Many laboratories have found that their procedure for determining lecithin/sphingomyelin (L/S)ratios gives an unacceptably high proportion of false predictions of fetal lung immaturity. We investigated each step in the procedure, in an attempt to improve the clinical performance of the test and to make the method more amenable to standardization between laboratories. L/S ratios were determined by the new procedure in amniotic fluid from 147 pregnancies, collected within two days of delivery of the infant. Four cases had an L/S ratio less than 2; all developed hyaline membrane disease. No other cases of hyaline membrane disease were encountered in this study. For 106 of these pregnancies, the L/S ratio was also determined by the procedure previously in use. This predicted lung immaturity for 16 infants, only four of whom developed hyaline membrane disease. All 12 cases incorrectly predicted as immature by the old procedure were correctly classified by the new procedure.
Methylene unit calibration for analysis of organic acids by gas--liquid chromatography/mass spectrometry.
Authors: Bromberg I L , Heininger J A , Cherian A G
Published: January 2020

We describe a method for automatic computation of "methylene unit" retention-time indices in real time on a computer-controlled gas chromatograph/mass spectrometer system. The calculation is based on the exponential relationship between the methylene unit values of hydrocarbon reference standards and the oven temperature at their elution. Using this mathematical relationship, we calculated calibration factors, which eliminates the need to include a set of methylene unit reference standards with each run and simplifies and speeds identification of unknown peaks in chromatograms. Long-term stability and good precision of the calibration factors were observed during the six-month period of study.
A variant alkaline phosphatase in renal cell carcinoma.
Authors: Whitaker K B , Eckland D , Hodgson H J , Saverymuttu S , Williams G , Moss D W
Published: January 2020

We report a case of renal cell carcinoma in which up to 32% of the abnormally increased alkaline phosphatase activity in serum was contributed by a variant alkaline phosphatase originating in the primary tumor and its secondary deposits. The variant enzyme was probably an altered form of normal renal alkaline phosphatase. The rest of the alkaline phosphatase activity in the serum was of hepatic origin, but no abnormality of the liver was discovered at autopsy.
Comparing multivariate and univariate subject-specific reference regions for blood constituents in healthy persons.
Authors: Harris E K , Yasaka T , Horton M R , Shakarji G
Published: January 2020

We examined the comparative behavior of subject-specific multivariate and univariate reference regions, using both computer-generated data and serial (semi-annual) measurements of selected analytes in subjects from a large health-maintenance program. Univariate studies under both homeostatic and random-walk time-series models were helpful in defining expected results, but only the homeostatic model was used in multivariate as well as univariate forms. Analysis of the computer-generated data and the real biochemical series produced similar findings, which showed the multivariate subject-specific reference region to be much more conservative than corresponding univariate intervals. That is, a multidimensional point of p correlated observations is quite likely to lie within the individual's multivariate reference region (based on past observation vectors), even when one or more of the observations lie outside their separate reference intervals for that individual. One consequence of this high specificity against univariate false positives in a large surveillance program is a higher than expected proportion of positive multivariate vectors in which none of the values lie outside their univariate ranges. Thus, although the development of multivariate reference regions should be encouraged, they should be used in conjunction with, not instead of, univariate ranges.
Multivariate and univariate optimization studies of liquid-chromatographic separation of steroid mixtures.
Authors: Fast D M , Culbreth P H , Sampson E J
Published: January 2020

We developed a high-performance liquid-chromatographic separation of five steroids (estriol, estradiol, cortisol, progesterone, and testosterone), eluting with a water-acetonitrile gradient from a reversed-phase (C18) column. By applying a simplex search algorithm to maximize a chromatographic-response function, we sought to optimize the original conditions of the chromatographic analysis, which did not separate two pairs of overlapping peaks. Our chromatographic-response function incorporated both peak separation and total time of analysis. Three factors were varied simultaneously to maximize this function: flow rate, column temperature, and gradient shape. From the simplex optimization, we selected a flow rate of 1.50 mL/min, a temperature of 52 degrees C, and a linear gradient for our analysis. Subsequent univariate studies of the initial mobile phase composition showed that acetonitrile/water (20/80 by vol) gave an adequate separation.
Quantitative liquid-chromatographic technique for the simultaneous assay of tricyclic antidepressant drugs in plasma or serum.
Authors: Sonsalla P K , Jennison T A , Finkle B S
Published: January 2020

A quantitative "high-pressure" liquid-chromatographic assay for tricyclic antidepressant drugs in plasma or serum is described in which amitriptyline, nortriptyline, imipramine, desipramine, doxepin, and desmethyldoxepin are separated with a 10-micrometer particle size silica column and a methanol/NH4OH/NH4NO3 solvent system. The drugs and two internal standards are extracted with hexane/isoamyl alcohol, the solvent layer is evaporated at 40 degrees C, and the drugs are detected at 254 nm. Drug concentrations are linear with absorbance from 25 to 1000 micrograms/L; within-assay and between-assay CVs are less than or equal to 10%.
Sample preparation and liquid-chromatographic analysis for tricyclic antidepressants in serum.
Authors: Koteel P , Mullins R E , Gadsden R H
Published: January 2020

We report two separate sample-preparation procedures and a "high-pressure" liquid-chromatographic method for quantitating nine tricyclic antidepressant drugs: trimipramine, doxepin, amitriptyline, imipramine, desmethyldoxepin, nortriptyline, desipramine, maprotiline, and protriptyline. The drugs are extracted from 1 mL of serum by a single manual hexane extraction or by using DuPont's PREP I Automated Sample Processor. The drugs from either process are chromatographed on a mu-Bondapak-CN column with a mobile phase of acetonitrile/methanol/Na2HPO4, 5 mmol/L (60/15/25 by vol), pH 7.0, at a flow rate of 2.0 mL/min. Absorbance at 254 nm is linear with drug concentration from 25 to 1000 mg/L for each drug. With the manual extraction procedure the detection limit was 10 mg/L, absolute recovery 20-44%, within-run precision (CV) 10.1%, and run-to-run precision 11.1%. The performance of automated sample extraction was better, with a detection limit of 5 mg/l, absolute recovery 72-97%, within-run precision 6.1%, and run-to-run precision 6.6%.
Two-dimensional electrophoretic analysis of human leukocyte proteins from patients with rheumatoid arthritis.
Authors: Willard K E , Thorsrud A K , Munthe E , Jellum E
Published: January 2020

Human leukocyte proteins from more than 150 patients with rheumatoid arthritis, together with age- and sex-matched controls, were analyzed by use of the ISO-DALT technique in two-dimensional polyacrylamide gel electrophoresis. Patients with ankylosing spondylitis, polymyalgia rheumatica, psoriatic arthritis, calcium tendinitis, post-infectious arthritis, and asymmetrical seronegative arthritis were also included as positive controls. Synthesis of several proteins, referred to by number as members of the "Rheuma" set, is shown to increase in the leukocyte preparations from patients with classical rheumatoid arthritis. Several of these proteins are specific to monocytes or granulocytes; others are of unknown cellular origin, but appear to be unique to rheumatoid arthritis. The Rheuma proteins appear to be indicators of disease activity, because their increased synthesis can be correlated with sedimentation rate and other clinical indices of rheumatoid disease activity.
A two-dimensional electrophoretic analysis of the heat-shock-induced proteins of human cells.
Authors: Anderson N L , Giometti C S , Gemmell M A , Nance S L , Anderson N G
Published: January 2020

Using two-dimensional electrophoresis, we have investigated the responses of human cells in culture to heat shock and to various chemical agents producing a similar effect. These treatments result in the induction of increased synthesis of several specific proteins. One (HShock:1, SDS-molecular mass about 65000) is increased by about 350-fold over the amount in untreated cells. Computer analysis of time-course studies indicates, however, that rates of synthesis of various proteins other than the classical "heat shock proteins" are affected, some of these alterations following time courses quite different from the main (HShock) inductions. The heat shock effect is thus much more complicated than previously realized. We purified the HShock:1 protein from heat-shocked human lymphoblastoid cells, and prepared a rabbit antiserum specific for HShock:1 on nitrocellulose two-dimensional gel transfers of total lymphoblastoid cell protein. A survey of mouse tissues shows high concentrations of an HShock:1-like protein in the testis, and human testes also appears to contain substantial (though lower) concentrations. These results are consistent with the hypothesis (derived from the tissue-culture studies) that the heat shock effect is a general response to the need for increased protein catabolism within the cell. Increased concentrations of HShock:1 are also observed in preparations of blood leukocytes collected from patients after surgery, indicating that some types of physiological trauma may induce the heat shock proteins in man. Using the antiHShock:1 antibody in an immunoassay, it will be possible to systematically examine HShock:1 concentrations in plasma and leukocytes, thereby opening up the possibility of a clinical test based for the first time upon an inducible aspect of cellular gene expression.
beta-Glucosidase assays in the diagnosis of Gaucher's disease.
Authors: Daniels L B , Glew R H
Published: January 2020

The description in 1965 of glucocerebroside: beta-glucosidase as the enzymic defect in Gaucher's disease stimulated considerable research interest and effort toward establishing rapid, reliable, and inexpensive enzymic assays for diagnostic purposes and carrier detection. Here, we consider some of the methods currently in use in which the substrate is the synthetic glucoside, 4-methylumbelliferyl-beta-D-glucopyranoside, and leukocytes and fibroblasts are the sources of enzyme. We also consider the concepts of the "acid beta-glucosidase" and multiple forms of beta-glucosidase that have been proposed to explain the effectiveness of the fluorometric assays. Finally, we analyze the limitations of each method and discuss the difficulties involved in instituting heterozygote screening programs in the general population.
Optimized bioluminescence assay of creatine kinase and creatine kinase B-subunit activity.
Authors: Lundin A , Jäderlund B , Lövgren T
Published: January 2020

Spectrophotometry of CK-B subunit activity based on immunoinhibition is rapid and convenient. However, the low sensitivity limits clinical applications to serum samples with CK-B activities considerably above normal. A sensitive, but not optimized, bioluminescent assay of CK-B, also based on immunoinhibition, has been described. We improved this method by the use of an ADP concentration saturating the CK reaction and by the combined use of AMP and diadenosine pentaphosphate for inhibition of adenylate kinase. The optimized bioluminescent method is similar to the corresponding spectrophotometric method (reaction conditions, analytical procedure, numerical results, etc.) and results by the two methods correlate well. However, the measuring time is shorter and, more important, the sensitivity of the bioluminescent method allows accurate determinations of CK-B, even in serum from healthy individuals (CV approximately 3%). Kits and instrumentation (manual and automatic) for determination of CK and CK-B are now commercially available.
Screening test for alpha 1-antitrypsin in dried-blood specimens.
Authors: Orfanos A P , Naylor E W , Guthrie R
Published: January 2020

We describe a fluorescent spot test for detecting alpha 1-antitrypsin activity in dried-blood specimens. The eluate of a blood disc is mixed with carbobenzoxy-L-arginine-7-amino-4-methylcoumarin amide and trypsin at the appropriate pH. In the absence of alpha 1-antitrypsin, aminomethylcoumarin, a strongly fluorescent compound, is released. The reaction mixture, when spotted on chromatography paper and viewed under ultraviolet light, exhibits a bright fluorescence only in the case of specimens with alpha 1-antitrypsin deficiency. alpha 1-Antitrypsin activity so estimated correlated well with quantitative assays of dried-blood spots and serum. The procedure is simple and inexpensive, and has the potential for use as a screening test.
Radioimmunoassay of serotonin (5-hydroxytryptamine) in cerebrospinal fluid, plasma, and serum.
Authors: Engbaek F , Voldby B
Published: January 2020

We describe a direct radioimmunoassay for serotonin (5-hydroxytryptamine) in cerebrospinal fluid, platelet-poor plasma, and serum. We raised antisera in rabbits against serotonin diazotized to a conjugate of bovine albumin and D,L-p-aminophenylalanine. Polyethylene glycol, alone or in combination with anti-rabbit immunoglobulins, is used to separate bound and unbound tritiated serotonin. The minimum concentration of serotonin detectable is 2 nmol/L in a 200-microL sample. Within-day precision (CV) is 4.3%, between-day precision 7.7%. Analytical recoveries of serotonin are 109% and 101% for cerebrospinal fluid and plasma, respectively. Tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol do not interfere with the assay. However, 5-methoxytryptamine and tryptamine cross react. Of samples of cerebrospinal fluid from patients with disc herniations (n = 21) or low-pressure hydrocephalus (n = 10), one-third had concentrations of 2-4 nmol/L and two-thirds were below the minimum detectable concentration. The observed range for the concentration of serotonin in plasma of 14 normal subjects was 5-14 nmol/L (mean +/- SD, 9 +/- 3 nmol/L). The observed ranges for serotonin in serum were: for 10 women 520-900 (mean +/- SD: 695 +/- 110) nmol/L and for 10 men 380-680 (520 +/- 94) nmol/L.
X-Ray fluorescence measurement of the zinc profile of a single hair.
Authors: Toribara T Y , Jackson D A
Published: January 2020

Many trace elements appear in hair in concentrations related to those in the blood, but the relationship for zinc is complicated by disorders that probably affect its state in the blood. A built-to-order x-ray fluorescence spectrometer was used to measure, nondestructively, the concentrations of 16 elements in a 1-mm interval of a single hair. A scan along a hair strand, together with the known growth rate, enables the zinc concentration in the hair to be correlated with the time the hair was formed. A comparison with the blood concentration at the same time may reveal possible bodily disorders that affect the availability of zinc to incorporation in the hair. The instrument has been carefully calibrated for zinc, and we studied the conditions under which there can be losses or gains in the hair after sampling. The sample chamber will accommodate 16 separate holders, and each sample may be automatically measured according to a program pre-selected for it. The average zinc content of hair samples from foreign countries, some ancient specimens, and an interesting profile of a strand from an Iraqi woman are shown.
Analysis of phosphate in serum with the phosphate redox electrode system.
Authors: Montalvo J G , Truxillo L A , Wawro R A , Watkins T A , Phillips A , Jenevin R M
Published: January 2020

A phosphate redox electrode system (PRES) was designed, constructed, and evaluated for analysis of inorganic phosphate in serum. This system is based on the observed phosphate ion potential of a chemically treated iron wire in a turbulent flow-through cell at constant oxygen tension. We analyzed 110 clinical samples by this technique and with the Technicon SMAC. Of the specimens analyzed, 32% contained abnormal phosphate concentrations. Each was assayed two to four times with the PRES and once with the SMAC. The PRES assay rate was 60 samples/h. Mean phosphorus concentrations (and SEM) were: PRES, range of values for replicate data sets, 30.5-31.0 (0.5-1.1) mg/L; SMAC, 30.8 (1.0) mg/L. Results for the PRES (y) correlated well with those by the SMAC: r = 0.968 to 0.980, estimated slope = 0.99 to 1.03, and estimated intercept = -1.1 to 0.1 mg/L. Differences in results by the two methods were not statistically significant.
Clinical studies of protein-bound calcium in various diseases.
Authors: Duncan P H , Wills M R , Smith B J , Savory J
Published: January 2020

A recently developed gel-filtration technique allows protein-bound calcium fractions to be separated and quantitated; the protein is separated under physiological conditions of pH, temperature, and concentrations of Na, Mg, and Ca to assure that the calcium-proteinate equilibrium is not disturbed. We used this gel-filtration technique to study the protein-bound calcium fractions in 18 patients with hyperparathyroidism, multiple myeloma, diabetes, osteoporosis, or liver cirrhosis. We calculated the amount of calcium bound per gram of protein for each of the three protein peaks and the intrinsic association constant (Ka) for calcium/albumin. Results with the multiple myeloma patients (three IgG, one IgA) indicated that IgG did not bind calcium appreciably, that IgA had about the same affinity as albumin for Ca, and that Ka was slightly low for one patient of the IgG type (79 L/mol) and normal for the other three myeloma patients (106, 90, and 91 L/mol). Results for patients with the other diseases were also essentially normal, except for the osteoporesis patients (two men, one woman), whose Ka values (69, 75, and 73 L/mol) were lower than normal.
Direct determination of the linoleate/oleate ratio in serum cholesterol esters by liquid chromatography.
Authors: Bernert J T , Akins J R , Miller D T
Published: January 2020

We describe a convenient method for the direct determination of the serum cholesterol linoleate/cholesterol oleate (L/O) ratio by reversed-phase "high-performance" liquid chromatography. After removal of phospholipids by silicic acid chromatography, a serum extract is analyzed on a 5-micrometers particle size Ultrasphere-ODS column, eluted isocratically with acetonitrile/isopropanol (30/70, by vol). Detection is at 200 nm. Cholesterol palmitoleate interferes with the measurement when the analysis is based on peak area, but not when peak height is used. The overall precision of L/O measurements by this method was very similar to that observed with a gas-liquid chromatographic procedure, in which the cholesterol esters are first isolated and transesterified to the methyl esters. In both cases, the within-run CV for six replicate analyses was less than 2%. Analysis of 53 human serum samples by both methods yielded very similar L/O ratios. A plot of the data (our method = y) vs the usual gas-liquid chromatographic procedure gave a correlation coefficient of 0.988 and a regression equation of y = 1.03x + 0.013. Furthermore, direct analysis of serum cholesterol ester L/O ratios by our liquid-chromatographic method is simpler, quicker, and more readily adaptable to automation.
Up-dated catalogue of HeLa cell proteins: percentages and characteristics of the major cell polypeptides labeled with a mixture of 16 14C-labeled amino acids.
Authors: Bravo R , Celis J E
Published: January 2020

A total of 1357 polypeptides [946 acidic (isoelectric focusing) and 411 basic (nonequilibrium pH-gradient electrophoresis)] from human HeLa cells have been separated and catalogued with use of high-resolution two-dimensional gel electrophoresis. Of these polypeptides, 1266 were detected by labeling cells with [35S]methionine, while the rest were revealed by silver staining or by labeling with a mixture of 16 14C-labeled amino acids. For convenience, all these polypeptides have been numbered and are indicated in a large fold-out protein map. The percentages of some of the major 14C-labeled proteins have been determined, and for some we list a few characteristics such as: variation during the cell cycle; cellular distribution in cytoplasts and karyoplasts; presence in Triton- and salt-extracted cytoskeletons; and phosphorylation and sensitivity to neoplastic transformation.
Two-dimensional gel electrophoresis of human brain proteins. III. Genetic and non-genetic variations in 145 brains.
Authors: Comings D E
Published: January 2020

To determine the frequency of genetic mutations, polymorphisms, and non-genetic variation in the major human brain proteins, I examined, by equilibrium two-dimensional gel electrophoresis, 145 brains from patients dying of a wide variety of psychiatric, neurological, and non-neurological disorders. Of 176 polypeptides screened, there was one polymorphism of glial fibrillary acidic protein (GFAP-Duarte). Chi square analysis indicated it was non-randomly distributed among different diseases. A possible mutation associated with Joseph's disease is being further investigated. Three examples of a possible mutation of protein 8c:1 were noted. No other genetic mutations were observed. This low frequency of polymorphisms is consistent with results for two-dimensional gel analysis of other tissue and species. The numerous non-genetic variations are described.
Two-dimensional gel electrophoresis of human brain proteins. IV. Disorders of glial proliferation and a polymorphism of glial fibrillary acidic protein--GFAP Duarte.
Authors: Comings D E
Published: January 2020

Several genetic and non-genetic neurological disorders are characterized by neuronal cell death, followed by astrocytic glial cell proliferation. Because the major intermediate filament in astrocytes is glial fibrillary acidic protein (GFAP), large amounts of this protein accumulate. The two-dimensional gel-electrophoretic pattern of gliosed tissue is described. It shows a massive accumulation of the 50 000-dalton GFAP charge isomers (8e:1--8) plus a series of breakdown products of lower molecular mass and more-acid charge. In addition, I describe a genetic polymorphism, GFAP-Duarte, presenting as an additional, basic charge isomer of GFAP. The possibility that this or other mutations of GFAP may predispose certain neurons to premature cell death is discussed.
Actin mutations in a human fibroblast model for carcinogenesis.
Authors: Leavitt J , Goldman D , Merril C , Kakunaga T
Published: January 2020

We assessed the modulation of gene expression accompanying neoplastic transformation by computerized microdensitometry of autoradiographic patterns of [35S]-methionine-labeled polypeptides separated by two-dimensional polyacrylamide gel electrophoresis. Nearly 1000 polypeptide species of parent diploid human fibroblasts (KD strain) and clonally-derived malignant fibroblasts (HUT-14 strain) were compared. We found that the neoplastic HUT-14 fibroblasts express a mutation in one of the two functional beta-actin genes. In addition, of the 700 more-abundant polypeptides measured, 13 were lost and 14 new ones gained after this neoplastic transformation. We estimate that although 2% of fewer of the genes expressing abundant polypeptides were either activated or shut off, at least 32% were modulated quantitatively. A substrain of HUT-14--HUT-14T--shows increased tumorigenicity, producing larger, faster-growing fibrosarcomas in the nude mouse than does the present parent HUT-14 strain, and with fewer inoculated cells. This increase in tumorigenicity is accompanied by three subsequent changes in the mutant beta-actin polypeptide expression. A more variant mutant actin species is synthesized in HUT-14T, which differs from the original mutant polypeptide by (i) one additional negative net charge, (ii) a short half-life in the cell, (iii) a greatly diminished ability to incorporate into the detergent-resistant cytoskeleton, (iv) a decrease in affinity for deoxyribonuclease I (EC 3.1.21.1), and (v) a faster rate of synthesis. Our results suggest that a second-site mutation in the mutant beta-actin of HUT-14 was selected for during a subcloning step in the presence of 6-thioguanine before derivation of the HUT-14T substrain. This apparent mutation and two subsequent defective beta-actin expressions are accompanied by incremental increases of malignant potential.
Two-dimensional gel electrophoresis of serum specimens from patients with monoclonal gammopathies.
Authors: Tracy R P , Currie R M , Kyle R A , Young D S
Published: January 2020

We modified the ISO-DALT two-dimensional gel electrophoresis system to allow the routine examination of serum specimens from patients with monoclonal gammopathies. This system, MC-Iso 1, is characterized by a broad pH gradient for resolving the basic immunoglobulin heavy and light chains. The increased resolution of basic proteins may be explained on theoretical grounds by an increase in voltage in this region of the cell. Ancillary techniques, such as those for albumin removal and pI assignment through use of charge standards, have also been implemented. The locations of immunoglobulin heavy chains have been confirmed by examination of over 250 serum samples as well as by "electro-blotting," with use of specific antisera. IgG subclass may also be predicted by location, but not with perfect accuracy. Differentiation of kappa and lambda light chains by relative mobility has been examined; the predictive value for correct identification of kappa chains is 83%, that for lambda chains 69%. Several unknown proteins have been observed in macroglobulinemia, related to mu heavy chain. Finally, we have determined that there is excellent correlation between non-denaturing isoelectric focusing and our system for pI assignment of light chains. This has importance due to reports of the potential importance of light-chain pI in the development of renal disease in patients with monoclonal gammopathies.
Two-dimensional electrophoretic analysis of erythrocyte membranes.
Authors: Rosenblum B B , Hanash S M , Yew N , Neel J V
Published: January 2020

In an effort to maximize the amount of genetic information that can be extracted from a blood sample, we investigated the use of two-dimensional polyacrylamide-gel electrophoresis (PAGE) to resolve the protein constituents of the erythrocyte membrane. Lyophilized membranes were dissolved in various concentrations of urea, NP-40 detergent, and mercaptoethanol and subjected to two-dimensional PAGE by a modification of the O'Farrell procedure, with use of the ISO-DALT apparatus. More than 600 spots were visible in silver-stained gels under conditions that excluded specific cytoskeleton protein components, including spectrin and actin. The reproducibility of the pattern depended highly on the precise composition of the solubilization mixture. Poor resolution was observed in the presence of actin and other proteins of high molecular mass (spectrin bands 1 and 2) when we used high urea concentrations that solubilized the entire erythrocyte membrane. The large number of polypeptides observed could not be attributed to proteolysis, because addition of proteolytic inhibitors to the membrane wash solutions did not alter the pattern on the gel. The pattern also did not appear to include erythrocyte cytosol proteins because, except for globin, none of five purified erythrocyte lysate proteins was visible in the erythrocyte membrane gels. We conclude that two-dimensional electrophoresis provides a powerful tool for the study of non-cytoskeletal erythrocyte membrane proteins.
Analysis of protein patterns in two-dimensional gels of cultured human cells with trisomy 21.
Authors: Klose J , Zeindl E , Sperling K
Published: January 2020

The occurrence of one chromosome of the cell in triplicate (trisomy, Ts) should increase the amount of all cell proteins coded by genes located on this chromosome. Many other proteins should be altered in their quantity by regulator genes, present in a threefold dosage, and by several indirect effects of the trisomy. We used two-dimensional electrophoresis to investigate the effect of the human Ts 21 on the proteins. Cells from different individuals with Ts 21 were cultured. Seven cell lines were derived from skin tissue and four from cells in amniotic fluid, and two cell clones were raised from fetal lung with Ts 21 mosaic. Stained two-dimensional protein patterns from these cell lines were compared with control patterns, and clearly visible differences in the staining intensity of corresponding polypeptide spots were evaluated. A few quantitatively variant polypeptides occurred in all trisomic cell lines investigated that were of a particular cell type, but no variants were consistently present in all of the 13 cell lines investigated. However the total number of variants (including variants not found in all individual cell lines) was considerably higher in trisomic cells than in normal cells.
Two-dimensional gel electrophoresis can be used to examine chromatographic effluent fractions from displacement column chromatography.
Authors: Torres A R , Krueger G G , Peterson E A
Published: January 2020

We show how two-dimensional gel electrophoresis can be used to monitor protein components in effluent fractions from a displacement column. A minor protein in human serum, of interest in studies on psoriasis and highly enriched by using carboxymethyldextrans as displacers on DEAE-Sephacel, was easily detected in the effluent fractions with the two-dimensional gel method because its concentration was sufficiently high and there was no interference by the carboxymethyldextrans or salt.
Clinical applications of protein determinations in biological fluids other than blood.
Authors: Killingsworth L M
Published: January 2020

Over the past decade, significant improvements in immunochemical and electrophoretic techniques have enabled collection of heretofore unavailable data on proteins in biological fluids, greatly increasing our understanding of protein physiology in the various body compartments and providing the foundation for clinical use of protein analysis in body fluids. The most striking advance has been in the diagnosis of demyelinating disease through the use of serum/cerebrospinal fluid protein ratios and the morphological evaluation of immunoglobulin banding patterns. These laboratory tests are now considered obligatory for any patient in whom demyelinating disease is suspected as the cause of neurological dysfunction. Cerebrospinal fluid protein data can also be helpful in quantitating the permeability of the blood/cerebrospinal fluid barrier in many inflammatory or infectious central nervous system disorders. Assays of individual proteins in urine can help distinguish between different types of proteinuria, and can give quantitative data on the selectivity of the glomerulus and the reabsorbing capacity of the tubules. The protein content of saliva, synovial fluid, and milk has also been well characterized, and is clinically applicable to a wide range of disorders.
Interference of immunoglobulins in two glucagon radioimmunoassays.
Authors: von Schenck H , Grubb A O
Published: January 2020

Radioimmunoassays of glucagon in plasma may be complicated by interaction with other substances of high molecular mass. Precipitates of such substances with ammonium sulfate showed, after isoelectric focusing, two fractions having glucagon immunoreactivity. One fraction (pl approximately 10) evidently is associated with the Fc portion (but not the Fab portion) of purified polyclonal immunoglobulin G (IgG). Equal amounts of purified monoclonal IgG of various subclasses, especially IgG 1, gave different "glucagon" readings, suggesting that some IgG may interfere more strongly than others. The other fraction (pI 5-6) appeared less consistently, and on gel chromatography appeared to be slightly larger than IgG. Together these fractions add about 50-100 ng/L to the immunoreactive glucagon values in plasma. Therefore methods in which glucagon is extracted before assay should be used for determining the concentration of glucagon present physiologically.
Radioimmunoassay and lipid-chromatographic analysis for free cortisol in urine compared with isotope dilution--mass spectrometry.
Authors: Lantto O
Published: January 2020

I compared results by three different routine methods for analysis for urinary cortisol with those by a highly specific reference method based on isotope dilution--mass spectrometry (i). A "high-performance" liquid-chromatographic method (II) gave the most comparable results (regression coefficient 0.86, intercept 9 nmol/L). For some urines much lower values were obtained by I than by II. Two radioimmunoassay (III) methods, one involving direct assay and one involving extraction, gave less-accurate results (regression coefficients of 1.87 and 1.52 and intercepts of 86 and 12 nmol/L, respectively, although values obtained by III and by I correlated well (r = 0.95-0.99), indicating a relation between the free cortisol and the compounds interfering in III. The apparent accuracy for the extraction method was previously assayed by I (regression coefficient 0.90, intercept 6 nmol/L). All four methods investigated showed a statistically significant sex-related difference in 24-h urinary cortisol excretion; evidently such a finding should be a prerequisite in any such method proposed for routine use.
Liquid chromatography of codeine in plasma with fluorescence detection.
Authors: Tsina I W , Fass M , Debban J A , Matin S B
Published: January 2020

This analytical method for easier determination of codeine in human plasma is based on "high-performance" liquid chromatography for separation and the natural fluorescence of codeine for detection. Codeine is extracted from alkalinized plasma with a mixture of hexane and dichloromethane, and the extract is further purified and chromatographed. The method can be used for routine assay of codeine at the concentrations of 10 micrograms/L or greater in human plasma. As little as 4 micrograms/L can be detected. Coefficients of variation for the assay of codeine in the concentration range of 10 to 100 micrograms/L were 2.2-7.4% (n = 6). We used this method to establish a concentration/time profile for plasma from a human volunteer after a 60-mg oral dose of codeine sulfate.
Dry film for the enzymic determination of total cholesterol in serum.
Authors: Dappen G M , Cumbo P E , Goodhue C T , Lynn S Y , Morganson C C , Nellis B F , Sablauskas D M , Schaeffer J R , Schubert R M , Snoke R E , Underwood G M , Warburton C D , Wu T W
Published: January 2020

We have prepared a dry film for the enzymic determination of total serum cholesterol. It consists of a transparent support bearing a buffered gelatin layer, and a white reflective spreading layer that contains all of the necessary components for the detection of cholesterol. The method is based on (a) hydrolysis of cholesterol esters to cholesterol by cholesterol ester hydrolase (EC 3.1.1.13), (b) oxidation of cholesterol to cholest-4-en-4-one and hydrogen peroxide by cholesterol oxidase (EC 1.1.3.6), and (c) oxidation of a triarylimidazole leuco dye with hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) to produce a dye with maximum absorption at about 650 nm. For use over a wider range of concentration, the dye density is read at 540 nm. With reflection densitometry and appropriate mathematical transformation, readings and cholesterol concentrations are linearly related to 5500 mg/L. Results correlate well with those by the Abell-Kendall comparison method (slope 0.97, intercept 92.5, correlation coefficient 0.974, Sy.x = 250.7), and the method is precise (CV of 1.2-2.3% for a control fluid and patients' samples) and relatively free of interferences.
Simultaneous quantitation of quinidine, procainamide, and N-acetylprocainamide in serum by gas-liquid chromatography with a nitrogen-phosphorus selective detector.
Authors: Kessler K M , Ho-Tung P , Steele B , Silver J , Pickoff A , Narayanan S , Myerburg R J
Published: January 2020

We describe a single-run method for quantitating quinidine, procainamide, and N-acetylprocainamide, involving gas-liquid chromatography with a nitrogen-phosphorus selective detector. Within-run precision (CV) was 3% (x = 2 mg/L, n = 20), 6.9% (x = 4 mg/L, n = 10), and 1.5% (x = 8 mg/L, n = 8) for quinidine; 7.7% (x = 4 mg/L, n = 14), 1.6% (x = 8 mg/L, n = 16), and 2.3% (x = 12 mg/L, n = 12) for procainamide; and 6.3% (x = 5 mg/L, n = 6), 3.6% (x = 10 mg/L, n = 20), and 4.0% (x = 20 mg/L, n = 10) for N-acetylprocainamide.l Between-run precision was 3.0%(x = 2 mg/L, n = 20), 7.0% (x = 4 mg/L, n = 9), and 2.8% (x = 8 mg/L, n = 9) for quinidine; 4.7% (x = 4 mg/L, n = 10). 3.3% (x = 8 mg/L, n = 20), and 1.9% (x = 12 mg/L, n = 10) for procainamide; and 9.3% (x = 5 mg/L, n = 6), 4.3% (x = 10 mg/L, n = 20), and 3.8% (x = 20 mg/L, n = 10) for N-acetylprocainamide. Tube stoppers that contain a rubber plasticizer interfere with the technique. Clinical application and correlation with drug concentrations by this technique are discussed.
Homogeneous nonisotopic assay for phenytoin evaluated.
Authors: Dugan S , Bailey J , Wu J W , Hoskin S , Riebe S , Gifford J , O'Neill S P
Published: January 2020

We evaluated a new homogeneous immunoprecipitation assay for phenytoin in human serum. No sample dilution or pretreatment is required. The new method is based on spectrophotometry of the inhibition by free phenytoin of the precipitating reaction between anti-phenytoin antibody and a phenytoin-human serum albumin conjugate. A serum test sample is simultaneously mixed with the phenytoin-albumin conjugate and rabbit antiserum to phenytoin in a centrifugal analyzer, and the subsequent reaction is monitored at 3 min. Within-run and between-run coefficients of variation were well below 7%. The relation between results for patients' test sample as determined by immunoprecipitation assay (y) and an enzyme immunoassay (x) can be expressed as y = 1.10x + 1.1 (r = 0.966, n = 66).
Diagnostic performance of enzymes in the discrimination of myocardial infarction.
Authors: Werner M , Brooks S H , Mohrbacher R J , Wasserman A G
Published: January 2020

We apply rigorous statistical methods to assess the diagnostic value of creatine kinase, creatine kinase-MB, aspartate aminotransferase, lactate dehydrogenase, and alpha-hydroxybutyrate dehydrogenase in realistic circumstances. The trade-off between false positives and false negatives obtained with various decision rules is examined with the receiver-operator characteristic function and with discriminant analysis. During the three days after an infarction, lactate and alpha-hydroxybutyrate dehydrogenase can provide diagnostic thresholds of constant sensitivity and specificity. By contrast, sensitivity progressively decays for creatine kinase, creatine kinase-MB, and aspartate aminotransferase. The diagnostic uncertainty introduced by the infarction's varying age at hospitalization is evaluated by subjecting the mixed patient population to discriminant analysis. For some enzymes, repeating the same assay on the second day contributes to sensitivity, while adding a different assay on the first day enhances specificity. The effects of lower or higher infarction prevalences on sensitivity and specificity are most favorable when creatine kinase-MB is combined with lactate or alpha-hydroxybutyrate dehydrogenase. Adding a third assay is ineffective. The further differentiation according to infarction stage produces only 81% correct classifications, even when five different assays are used on two consecutive days. As a general strategy, we recommend the assay of two enzymes on two consecutive days.
beta 2-Microglobulin instability in pathological urine.
Authors: Davey P G , Gosling P
Published: January 2020

Urinary excretion of beta 2-microglobulin (beta 2M) is a widely used test of renal proximal tubular function. beta 2M is known to be unstable in normal urine with a pH less than 5.5. The results of a study of beta 2M excretion in patients receiving gentamicin suggested that beta 2M might be more unstable in pathological urine. A series of experiments was designed to define the extent, variability, and mechanism of its instability in pathological urine. It was unstable in pathological urine with a pH of 6.0 at 37 degrees C and pH 5.5 at room temperature, but this varied in urines from different patients. The instability was abolished by heating the urine to 80 degrees C. These results suggest that the most likely explanation for the instability is enzymic degradation. Because enzymuria will vary among patients, investigators should test the stability of beta 2M in urine from the patients they wish to study before trying to define conditions for urine collection.
Normalization and reproducibility of mass profiles in the detection of individual differences from urine.
Authors: Marcuson R , Burbeck S L , Emond R L , Latter G I , Aberth W
Published: January 2020

The mass profiles of urine samples and their reproducibility (coefficient of variation) obtained by field-ionization mass spectrometry (volcano source) depend upon the choice of normalization method. We compared six different normalization techniques in terms of improved reproducibility and sensitivity. Several simple procedures markedly improved reproducibility while preserving the high degree of sensitivity. Although these procedures gave similar and satisfactory results, others were clearly inferior and are not recommended for clinical profiling.
Improved micromethod for assay of serum angiotensin converting enzyme.
Authors: Neels H M , Scharpé S L , van Sande M E , Verkerk R M , Van Acker K J
Published: January 2020

We describe conditions for determining angiotensin converting enzyme (EC 3.4.15.1) in serum, by liquid chromatography. Serum (10 microL) is assayed with the artificial substrate hippuryl-glycyl-glycine (30 mmol/L) in a 50 mmol/L "HEPES" buffer solution with a high salt content (300 mmol of NaCl and 400 mmol of Na2SO4 per liter), at pH 8.0. The resulting enzymic activity is ninefold that of the currently popular spectrophotometric assay of Cushman and Cheung as modified by Lieberman (Am. J. Med. 59: 365-372, 1975). The hippuric acid end product is separated from the substrate by reversed-phase liquid chromatography and measured spectrophotometrically at 228 nm. o-Methyl hippuric acid is used as internal standard. The mean value for 100 normal control subjects was 317 (SD 96) nmol of hippuric acid released per milliliter of serum per minute. The enzyme activity is greater in newborns (p less than 0.05) and has a tendency to decrease with age. This partly automated method, which is optimized with regard to activity and detection, can be used in clinical routine.