bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous measurement of plasma apolipoproteins A-I and B by electroimmunoassay.
Authors: Fruchart J C , Kora I , Cachera C , Clavey V , Duthilleul P , Moschetto Y
Published: January 2020

We describe a simplified electroimmunoassay for quantification of human apolipoproteins A-I and B on prepared plates. A solution of agarose at 55 degrees C, containing hydroxyethylcellulose and antibodies, is poured onto a plastic film and allowed to gel. Wells are punched in the gels and the plates are dried for storage. Before use, they are rehydrated and buffered. We use succinylated Sudan Black to prestain lipoprotein fractions of plasma. After electrophoresing samples of plasma or standards for 3 h at 4 degrees C at 12.5 V/cm, we measure the peak heights and read the results from a standard curve prepared by using calibrated sera of known apolipoprotein B and A-I content as secondary standard. The within- and between-assay coefficients of variation were less than 4% in all cases. Results correlated well with those obtained by classic electroimmunodiffusion. Subjects with confirmed atherosclerotic lesions had significantly (p less than 10(-9)) lower ratios of apolipoprotein A-I to apolipoprotein B, compared with ratios in controls.
Influence of erythrocyte iodothyronine-binding proteins on radioimmunoassay of thyroxin in dried blood spots.
Authors: Sadler W A , Lynskey C P
Published: January 2020

Three erythrocyte proteins, one identified as hemoglobin, bind thyroid hormones. Using a dextran/charcoal radioimmunoassay for thyroxin in dried blood spots, we demonstrate that such binding differs with the buffer used. Barbital, phosphate, and borate buffers significantly enhance the binding more than glycine and tris(hydroxymethyl)methylamine buffers. Binding is not affected by agents commonly used to inhibit thyroxin binding to serum proteins. A highly significant nonlinear direct relationship between sample storage (temperature and duration) and increased thyroxin-erythrocyte binding is documented, together with an associated decrease in assayed concentrations of thyroxin. However, concomitant serial measurement of thyroxin with polyethylene glycol and combined double-antibody/polyethylene glycol radioimmunoassays produced no evidence of interference by erythrocyte proteins in the radioimmune reaction. We conclude that erythrocyte proteins act only as low-affinity secondary binders in radioimmunoassay for thyroxin.
Development and validation of a new radioimmunoassay for parathyrin (PTH).
Authors: Kao P C , Jiang N S , Klee G G , Purnell D C
Published: January 2020

A radioimmunoassay for human parathyrin has been developed and characterized with whole-molecule (residues 1-84) human parathyrin and with the 1-34, 44-68, and 53-84 amino acid residue fragments of it. The antiserum used reacted with the whole molecule and with the 44-68 and 53-84 fragments, but not with the 1-34 fragment. Parathyrin concentrations in the serum of 118 normal subjects and of 112 patients with surgically proved primary hyperparathyroidism were determined with this assay. The mean results were 39 (SD 13) microL-Eq/mL for the normals and 111 (SD 77) microL-Eq/mL for the patients with primary hyperparathyroidism (p less than 0.0005). The upper 95% confidence limit of the normal range was 60 microL-Eq/mL. For 54 patients with primary hyperparathyroidism, the preoperative values for serum parathyrin, calcium, and phosphate--but not creatinine--were statistically different from the postoperative values (paired t-test, p less than 0.0005). Normal subjects showed significant (p less than 0.0005) differences in serum calcium concentrations but not in parathyrin concentrations, compared with concentrations found in cancer patients and patients who had thiazide-induced hypercalcemia. Phosphate concentration in serum, although not a specific indicator of disease, is a valuable clue to the diagnosis of primary hyperparathyroidism.
Hemoglobin A1C by isoelectric focusing.
Authors: Simon M , Cuan J
Published: January 2020

We separated the hemoglobins in an unwashed erythrocyte hemolysate according to their isoelectric points on a thin-layer horizontal polyacrylamide gel containing ampholyte (40 g/L) over a pH gradient of 6-8. We scanned the fixed, unstained gels by microdensitometry and calculated the percentage of hemoglobin A1C. The overall CV (between-run imprecision) for a normal hemolysate (mean, 5.2% of total hemoglobin) stored at -70 degrees C was 12.6%. An above-normal pooled specimen (mean 8.5%) showed an overall CV of 8.4%. We confirmed that hemoglobins S, C, and F do not interfere; acetylated F co-migrates with A1C. The mean A1C percentage in non-diabetic adults was 4.9%, the reference interval 3.9-6.4%. The mean values for diabetics in various degrees of control were 7.6% in the group rated "good," 9.9% in the "fair" group, and 12.2% in the "poor" group. Results for patients' samples (y) were compared with results by cation-exchange chromatography (x). The slope was 1.0 and the intercept was 2.2%. The percentage of hemoglobin A1C in erythrocytes remains constant for seven days in samples stored at 30 degrees C.
New spectrophotometric method for the determination of hemoglobin A1 compared with a microcolumn technique.
Authors: Wålinder O , Ronquist G , Fager P J
Published: January 2020

We compared a spectrophotometric kit method (Glycospec) for determination of glycosylated hemoglobin (HbA1) with a microcolumn kit method (Bio-Rad). The Glycospec method is based on the change in absorbance when phytic acid binds to hemoglobin A. With glycosylated hemoglobin there is no such change because the binding is blocked by the sugar moiety. Inter-assay CVs were 2-6% for both methods. In healthy subjects the mean (+/- SD) value for HbAl was about 1% higher with the spectrophotometric than the microcolumn method. For samples from 122 diabetics the correlation between values for HbAl obtained with the two methods was acceptable (r = 0.89), although the spectrophotometric technique yielded 2-4% higher values, a difference at least partly due to the absence of 2,3-diphosphoglycerate from the spectrophotometric standards. Adding 1.8 mmol of it per liter to these standards caused displacement of the standard curve; HbAl values then agreed well with those of the microcolumn method. The spectrophotometric procedure is easily automated, and therefore is well suited for large-scale analyses if problems with standards and calibration can be solved.
Evaluation and comparison of immunoinhibition and immunoprecipitation methods for differentiating MB and BB from macro forms of creatine kinase isoenzymes in patients and healthy individuals.
Authors: Wu A H , Bowers G N
Published: January 2020

We compared results for measurements of creatine kinase isoenzyme MB (CK-MB) by immunoinhibition vs immunoprecipitation, using sera from 53 normal healthy individuals, 55 patients with increased CK-MB associated with acute myocardial infarction, and 42 patients whose blood exhibited one or more abnormal forms of CK by electrophoresis. These last 42 patients, selected from a group of 91 cases exhibiting abnormal forms as detected in a screening of 5000 hospitalized and clinic patients, include: (a) CK-BB bound to IgG (macro CK type 1), (b) a polymeric complex of mitochondrial CK (macro CK type 2), (c) abnormally high activity of free CK-BB isoenzyme, and (d) persistent increases of CK-MB from patients without myocardial infarction. These abnormal forms occur in less than 2% of all patients and are exceedingly rare in patients with acute myocardial infarction. Therefore, the vast majority of CK-MB analyses can be performed rapidly and efficiently by immunoinhibition, which has analytical sensitivity, is associated with high clinical sensitivity, and is easily automated for a low cost per test. In contrast, immunoprecipitation is a more specific analytical measurement of CK-MB but is less efficient and more costly.
Serum calcium fractions in diabetes mellitus.
Authors: Fogh-Andersen N , McNair P , Møller-Petersen J , Madsbad S
Published: January 2020

Diabetes mellitus is associated with a decrease in bone mineral content and increased urinary excretion of calcium and phosphate. The purpose here was to elucidate the pathogenesis of these changes by comparing serum calcium fractions of diabetics and healthy controls. In a cross-sectional study, serum ionized calcium at pH 7.40, [Ca2+]7.4, of 46 insulin-treated diabetics was decreased in comparison with 44 healthy controls: mean 1.16 (SD 0.04) vs 1.21 (SD 0.03) mmol/L (p less than 0.001). The decreased [Ca2+]7.4 was associated with a higher concentration of complexed calcium in diabetic serum. The diabetics had higher concentrations of undetermined anions (p less tha 0.001) and this anion gap correlated negatively with [Ca2+]7.4 (r = -0.43, p less than 0.02). Serum [Mg2+] was decreased in the diabetics (p less than 0.001). Values for venous acid-base status, serum creatinine, total and ultrafiltrable calcium, parathyrin, and inorganic phosphate were the same in the two groups. [Ca2+]7.4, but not serum total calcium, had increased by 0.04 mmol/L (p less than 0.001) in the diabetics and by 0.02 mmol/L (p less than 0.01) in the controls by 90 min after breakfast, with and without subcutaneous insulin, respectively.
Four methods for determining uric acid compared with a candidate reference method.
Authors: Elin R J , Johnson E , Chesler R
Published: January 2020

Uric acid as measured in serum by three different uricase (EC 1.7.3.3) methods (aca, Ektachem, and SMAC) and by the SMAC method with phosphotungstic acid was compared with a candidate Reference Method for uric acid. Serum specimens from 83 patients (uric acid concentrations, 19 to 141 mg/L) were analyzed by all five methods. Results were compared by using linear regression analysis, and the mean difference between results by the candidate Reference Method and the four other methods was calculated. Compared with the candidate Reference Method, the aca method gave the smallest deviation from zero for the intercept and the smallest mean difference, and the SMAC phosphotungstic acid method showed a slope closest to unity. The SMAC uricase method had the largest intercept and greatest deviation of the slope from unity.
Total calcium and magnesium determined in serum with an automated stopped-flow analyzer.
Authors: Koupparis M A , Diamandis E P , Malmstadt H V
Published: January 2020

We describe the measurement of total calcium and magnesium in serum with an automated microcomputer-controlled stopped-flow analyzer. The calcium method is based on the cresolphthalein complexone procedure, with 2-amino-3-methyl-1-propanol as the alkalinizing agent. The assay, performed on 60-fold prediluted samples, requires 50 microL of serum. Absorbance is measured at 580 nm for 1 s, after a 5-s delay. Response is linearly related to concentration up to 5 mmol/L; analytical recovery averaged 97.8%. Within-day CVs were 0.7 to 1.5%, day-to-day CVs 1.8 to 2.5%. Results compared well with those by continuous-flow Technicon SMA II method. A sample throughput of as many as 260 samples per hour is possible. The magnesium determination, a complexometric procedure, involves magnesium/calmagite complex in an alkaline reagent mixture and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate calcium interference. Prediluted serum samples are used (100 microL of serum diluted 25-fold), and absorbance at 520 nm is linear with concentration to 50 mg/L. Within-run CVs were 0.5 to 1.1%, and day-to-day 1.3 to 3.8%; analytical recovery was 99.3%. Results compared well with those by atomic absorption spectrometry (r = 0.994). A delay time of 10 and a measurement time of 2.5 s allows for a throughput of as many as 180 samples per hour.
Liquid-chromatographic determination of amoxapine and 8-hydroxyamoxapine in human serum.
Authors: Tasset J J , Hassan F M
Published: January 2020

We describe a liquid-chromatographic procedure for amoxapine and 8-hydroxyamoxapine, its active metabolite, in serum. We used a mu-Bondapak C18 reversed-phase column and a mobile phase of acetonitrile/water (74/26 by vol) plus 26 microL of n-butylamine per liter. The compounds were measured at 254 nm, with 8-methoxyloxapine as internal standard. Necessary pre-analysis purification consisted of adsorbing the drug from serum onto extraction columns, eluting with 1-butanol/hexane (1/5 by vol), re-extracting into aqueous acid, and from that re-extracting again into the elution-solvent mixture. We prefer this procedure for monitoring both therapeutic and toxic concentrations of amoxapine, because parent drug and metabolite are measured separately.
Liquid-chromatographic determination of cyclosporine in serum with use of a rapid extraction procedure.
Authors: Yee G C , Gmur D J , Kennedy M S
Published: January 2020

In this procedure, reversed-phase liquid chromatography is used to measure cyclosporine, a fungal metabolite with immunosuppressive activity, in human serum. With a gradient elution, the retention times for cyclosporine and cyclosporin D were 14.1 and 15.7 min, respectively. Ultraviolet absorbance at 215 nm was used to detect cyclosporine; this wavelength improved assay accuracy without decreasing sensitivity, as compared with detection at 205 nm, which is near the absorption maximum. The major advantage of our procedure is the clean-up method, which involves use of disposable extraction columns. This extraction is simple and requires only 10 to 15 min per sample. Results by radioimmunoassay for cyclosporine were unpredictably greater than those measured by the present method. Dosing guidelines for cyclosporine need re-evaluation, based on more specific assay methods.
An improved ultrafiltration method for determining free testosterone in serum.
Authors: Vlahos I , MacMahon W , Sgoutas D , Bowers W , Thompson J , Trawick W
Published: January 2020

In this method, we use the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane in measuring free testosterone in serum. Two independent assays are combined: total testosterone and the ultrafiltrable fraction of added [3H]testosterone. The unbound fraction is determined in 0.15-0.5 mL ultrafiltrates of 0.6 to 1 mL of variably diluted serum that has been equilibrated with [3H]testosterone at 37 degrees C. The assay is rapid (less than 1 h), practicable (requires 0.6 mL of serum), and reproducible (CV 3.2% within assay, 3.9% between assays). Accuracy was evaluated as the fraction of free testosterone in the ultrafiltrate of dialyzed serum vs that in a prior dialysate; they were the same confirming the validity of the free testosterone measurement. Samples from ostensibly healthy men and women and from hirsute and pregnant women gave results that agreed with those obtained by equilibrium dialysis. Total testosterone concentrations for normal and hirsute women showed considerable overlap, but data on free testosterone concentrations in these populations were better resolved.
The Kodak Ektachem clinical chemistry slide for measurement of bilirubin in newborns: principles and performance.
Authors: Wu T W , Dappen G M , Powers D M , Lo D H , Rand R N , Spayd R W
Published: January 2020

In this slide, unconjugated bilirubin and its sugar conjugates interact with a cationic polymeric mordant to form spectrally enhanced complexes having similar absorptivities at approximately 400 nm. With reflection densitometry and appropriate mathematical transformation, readings at this wavelength are linearly related to bilirubin concentrations up to 260 mg/L. The slide requires 10 microL of serum, is precise (total CV less than 2% determined over 20 days for the analyte range 39-184 mg/L), gives results that correlate well with the Doumas et al. modification of the Jendrassik-Gróf method (slope 0.95, intercept 0.3, Sy . x 3.4, r = 0.991), and is relatively interference free. Also, the slide measures less loss of bilirubin after in vitro illumination of serum specimens than do diazo tests. An intermediate layer in the slide minimizes the spectral interference from hemoglobin and prevents the detection of the strongly protein-linked ("delta") bilirubin found in many jaundiced adults. The method is recommended for newborns (less than or equal to 14 days), in whom the incidence of delta bilirubin is negligible.
Radioimmunoassay for prostatic acid phosphatase helps discriminate patients with prostatic cancer.
Authors: Blick K E , Dick T T , Webb T A
Published: January 2020

We measured serum prostatic acid phosphatase in ostensibly normal controls and a selected patient population, using both a modified radioimmunoassay and an enzymic method with thymolphthalein monophosphate as substrate. The upper limit of normal for the radioimmunoassay was 2.2 micrograms/L; its sensitivity and specificity for prostatic cancer were 71 and 95%, respectively, vs 51 and 99% for the enzymic method. For both methods the correlation between clinical staging and values for acid phosphatase was poor. Our data suggest that adjunctive use of the radioimmunoassay may help further discriminate those patients requiring needle biopsy.
Mixture analysis by triple-quadrupole mass spectrometry: metabolic profiling of urinary carboxylic acids.
Authors: Hunt D F , Giordani A B , Rhodes G , Herold D A
Published: January 2020

In this new method for profiling urinary carboxylic acids, lyophilized urine samples are analyzed directly with a triple-quadrupole mass spectrometer. Extraction, derivatization, and lengthy gas-chromatographic separation procedures are obviated by this approach. Total sample-preparation, instrument, and data-analysis time per sample is about 15 min. More than 100 different organic acids can be detected in a typical urine sample. All components of the solid urine residue are volatilized into the ion source of the mass spectrometer and converted to (M-1)- ions by reaction with OH- under chemical ionization conditions. Quadrupole 1 is set to transmit ions of a particular m/z ratio, which in turn collide with molecular nitrogen in quadrupole 2 and dissociate to smaller charged fragments. Carboxylic acid (M-1)- ions uniquely lose either CO2 (44 atomic mass units) or both H2O and CO2 (62 atomic mass units). Quadrupole 3 is set to pass only ions that have lost either 44 or 62 atomic mass units. Accordingly, the instrument specifically detects carboxylic acids in the urine matrix and no other components. Direct analysis of polyethylene glycols and their acidic metabolites in urine and serum of a burn patient treated with an antimicrobial cream having a polyethylene glycol base is also discribed.
L-Phenylalanine inhibition of human alkaline phosphatases with p-nitrophenyl phosphate as substrate.
Authors: Komoda T , Hokari S , Sonoda M , Sakagishi Y , Tamura T
Published: January 2020

With p-nitrophenyl phosphate as the substrate, there reportedly is no organ-specific inhibition of alkaline phosphatase (EC 3.1.3.1) activity by L-phenylalanine. However, we found that at pH 10.0, with p-nitrophenyl phosphate as the substrate, L-phenylalanine obviously inhibits the alkaline phosphatase isoenzyme from human placenta, whereas there is little if any inhibition of the isoenzyme from human intestine. Because of the differing effects of substrates (p-nitrophenyl phosphate and phenyl phosphate) and their enzymic products (p-nitrophenol and phenol) for L-phenylalanine action on the placental alkaline phosphatase isoenzyme, we suggest that the isoenzyme--inhibitor--substrate complex and the effect of released phosphate on L-phenylalanine inhibition of the isoenzyme activity differ from each other.
Determination of selenium concentration and glutathione peroxidase activity in plasma and erythrocytes.
Authors: Pleban P A , Munyani A , Beachum J
Published: January 2020

We determined selenium concentrations and activities of the selenoenzyme, glutathione peroxidase (EC 1.11.1.9), in the plasma and erythrocytes of 38 apparently healthy women. We determined selenium concentrations directly by polarized Zeeman-effect flameless atomic absorption spectroscopy. Within-run precision studies for the assays gave CVs of 5.6% for a mean erythrocyte selenium concentration of 149.9 (SD 8.3) microgram/L (n = 10) and 6.4% for a mean plasma selenium concentration of 97.3 (SD 6.2) microgram/L (n = 12). For the women, mean selenium concentrations were 141.4 (SD 14.3) microgram/L of erythrocytes [0.49 (SD 0.07) microgram/g of hemoglobin and 96.3 (SD 14.2) microgram/L of plasma. Glutathione peroxidase activities were measured by a modification of the method of Paglia and Valentine (J. Lab. Clin. Med. 70: 158--169, 1967). Within-run precision studies for the glutathione peroxidase assays gave CVs of 12.8% for mean erythrocyte glutathione peroxidase activity of 77.2 (SD 9.9) U/g of hemoglobin (n = 13), and 8.1% for mean plasma activity of 312.5 (SD 25.2) U/L (n = 11). Mean enzyme activity was 78.7 (SD 12.9) U/g of hemoglobin for erythrocytes and 424 (SD 40) U/L for plasma. Erythrocyte selenium concentrations and glutathione peroxidase activities were positively, but poorly, correlated (r = 0.41, p less than 0.01).
FLow-injection analysis: a new approach to quantitative measurements in clinical chemistry.
Authors: Rocks B , Riley C
Published: January 2020

Flow-injection analysis, founded on an approach that is entirely different from continuous-flow analysis, involves use of three principles: sample "injection," controlled dispersion of sample (rather than a dispersion retarded with gas bubbles), and reproducible timing. The conditions governing the dispersion of the sample in the flowing carrier stream are considered, and we illustrate how the dispersion can be manipulated to suit particular analytical requirements. Instrumentation and practical aspects of flow-injection analyses are discussed, especially with regard to clinical chemistry applications, and the technique is compared with the more conventional gas-segmented-flow analysis system. We conclude that, because of its speed, economy, and simplicity, flow-injection analysis will eventually replace the gas-segmented approach for many clinical chemistry analyses.
Enzyme immunoassay for tartrate-resistant acid phosphatase.
Authors: Lam K W , Siemens M , Sun T , Li C Y , Yam L T
Published: January 2020

An immunochemical method for quantitative analysis of the tartrate-resistant acid phosphatase (EC 3.1.3.2), band 5, is presented. This method involves precipitation of the enzyme from the serum by the antibody specific to band 5 and by sheep anti-rabbit immunoglobulin, followed by analysis of the enzyme activity in the precipitate. The precipitation procedure eliminates the interferences of the tartrate-sensitive phosphatase of all tissues, of the tartrate-resistant phosphatase of erythrocytes, and of unknown substances that interfere with the colorimetric method. We compare the present method with previously described colorimetric and electrophoretic methods.
Rapid quantitation of serum immunoglobulins with a miniature centrifugal analyzer.
Authors: Koch T R , Adolf P K , Zimmerman D H , Knoblock E C , Leitz V M
Published: January 2020

For these quantitative turbidimetric assays for immunoglobulins IgG, IgA, and IgM in serum, the reagents used are commercially available in kit form. The two-point assays are performed with a miniature centrifugal analyzer (GEMENI, Electro-Nucleonics, Inc.). The reading taken 5 s after mixing is used to correct for specimen turbidity. Run-to-run precision (CV) was typically 5% (range: 3.0-8.7%). Excellent correlation with radial immunodiffusion was observed for each test (r greater than 0.98 for each test). The dynamic range of each test extends above and below the reference-interval ranges.
Methemoglobin in blood as determined by double-wavelength spectrophotometry.
Authors: Sakata M , Yoshida A , Haga M
Published: January 2020

We describe spectrophotometric determination of methemoglobin (Met-Hb) in blood by absorbance differences at two wavelengths (characteristic of the instrument used) in the 500-600 nm region. Blood is diluted 100- to 200-fold with a solution containing KCN and carbon monoxide to convert hemoglobin to carboxyhemoglobin and Met-Hb to cyanomethemoglobin (CN-Met-Hb). Carboxyhemoglobin has the same absorbance at the two wavelengths used, so that the differences in absorbance reflect only the CN-Met-Hb component. After measurement of absorbance differences, potassium ferricyanide is added to convert all hemoglobin derivatives to CN-Met-Hb; samples are then remeasured at the same wavelengths. The percentage of Met-Hb is determined from the ratio of the absorbance differences. Results agree satisfactorily with currently accepted procedures and with calculated theoretical results for mixtures of oxyhemoglobin, carboxyhemoglobin, and Met-Hb.
Measurement of magnesium by continuous-flow colorimetry.
Authors: Fowler R T , Abernethy M H , Walmsley T A , Taylor H W
Published: January 2020

We describe the application to the Technicon SMAC system of a micellar-improved calmagite method for the measurement of magnesium. In this continuous-flow method a dialyzer is not needed, because protein interference is negligible. Sample interaction was estimated to be 3%. The increased sensitivity of the method allows for a small sample volume (37 microL/min). Results obtained agreed well with those by an atomic absorption procedure (x): SMAC magnesium - 0.971 x + 0.030 mmol/L (n = 58). The estimated total error for the SMAC procedure was less than 50 mumol/L at two analyte concentrations: 0.50 and 1.50 mmol/L. The inclusion of magnesium estimation on SMAC should extend the screening function of this analyzer.
Two-dimensional gel electrophoresis of cell proteins in childhood leukemia, with silver staining: a preliminary report.
Authors: Hanash S M , Tubergen D G , Heyn R M , Neel J V , Sandy L , Stevens G S , Rosenblum B B , Krzesicki R F
Published: January 2020

We have investigated the potential of two-dimensional electrophoresis for identifying differences in protein patterns between acute lymphocytic (ALL) and acute myelogenous leukemia (AML). We studied blood cells directly isolated from seven children with non-T, non-B ALL and five with AML. Solubilized proteins were electrophoresed with the ISO-DALT system and made visible by silver staining. More than 800 spots were routinely detected in individual gels. Patterns for multiple runs of the same sample were indistinguishable. Most of the prominent spots were present in gels from all 12 of the individuals studied. However, substantial variability in spot intensity was observed, even among patients in the same group. Five spots were consistently present in gels of all the AML patient, and either completely absent or substantially decreased in ALL gels. Three other spots were present in ALL but not AML gels. We conclude that this may be a useful tool for characterizing leukemic cells.
Proteins of human milk. I. Identification of major components.
Authors: Anderson N G , Powers M T , Tollaksen S L
Published: January 2020

Traditionally, human milk proteins are identified largely by reference to bovine milk. Hence, to identify the major proteins in human milk, we subjected human and bovine milk, in parallel, to high-resolution two-dimensional electrophoresis. Isoelectric precipitation at pH 4.6 was our criterion for distinguishing whey proteins from those of the casein complex. The alpha- and beta-caseins were identified on the basis of relative abundance, relative molecular mass, and relative isoelectric points. Kappa casein was identified as a series of four spots, which disappear from bovine skim milk treated with rennin (chymosin; EC 3.4.23.4) during the clotting process. Para kappa-casein does not appear on the standard ISO-DALT pattern after treatment of bovine milk with rennin, but does appear in BASO-DALT pattern, indicating its high isoelectric point. No protein disappeared from ISO-DALT patterns of human milk after rennin treatment, and no new protein comparable to bovine para kappa-casein appeared in the BASO-DALT patterns; this suggests that kappa-casein is absent from human milk. The proteins identified in human milk patterns include the alpha and beta casein families, lactalbumin, albumin, transferrin, IgA, and lactoferrin. Numerous additional proteins seen in patterns for human milk remain to be identified.
Hair as an indicator of excessive aluminum exposure.
Authors: Yokel R A
Published: January 2020

To determine if excessive systemic exposure to aluminum would be reflected in increased aluminum concentration in hair, rabbits were given a series of aluminum lactate injections. Hair was collected before the aluminum lactate administration from the site of injections and twice after the injections from this site as well as from an area adjoining the injection site. Aluminum was determined by flameless atomic absorption analysis of acid-digested samples. The concentration of aluminum in the hair increased after the injections in samples taken at both times from both sites. Considerable variability in hair aluminum was found before excessive exposure, as has been reported in humans, and in response to the exposure. The increase in hair aluminum did not correlate with the amount of hair produced. Nevertheless, because some subjects exposed to excessive aluminum showed a very large increase in hair aluminum, hair may be a useful indicator of aluminum body burden in such aluminum-induced conditions as dialysis encephalopathy.
Standardization of serum cholesterol assays by use of serum calibrators and direct addition of Liebermann-Burchard reagent.
Authors: Katan M B , van der Haar F , Kromhout D , Schouten F J
Published: January 2020

Serum cholesterol concentrations of subjects in epidemiological studies were measured after direct addition of Liebermann-Burchard reagent; results were calibrated with human serum pools assayed according to Abell et al. (J. Biol. Chem. 195:357-366, 1952). Accuracy and precision were monitored for six years by analysis of internal-control pools and blind external-control pools. For various internal-control pools, the imprecision (CV) of the long-term averages of run means ranged from 0.5 to 0.9%. The within-run CV for internal control and patients' sera was about 1%. For blind control sera with different concentrations (provided by the Centers for Disease Control, Atlanta, GA, over the same period), the average difference per three-month period between the values found and the target values was usually between -0.5% and +0.7% for medium-concentration pools and between -2% and +2% for low- and high-concentration pools (extreme values: -2.4% and +2.5%). The CV per three-month period ranged from 0.6 to 2.7%. Sera from subjects on diets of high or low linoleic acid content were analyzed to study the effect of the fatty acid portion of serum cholesterol esters; the differences between values obtained with the comparison method and the direct method was insignificant on both diets. We conclude that the use of serum calibrators eliminates the bias inherent in the direct method.
Liquid-chromatographic analysis for serum theophylline in less than 70 seconds.
Authors: Kabra P M , Marton L J
Published: January 2020

We describe a sensitive, specific, and very fast liquid-chromatographic assay for serum theophylline, involving a commercially available high-speed reversed-phase column and a micro-flow-cell-equipped detector. Each analysis requires only 100 microL of serum (as little as 25 microL may be used when necessary), and chromatography is complete in less than 70 s. Analytical recovery of theophylline added to serum ranged from 97 to 102%. Between-run precision (CV) ranged from 2.1 to 3.5%. The lower limit of detection for theophylline is 0.5 mg/L, and linearity extends to 50 mg/L. Numerous drugs and xanthine metabolites tested do not interfere.
Three radioactivity detectors for liquid-chromatographic systems compared.
Authors: Frey B M , Frey F J
Published: January 2020

Three radioactivity detectors coupled to a "high-performance" liquid-chromatography system are compared with regard to static efficiency, dynamic efficiency, background measurements, and within- and between-day variabilities. Their advantages and disadvantages are discussed.
Liquid-chromatographic assay for retinol (vitamin A) and retinol analogs in therapeutic trials.
Authors: McClean S W , Ruddel M E , Gross E G , DeGiovanna J J , Peck G L
Published: January 2020

A "high-performance" liquid-chromatographic separation of retinoids (retinol, isotretinoin, all-trans retinoic acid, retinal, etretinate, and retinyl acetate) in serum is described. The separation was used in developing a quantitative assay for retinol (vitamin A) and two therapeutic analogs, isotretinoin (13-cis-retinoic acid) and etretinate (Ro 10-9359). The procedure requires 1 mL of serum. Overall analytical recovery for retinol, isotretinoin, and etretinate from serum was 100% (SD 7%). The between-day coefficient of variation for specimens with concentrations ranging from 0.70 to 0.95 mg/L was less than 4%. Normal reference intervals for serum retinol in men and women are 0.61 to 1.33 and 0.44 to 1.19 mg/L, respectively.
A new chromogenic amylase method compared with two established methods.
Authors: Fenton J , Foery R , Piatt L , Geschwindt K
Published: January 2020

We tested a new amylase reagent involving chromogenic substrates (Pantrak, Calbiochem-Behring) for precision, dynamic range, and susceptibility to potential interferences. Two p-nitrophenyl-alpha-maltaosides are used as substrates, which amylase cleaves to shorter-chain p-nitrophenyl maltaosides, the latter then yielding p-nitrophenol from the activity of alpha-glucosidase. A series of 100 specimens were tested by Pantrak and two established methodologies. In both cases, correlation was excellent. Precision was good for all methods; CVs for Pantrak were 3.0 to 4.5%. The dynamic range of Pantrak extended to 800 U/L. Above-normal quantities of triglyceride, hemoglobin, or bilirubin did not cause spurious results, but anticoagulants that bind divalent cations should not be used. The Pantrak method has desirable analytical features and is easy to use.
How many proteins are there in a typical mammalian cell?
Authors: Duncan R , McConkey E H
Published: January 2020

There is a major disparity between the number of polysomal mRNA species found in mammalian cells and the number of polypeptides detected by high-resolution two-dimensional polyacrylamide gel electrophoresis. Here we show that technical factors are not responsible for the relative paucity of proteins, and that the translation products of rare mRNAs would be easily detectable if all mRNAs were translated in proportion to their abundance. We conclude that a large majority of rare mRNAs are translated at no more than a tenth the average translation rate, if they are translated at all. There may be no more than 2000 physiologically significant primary gene products (polypeptides) in a typical mammalian cell.
Two-dimensional gel electrophoresis of human brain proteins. I. Technique and nomenclature of proteins.
Authors: Comings D E
Published: January 2020

To understand at a molecular level the basis of the normal and pathological genetic differences between individuals it is necessary to begin a detailed two-dimensional gel electrophoretic mapping of the proteins of the human brain in normal individuals and those with various genetic neurological disorders. The present study is an examination of the polypeptide patterns of extracts of the human brain made with 9 mol/L urea solution. Details of the technique and the nomenclature of the patterns obtained are presented. the gels are separated into 20 sub-sections, based on standards with known molecular masses and isoelectric points. Groups of polypeptides within these sub-sections are identified by a number and a letter; the individual proteins are identified by a number. Thus, protein 1 in subsection 8, group b, would be designated 8b: 1. Subsequent papers in this series identify many of these proteins; show which proteins belong to the cytosol, synaptosome, myelin, and other brain fractions; show how these patterns vary between normal individuals and those with different neurological and psychiatric conditions; examine the effect of severe gliosis; and present the results of non-equilibrium gel electrophoresis for the more basic proteins.
Microscale multisample two-dimensional electrophoresis of proteins in human serum, cerebrospinal fluid, and urine.
Authors: Manabe T , Hayama E , Okuyama T
Published: January 2020

In this technique, in which no denaturing agent is used, proteins in human serum, cerebrospinal fluid, and urine are separated by isoelectric focusing in cylindrical 40 g/L polyacrylamide gels of capillary size (1.3 x 35 mm) for 40 min, followed by electrophoresis in 40--170 g/L polyacrylamide linear gradient gel, with use of 38 x 35 x 1 mm slab gel, for 1 h. Only 2 microL of untreated human serum is required to obtain clear protein-distribution patterns, made visible by Coomassie Blue staining. By use of silver staining, proteins in unconcentrated cerebrospinal fluid can be made visible. An apparatus we devised for microscale two-dimensional electrophoresis enables us to analyze eight protein samples simultaneously.
A two-dimensional gel analysis of autologous T and B lymphoblastoid cell lines.
Authors: Lester E P , Lemkin P , Lipkin L
Published: January 2020

We sought polypeptide markers of pathways of lymphoid differentiation by comparing the protein-synthesis patterns of a pair of autologous human lymphoblastoid cell lines: the B-cell line CCRF-SB and the T-cell line CCRF-HSB-2. Cells were cultured in standard medium for 24 or 120 h, then biosynthetically radiolabeled with [3H]leucine. Whole cell lysates were subjected to two-dimensional gel electrophoresis and patterns of protein synthesis were made visible by fluorography. Replicate cultures showed highly reproducible patterns, but differing culture durations produced alterations in the relative synthetic rates of some proteins. Protein synthetic patterns were analyzed visually and with the GELLAB system for computer analysis of two-dimensional gel images. Qualitative and quantitative differences were found between the T- and B-cell patterns. GELLAB detected and accurately paired most of the well-defined spots in each image and constructed a unified data base from data on our initial gels. Statistical analysis of this data revealed several marker proteins, the relative synthetic rates serving to distinguish the T-cell line from the B-cell line. Such computerized analysis of protein synthetic patterns of malignant cells appears likely ultimately to provide important diagnostic information regarding human malignancies.
Design and implementation of a prototype Human Protein Index.
Authors: Taylor J , Anderson N L , Scandora A E , Willard K E , Anderson N G
Published: January 2020

This paper describes information-handling aspects of the TYCHO I analysis system (Clin, Chem. 27: 1807--1820, 1981), which analyzes two-dimensional electrophoresis gels, matches the individual protein spots with those in a reference pattern, and stores various information--including spot measurements, identifications, treatment profiles, set memberships, and comments--in a computerized database. This and additional information such as amino acid composition and cellular localization is then accessible from an interactive program that includes a pictorial user interface and presents much of the data in graphical form. Use of the TYCHO I system is illustrated by examples drawn from analyses of gel patterns from human leukocytes.
Clinical applications of two-dimensional electrophoresis.
Authors: Jellum E , Thorsrud A K
Published: January 2020

High-resolution two-dimensional electrophoresis has been used in our institute to study sera from patients with multiple myelomas; cerebrospinal fluid from patients with different neurological disorders; blister fluid, dermis, and epidermis from patients with skin diseases; and biopsies from brain tumors; and to search for tumor-associated proteins in cancer biopsies and pre-cancer sera. The method differentiates between all main types of immunoglobulins and gives rise to protein patterns for cerebrospinal fluid that may carry diagnostic information. The pattern of proteins in serum and blister fluid is almost identical, confirming that the latter is a filtrate of serum. We also analyzed sera collected from patients several years before it was clinically recognized that they had cancer (JANUS serum bank), in an attempt to find tumor-associated proteins in serum at an early stage of the disease. We encountered difficulties in detecting trace proteins, due to masking effects of such major serum-protein constituents as albumin. A combination of computerized gas chromatography-mass spectrometry and high-resolution two-dimensional electrophoresis is capable of mapping compounds of both low and high molecular mass. Used in combination with pattern-recognition analysis, the complete multicomponent analytical system opens the possibility of diagnosing diseased cells solely on the basis of differences in their biochemical composition.
Demonstration of basic human-brain-specific proteins by the BASO-DALT system.
Authors: Jackson P , Thomson V M , Thompson R J
Published: January 2020

We used the BASODALT system (Cell 12: 1133--1142, 1977) of high-resolution two-dimensional gel electrophoresis to resolve proteins in extracts of human brain and, for comparison, those in nine other human organs. At least 180 protein-staining spots are visible on a BASODALT electrophoretogram of water-soluble human brain proteins. By electrophoresing extracts of different human organs, separately and mixed with brain extract, we could detect 14 basic spots that appear to be present in brain in concentrations at least 20-fold those in any other organ. Measurement of these new basic brain-predominant proteins may provide useful diagnostic information in neurological disorders.
Some relatively simple steps toward a computer system for the analysis of two-dimensional gel-electrophoresis autoradiographs.
Authors: Fox S H
Published: January 2020

A computerized system for the quantitative comparison of two-dimensional polyacrylamide gel autoradiographs is being developed that requires relatively limited hardware resources. Two fast, simple, and stable programs--one for background and streak subtraction and one for peak detection--have been developed and tested. Two methods developed by others--one for image smoothing and one for peak matching--also have been tested. A very simple spot-density integration program that works on isolated spots has been written and is being developed further.
Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis.
Authors: Giometti C S , Willard K E , Anderson N L
Published: January 2020

Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. Three groups (Cytoskf:8--10, :14--16, and :17--18) showed qualitative differences, and the fourth group (Cytoskf:11 and :13) showed quantitative differences. In addition, surface labeling of GM607 and human fibroblasts with 125I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. Cytoskf:11 and :13 in fibroblast preparations were also labeled with the 125I. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.
Computer programs for adapting two-dimensional gels to the study of mutation.
Authors: Skolnick M M , Sternberg S R , Neel J V
Published: January 2020

We are attempting to adapt the two-dimensional polyacrylamide gel technology to the search for mutations that after the electrophoretic mobility of proteins. These events are expected to have a frequency on the order of 10(-6). To this end, programs that run on the CytocomputerTM are being developed for comparing the pattern of the gel derived from a child's blood sample with those of the gels of corresponding samples from its parents. We anticipate examining thousands of such trios. Programs are described that perform background normalization, noise filtering, spot detection, gel registration, and, finally, compare the father/mother/child gel trios for the presence of unique protein moieties in the child that are consistent with a mutational event.
An approach to completely automatic comparison of two-dimensional electrophoresis gels.
Authors: Skolnick M M
Published: January 2020

The problem addressed is that of determining the similarities and differences appearing in a sequence of two-dimensional polyacrylamide gels where we have no a priori knowledge of the intensity and spatial distribution of the protein spots. It is assumed that the gels are not in precise registration. An attempt is being made to develop a completely automatic program for use in genetic studies, which will compare a sequence of three gels run on samples from father, mother, and child. The program constructs a graph by using as nodes those spot cues that exceed a given intensity threshold. The graphs are then compared to determine an initial subset of spots that are common to all three gels. From this subset of common spots the program then determines whether the remaining graph differences are real or result from quantitative variation causing spots to fall below threshold.
Latex immunoassay of retinol-binding protein.
Authors: Bernard A M , Moreau D , Lauwerys R R
Published: January 2020

Latex immunoassay, a sensitive method based on latex particle agglutination, is used here for the determination of retinol-binding protein in human biological fluids. The assay, similar to that described previously for beta 2-microglobulin (Clin. Chem. 27:832-837, 1981), consists of incubating the sample at 37 degrees C for 30 min with antibody-coated particles, then quantifying the resulting agglutination by particle-counting or turbidimetry. Latex particles coated with antibody are stabilized just before use by dispersing them in a solution of bovine serum albumin at pH 10. The standard curve ranges from 0.5 to 32 micrograms/L; recovery averages 102% in urine and 93.5% in serum; between- and within-assay CVs range from 5.1 to 11.7%. The correlation coefficients of latex immunoassay with rocket immunoelectrophoresis for analysis of retinol-binding protein in 26 urines and with radial immunodiffusion in 30 sera are 0.99 and 0.91, respectively. In healthy subjects, the mean urinary excretion of retinol-binding protein is 52.5 micrograms/g of creatinine (SD = 59.2 micrograms/g of creatinine; n = 150) and the concentration in serum averages 46 mg/L (SD = 10.4 mg/L, n = 22).
Rapid colorimetric assay for acetaminophen without salicylate or phenylephrine interference.
Authors: Swanson M B , Walters M I
Published: January 2020

A simple rapid, and economical procedure is presented for the quantitative determination of acetaminophen in serum. Acetaminophen is extracted into solvent, and, when back-extracted into base, is simultaneously reacted with Folin-Ciocalteau reagent to form a colored complex. The potential interference of bilirubin is eliminated by treatment with p-diazobenzene sulfonic acid. Results obtained with this method agree with those obtained by "high-pressure" liquid chromatography.