bluuebunny/crossref_metadata_2025 huggingface dataset:
Applicability of various quality-control sera to assay of high-density lipoprotein cholesterol.
Authors: Bullock D G , Carter T J , Hughes S V
Published: January 2020

Effective internal quality control and external quality assessment of high-density lipoprotein cholesterol assay is made difficult by analyte instability, and the suitability of quality-control sera for this purpose has not been studied. We have therefore investigated the properties of 25 different control sera from 15 suppliers by estimating within-batch precision for the two precipitation procedures used most widely (phosphotungstate/Mg2+ and heparin/Mn2+ with enzymic measurement of cholesterol. Some sera had properties similar to those of fresh human serum, but others demonstrated poor precision for one or both procedures or contained apparent high-density lipoprotein cholesterol in unphysiological concentrations. A study of six sera indicated that between-batch precision was consistent with the within-batch findings. We found that eight of the 25 batches of quality-control serum we investigated may be used for internal quality control and external quality assessment of high-density lipoprotein cholesterol assay.
Liquid-chromatographic assay of urinary 5-hydroxy-3-indoleacetic acid, with electrochemical detection.
Authors: Shihabi Z K , Scaro J
Published: January 2020

After extraction with two organic solvent mixtures, urinary 5-hydroxy-3-indoleacetic acid can be assayed by "high-performance" liquid chromatography on a reversed-phase column, with electrochemical detection. Compared to the nitrosonaphthol method (J. Biol. Chem. 216: 499, 1955), this method is more specific for detection of patients with carcinoid tumors.
Improvements in hemoglobin AI determination by rapid cation-exchange chromatography.
Authors: Rand P G , Nelson C
Published: January 2020

The accuracy of many convenient methods for determining the proportion of hemoglobin AI to total hemoglobin by ion-exchange chromatography is in question because those methods have not been evaluated against a reference method. In addition, several if not all of these tests are influenced by laboratory temperature. We modified the chromatographic conditions usually used, to arrive at a method that gives results that agree well with those by the Trivelli method (N. Engl. J. Med. 284, 353, 1971) and is less influenced by ambient temperature than are results obtained with the commercially available Helena and Isolab kits. We accomplished this by lowering the pH of the fast-hemoglobin eluting buffer, with a corresponding adjustment in the ionic strength. We also use a larger elution volume, which assures more nearly complete elution of the hemoglobin A1 fraction. The final method (y) agreed being y = 0.98x + 0.63 r2 = 0.887. Average results from the Helena and Isolab methods were lower than with the reference method by 1.58 and 3.03% hemoglobin A1, respectively.
Composition of serum very-low-density and high-density lipoproteins in diabetes.
Authors: Gabor J , Spain M , Kalant N
Published: January 2020

We examined the cholesterol/protein ratio and the apoprotein composition of serum lipoproteins in a randomly selected population of maturity-onset diabetics and in a group of nondiabetics of similar age. We found no differences in cholesterol distribution between the groups as a whole, but diabetics with above-normal low-density lipoproteins (LDL) had decreased concentrations of high-density lipoprotein (HDL) cholesterol. In the diabetics as a whole, there was an increase in the cholesterol/protein ratio in HDL, a negative correlation between the amounts of LDL cholesterol and HDL cholesterol, an increase in the proportion of apolipoprotein C in very-low-density lipoprotein (VLDL), and a decrease in the proportion of the apolipoprotein AI component of HDL. In diabetic subjects with increased VLDL, there was an increase in the relative amount of apolipoprotein CIII, and a consequent decrease in the ratio of apolipoprotein CII/apolipoprotein CIII in the VLDL. In both diabetic and control subjects, apolipoprotein E and cholesterol content of VLDL were linearly related.
Enzymic determination of 3 alpha-, 7 alpha-, and 12 alpha-hydroxyl groups of fecal bile salts.
Authors: Crowell M J , Macdonald I A
Published: January 2020

We used microbial 3 alpha, 7 alpha-, and 12 alpha-hydroxysteroid dehydrogenases to quantify 3 alpha-, 7 alpha-, and 12 alpha-hydroxyl groups in human feces. Samples collected over a 48-h period were lyophilized, extracted, and reconstituted by a modification of the method of DeWael et al. (clin. Chim. Acta 79: 465--470, 1977), then quantified enzymically by use of three group-specific hydroxysteroid dehydrogenase preparations. Total values for the three hydroxyl groups are given for 18 healthy humans. The ratio of 3 alpha-OH groups/7 alpha-OH groups/12 alpha-OH groups was 34.4 (SD 5.9)/25.2 (SD 6.4)/40.4 (SD 7.7) for the 18 samples.
Human conjugated bilirubin--isolation, biosynthesis, and direct molecular characterization.
Authors: Wu T W , Zumbulyadis N , Gross S , Gohlke R S
Published: January 2020

We prepared human conjugated bilirubin by isolation from fresh gall bladder bile and by biosynthesis using liver homogenates. The isolation protocol was modified after Lucassen (doctoral thesis, Univ. Utrecht, 1961), and the in vitro synthesis was done with fresh liver homogenates in the presence of D-glucaro-1,4-lactone (a glucuronidase inhibitor). From direct analyses of these preparations with 270-MHz proton nuclear magnetic resonance and field-desorption mass spectrometry, we deduced that a major conjugated bilirubin species in bile is a diglucuronide, whereas in liver biosynthesis it is a diconjugate containing glucuronic acid and possibly glucuronolactone co-esterified to the bilirubin backbone. If the glucuronolactone is indeed present in the native biosynthetic material, we do not know whether it derives from glucuronic acid and, if so, whether lactonization occurs before or after the acid has been esterified to form the conjugate.
Melanoma detection by enzyme-radioimmunoassay of L-dopa, dopamine, and 3-O-methyldopamine in urine.
Authors: Faraj B A , Lawson D H , Nixon D W , Murray D R , Camp V M , Ali F M , Black M , Stacciarini W , Tarcan Y
Published: January 2020

This enzyme-radioimmunoassay for the measurement of L-dopa, dopamine, and 3-O-methyldopamine is based on the incubation of urine in the presence of catechol-O-methyltransferase, aromatic-L-amino-acid decarboxylase, and S-adenosylmethionine. The O-methylated dopamine metabolite formed, 3-O-methyldopamine, was characterized by radioimmunoassay. To evaluate the role of L-dopa metabolism in melanoma, we used the enzyme-radioimmunoassay to assess concentrations of L-dopa, dopamine, and 3-O-methyldopamine in urine from 10 healthy subjects, 10 hospitalized patients without melanoma and 28 patients with different degrees of melanoma. The effect of surgery for melanoma on urinary output of these catechols of melanoma patients was also evaluated. No significant difference in urinary L-dopa, dopamine, and 3-O-methyldopamine excretion rates was seen between normal subjects (L-dopa 1.3 +/- 0.3, dopamine 147 +/- 38, and 3-O-methyldopamine 31.4 +/- 13.6 microgram/24 h), hospitalized patients without melanoma, and amelanotic melanoma patients. However, the excretion rates for these metabolites in melanotic melanoma (L-dopa 5.6 +/- 1.2, dopamine 555 +/- 121, and 3-O-methyldopamine 178 +/- 40.3 microgram/24 h) were significantly (p < 0.005) higher than in control or amelanotic melanoma subjects. After surgery, there was a substantial decrease in urinary output of L-dopa and its metabolites by these patients.
Gas-chromatographic assay for thiopental in plasma, with use of a nitrogen-specific detector.
Authors: Jung D , Mayersohn M , Perrier D
Published: January 2020

An accurate, sensitive, and specific gas-liquid-chromatographic procedure is described for determining concentrations of thiopental in human plasma. After a double extraction of 0.2 or 1.0 mL of plasma containing phenobarbital as an internal standard, thiopental and the internal standard are derivatized in a polar non-aqueous solvent system with iodomethane. The reaction mixture is then evaporated, the residue reconstituted with ethyl acetate, and 20 microL injected into a 3% OV-17 column of a gas chromatograph equipped with a nitrogen-phosphorus detector. Linearity and reproducibility over the concentration range 25 microgram/L to 10 mg/L in plasma are excellent. The sensitivity and wide range of linearity exhibited by this method permit thorough characterization of the disposition of thiopental after the usual induction doses of 3-4 mg/kg of body weight.
Direct quantitation of glycosaminoglycans in 2 mL of urine from patients with mucopolysaccharidoses.
Authors: Burlingame R W , Thomas G H , Stevens R L , Schmid K , Moser H W
Published: January 2020

Glycosaminoglycans in urine from patients representing the major different mucopolysaccharidoses were separated and measured by use of a procedure that requires only 2 mL of urine. The compounds were resolved by two-dimensional electrophoresis on cellulose acetate plates and made visible by staining with Alcian Blue. They were identified by co-migration with standard glycosaminoglycans, by digestion with specific glycosidases, and by specific degradation with HNO2. They were quantitated by comparing the absorbance of eluates of the stained spots to appropriate standard curves for each glycosaminoglycan. This study revealed additional findings. About half of the patients excreted small amounts of heparin. Further, the keratan sulfate in samples from Morquio's disease patients migrated differently from authentic keratan sulfate unless digested with chondroitinase ABC. Our results for these diseases are in harmony with earlier reports.
Optimizing determination of plasma albumin by the bromcresol green dye-binding method.
Authors: Robertson W S
Published: January 2020

Some modifications of the conditions of the reaction between plasma and bromcresol green have led to an improved method for determination of plasma albumin with the Vickers M300 multichannel analyzer. Dye concentration and reaction time are the factors principally influencing method specificity, but variable protein composition of human plasma also affects it, so that optimal reaction conditions vary from specimen to specimen. Thus a compromise must be reached such that the best conditions for determining plasma albumin over a range of different protein concentrations are achieved. In the proposed method for the Vickers M300 a reaction time of 12 s (the minimum possible) is used. Comparison with "rocket" immunoelectrophoresis gave the following linear regression: y = 10 + 0.79 x (n = 91; r = 0.96).
The "ultra-free" ultrafiltration technique compared with equilibrium dialysis for determination of unbound thiopental concentrations in serum.
Authors: Jung D , Mayersohn M , Perrier D
Published: January 2020

We compare a new ultrafiltration technique, involving a unique Millipore membrane, with the classical method of equilibrium dialysis for determining the fraction of thiopental not bound to serum proteins. This fraction, as determined by equilibrium dialysis at 37 degrees C, ranged between 12 and 16% for total concentrations at 50 microgram/L to 10 mg/L of serum. In contrast, ultrafiltration at 37 degrees C yielded a 49% higher value for unbound thiopental: 26.3 (SD 2.6)%. Determined at room temperature (24 degrees C), there was no statistically significant difference for results by the two methods: 14.2 and 15.9%, respectively. The discrepancy between results at 37 degrees C may partly be explained by serum proteins penetrating the Ultra-Free filter. For the routine clinical measurement of unbound drug concentrations, the ultrafiltration membrane at room temperature appears to be sufficiently accurate and less time-consuming than equilibrium dialysis.
Sensitive and specific radioimmunoassay for creatine kinase BB isoenzyme in serum, with use of an autoantibody.
Authors: Urdal P , Kierulf P
Published: January 2020

We have developed an equilibrium radioimmunoassay for creatine kinase BB (CK-BB) isoenzyme in serum. We used as antibody a CK-BB binding immunoglobulin G isolated from serum of a patient with macro CK-BB. Free and antibody-bound tracer were separated by anion-exchange chromatography. The affinity constant, Keq, for the binding of CK-BB to immunoglobulin was calculated as 1.4 X 10(11) L/mol, which suggests that the immunoglobulin was an autoantibody. The assay may detect 1 microgram of enzyme per liter in a 100- microL sample. CK-MM and CK-MB isoenzymes did not cross react in the assay. Within-assay precision (CV) was 5%; between-assay precision was 14 and 15% at 6 and 14 microgram of CK-BB per liter, respectively. Of 45 sera from blood donors, 95% contained < 1.8 microgram of CK-BB per liter, whereas of 19 sera from unselected patients the corresponding figure was 2.8 microgram/L.
An instrument for digital matrix photometry.
Authors: Neeley W E , Epstein D , Zettner A
Published: January 2020

A new instrument was conceived and designed for quantitative measurement of chromophoric areas or colored spots such as are produced in (e.g.) thin-layer chromatography. The areas to be measured are subdivided grid-like into small subunits, and the absorbance of each of these is measured. The sum of absorbances for all subunits is directly proportional to the total amount of light-absorbing substances in a spot. The absorbances of the subunits are measured with a photodetector that contains hundreds of microscopically small photodiodes, arranged in a precise geometric array. The photodiode array is interfaced with a computer via an analog-to-digital converter for numerically integrating the individual signals from each photodiode. With this analytical system, quantitation of light-absorbing substance is accurate and precise for areas of different sizes, shapes, and internal irregularity.
Liquid-chromatographic assay of angiotensin-converting enzyme in serum.
Authors: Shihabi Z K , Scaro J
Published: January 2020

This assay of angiotensin-converting enzyme is based on release of hippuric acid from a synthetic tripeptide, hippuryl-L-histidyl-L-leucine. Hippuric acid is separated and quantitated on a reversed-phase column under conditions similar to those used for assay of theophylline and acetaminophen. The method is rapid and free from interferences.
Influence of hemolysis on the serum lactate dehydrogenase-1/lactate dehydrogenase-2 ratio as determined by an accurate thin-layer agarose electrophoresis procedure.
Authors: Leung F Y , Henderson A R
Published: January 2020

The accuracy of our lactate dehydrogenase (EC 1.1.1.27) isoenzyme procedure, based on electrophoresis in thin-layer agarose, has previously been assessed (Clin. Chem. 22: 1995, 1976). We now show that the method is similarly accurate and precise for the specific measurements of lactate dehydrogenase isoenzyme-1 and -2. We have also determined the lactate dehydrogenase isoenzyme pattern in peripheral erythrocytes and have found that lactate dehydrogenase-1 is usually the isoenzyme present in greatest concentration. Therefore, hemolysis may increase lactate dehydrogenase-1 in serum, affecting the lactate dehydrogenase isoenzyme ratio used in the diagnosis of myocardial infarction. This conjecture has been confirmed by in vitro experiments, and in a small group of patients with intra-vascular hemolysis.
Enzyme immunoassay for thyroxine and triiodothyronine in human serum, with use of a covalent chromatographic separation method.
Authors: Yamamoto R , Hattori S , Inukai T , Matsuura A , Yamashita K , Kosaka A , Kato K
Published: January 2020

A practicable enzyme immunoassay for measurement of thyroxine and triiodothyronine in serum was developed, involving a separation method based on the thiol-disulfide interchange reaction. Serum samples at alkaline pH were incubated for 1 h with antibodies and antigens labeled with beta-D-galactosidase. Each reaction mixture was then passed through a 0.1-mL column containing (anti-IgG) antibody immobilized on Sepharose 4B, to which the anti-IgG antibodies were coupled by means of a disulfide bond (3 g of IgG fraction per liter). The column was washed and the bound form of the label then was eluted from the column with a buffer containing dithiothreitol (25 mmol/L) by splitting the disulfide bonds between the anti-IgG antibody molecules and Sepharose matrix. From the enzyme activity in the eluate, concentrations of thyroxine or triiodothyronine in serum could be determined. Values obtained by this method and those obtained by a radioimmunoassay correlated well (thyroxine, r = 0.97, slope = 1.1 y-intercept = -0.94 microgram/L, n = 70; triiodothyronine, r = 0.98, slope = 0.91, y-intercept = 0.067 microgram/L, n = 77.
Use of N-methyl-D-glucamine as buffer in the determination of serum alkaline phosphatase activity.
Authors: Chromý V , Zahradnícek L , Voznícek J
Published: January 2020

We describe a method for determining serum alkaline phosphatase activity with use of N-methyl-D-glucamine buffer, Na+ is a definite activator, whereas NH4+ and Li+ inhibit enzyme activity. Optimum reaction conditions are: methylglucamine buffer, 0.35 mol/L, pH 10.2 +/- 0.1 (30 degrees C); NaCl, 70 mmol/L; MgCl2, 0.5 mmol/L; disodium 4-nitrophenyl phosphate, 15 mmol/L; reaction temperature, 30 degrees C; reaction time, 2 min. The assay conditions are optimum for all human serum isoenzymes.
Total-calcium measurement in serum from neonates: limitations of current methods.
Authors: Brett E M , Hicks J M
Published: January 2020

In the routine analysis for calcium in serum from neonates we noted large discrepancies in icteric samples analyzed by several different methods. The present investigation was undertaken to demonstrate the magnitude of the problem of bilirubin interference in calcium analysis and to assess the specific applicability of commonly used micro-methods in the pediatric or neonatal laboratory. The intention is not to advocate any single methodology but rather to point out inadequacies that must be considered. Colorimetric and fluorometric methods correlated poorly with an atomic absorption spectroscopic method for samples from pediatric patients. In addition, we noted significant interference proportional to the concentration of bilirubin. A fluorescence titration method was shown to be unacceptable for pediatric use because of the variable and unpredictable response to bilirubin interference. The study emphasizes the need for thorough invesigation of methods before they are put to use in the specialized pediatric setting.
Plasma lipid-bound sialic acid and carcinoembryonic antigen in cancer patients.
Authors: Dnistrian A M , Schwartz M K
Published: January 2020

We evaluated lipid-bound sialic acid as a "marker" in cancer patients and assessed the individual and combined value of lipid-bound sialic acid and carcinoembryonic antigen determinations in these patients. Plasma was sampled from 62 normal subjects and 125 cancer patients. Lipid-bound sialic acid was determined by the resorcinol method after total lipid extraction and isolation of the sialolipid fraction from plasma. Neither marker was increased in many breast cancer patients. Carcinoembryonic antigen was increased more commonly and to a greater degree in colon cancer patients and seems to be the preferred marker. Both markers were increased in lung cancer patients and their combined evaluation improved the rate of detection. Lipid-bound sialic acid was increased in more patients with leukemias, lymphomas, Hodgkin's disease, and melanomas, suggesting that it may be a useful biochemical marker in these types of cancer.
Comparison of countercurrent immunoelectrophoretic assay with commercial radioimmunoassay kits for measuring prostatic acid phosphatase.
Authors: Wright G L , Schellhammer P F , Brassil D N , Sieg S M , Leffell M S
Published: January 2020

We evaluated and compared five commercial radioimmunoassay kits with a standard counter-immunoelectrophoretic assay for the measurement of prostatic acid phosphatase in serum. Four of the five radioimmunoassays performed as described by the supplier with respect to sensitivity, stability, precision, linearity, analytical recovery, and expected values for the normal male population. None of the radioimmunoassays was more clinically sensitive then the counter-immunoelectrophoretic assay for detecting increased prostatic acid phosphatase in serum. Results obtained by counter-immunoelectrophoretic assay agreed with results obtained by radioimmunoassay in 96% of the tests. The proportion of positive results in patients with confirmed prostatic adenocarcinoma increased with disease progression. The fewer positive tests in localized adenocarcinoma (Stages A and B) suggests that neither the counter-immunoelectrophoretic assay nor the radioimmunoassay procedures are useful for screening unselected populations for adenocarcinoma of the prostate. The high percentage of normal values found in those patients clinically free of disease after treatment is encouraging and supports the use of the prostatic acid phosphatase immunoassays in prospectively monitoring the treatment of prostatic cancer patients.
Factitious hypophosphatemia related to mannitol therapy.
Authors: Donhowe J M , Freier E F , Wong E T , Steffes M W
Published: January 2020

We observed factitious hypophosphatemia in a patient who was receiving large amounts of intravenous mannitol. Mannitol concentrations as low as 25 mmol/L inhibited phosphorus measurement by the Dupont aca endpoint method; a kinetic method was unaffected. The mechanism of the mannitol interference was binding to molybdate in the reaction, decreasing both the rate of color development and the endpoint measurement. We conclude that the molybdate concentration in the DuPont method is suboptimal for measuring phosphorus in specimens containing mannitol.
The TYCHO system for computer analysis of two-dimensional gel electrophoresis patterns.
Authors: Anderson N L , Taylor J , Scandora A E , Coulter B P , Anderson N G
Published: January 2020

We describe here a computer system for the analysis of high-resolution two-dimensional gel-electrophoresis patterns, with some initial applications. The system (called TYCHO) comprises programs for image acquisition, background subtraction and smoothing, spot detection, gaussian spot modeling, and pattern matching and comparison. It is based on a conventional minicomputer, but makes extensive use of a high-speed array processor in the image-processing and -modeling steps. Used in concert with the ISO-DALT two-dimensional electrophoresis system (Anal. Biochem. 85:331-354, 1978), TYCHO allows quantitative measurement of hundreds of proteins in complex biological samples, and constitutes the initial data-reduction system required for work towards a Human Protein Index.
Heterogeneity of corticotropin-immunoreactive compounds in human body fluids.
Authors: Schöneshöfer M , Fenner A , Molnar I
Published: January 2020

Using reversed-phase "high-performance" liquid chromatography and hydrophilic ion-pairing reagents, we studied the chromatographic profiles of corticotropin immunoreactivities in human plasma, cerebrospinal fluid, and urine. The chromatographic system we applied provides excellent separation of peptide molecules, with reproducibility (CV) of retention times better than 0.5%, and makes feasible the subsequent immunological quantitation of eluted fractions. Profiles of corticotropin immunoreactivities exhibited several chemically different compounds in each of the three body fluids studied. A considerable portion of these immunoreactive compounds is more hydrophilic and probably of lower molecular mass than the human corticotropin 1-39 molecule itself. These hydrophilic molecules are more predominant in urine and plasma than in cerebrospinal fluid.
Improved radioimmunoassay for creatine kinase isoenzymes in plasma.
Authors: Ritter C S , Mumm S R , Roberts R
Published: January 2020

We describe convenient and relatively rapid procedures for purifying creatine kinase isoenzymes MM, BB, and MB, and their use in an improved radioimmunoassay for creatine kinase isoenzymes in plasma. The modifications include use of: (a) BB with a specific activity of 400 kU/g, which can be labeled with a specific radioactivity of 20 Ci/g; (b) albumin-free purified MB as inhibitor; (c) antiserum to MB creatine kinase; and (d) a second-antibody technique that necessitates only a 15-min incubation. The radioimmunoassay for MB has a sensitivity of 0.2 microgram/L (80 mU/L) and a CV of less than 5%. Plasma MB average 22 (SD 12) microgram/L in 200 normal subjects; 24 (SD 12) microgram/L in 200 patients with chest pain without infarction; and 23 (SD 7) microgram/L in 43 patients with renal disease, whether measured before or after dialysis. Peak values for plasma MB averaged 191 (SD 86) microgram/L in 325 patients with documented myocardial infarction; BB was negligible. Extensive clinical experience indicates the radioimmunoassay to be suitably rapid, highly sensitive, and reliable as a diagnostic assay for MB in plasma.
Reversed-phase liquid chromatography and gas chromatography/mass fragmentography compared for determination of tricyclic antidepressant drugs.
Authors: Breutzmann D A , Bowers L D
Published: January 2020

We compare measurement of the tricyclic antidepressants desipramine, imipramine, nortryptaline, and amitryptaline by reversed-phase "high-performance" liquid chromatography and by gas chromatography/mass fragmentography. The liquid-chromatographic procedure does not require ion-pairing reagents, and involves detection at 215 nm. The within-day and between-day CVs for this method were less than 5% and less than 7%, respectively, and linear-regression analysis of peak height vs concentration for each of the drugs showed excellent correlation (r greater than 0.97) over the range 0-250 microgram/L. Potential interferences with the internal standard (N-desmethyldoxepin) and nortryptaline were oxazepam and N-desmethyldiazepam, respectively, as shown by mass spectrometry. The interference could be circumvented by increasing the column temperature to 50 degrees C. We conclude that reversed-phase liquid chromatography with detection in the far-ultraviolet wavelengths provides an alternative to gas-chromatographic procedures for the tricyclic antidepressants, provided the analyst has prior knowledge of the patient's other medications.
16 Alpha-[125I]-beta-estradiol compared with [3H]-beta-estradiol as the tracer in estradiol receptor assays.
Authors: Tercero J C , Nelson J C , Chi E , Broughton A
Published: January 2020

We assessed the potential usefulness of 16 alpha-[125I]-beta-estradiol in estradiol receptor assays as compared with a long-used [3H]-beta-estradiol dextran-coated charcoal method, measuring 472 consecutive human breast-cancer cytosols by both procedures. Six different preparations of 16 alpha-[125I]-beta-estradiol were used. Nonspecific binding in five batches was similar and comparable to [3H]-beta-estradiol. Although the sixth batch had increased nonspecific binding, this did not affect results. Dissociation constants were virtually identical (r = 0.94). Results were concordant for 98.5% of cytosols: 161 were negative and 304 positive by both methods and seven were positive by one method, negative by the other. Four were positive with the 125I procedure and undetectable with [3H]-beta-estradiol. Three were measurable by both methods, but were above the cut-off value in one and below it in the other. We find 16 alpha-[125I]-beta-estradiol to be an adequate substitute for [3H]-beta-estradiol in estradiol receptor assays.
Muscle protein analysis. III. Analysis of solubilized frozen-tissue sections by two-dimensional electrophoresis.
Authors: Giometti C S , Anderson N G
Published: January 2020

Proteins from frozen histological sections of human muscle were analyzed by two-dimensional gel electrophoresis. Patterns so obtained were identical to those from whole homogenates of muscle prepared from frozen tissue powders that had much higher protein concentrations. To increase the number of proteins visible on gels of samples low in protein content, the gels were silver stained, or the proteins were labeled with [14C]iodoacetamide before electrophoresis and the gels were fluorographed. The latter method allow use of a single frozen-tissue section for two-dimensional electrophoretic analysis and brings the technique closer to practicable clinical use.
Relative value of three laboratory methods in the diagnosis of multiple sclerosis.
Authors: Bloomer L C , Bray P F
Published: January 2020

We compared three methods of analysis for IgG in cerebrospinal fluid, using samples from 158 patients with clinically suspected multiple sclerosis and from 200 neurological controls. The tests were: search for oligoclonal bands, calculation of rate of synthesis of IgG in the cerebrospinal fluid, and determination of the IgG/albumin ratio. Paired cerebrospinal fluid and serum samples were collected and their IgG and albumin concentrations measured. Oligoclonal bands were detected by electrophoresis on agarose. Positive results were obtained in 94, 75, and 67% of patients with probable or definite multiple sclerosis by the three respective methods. In contrast, for patients for whom the clinical diagnosis of multiple sclerosis was considered possible, positive results were obtained in 10, 43, and 13%, respectively. Evidently, detection of oligoclonal bands remains the best single test for the presence of abnormal IgG in suspected multiple sclerosis patients. A combination of the first two tests is most sensitive for both probable and definite multiple sclerosis (97%) and possible multiple sclerosis (50%). Some infectious or immunologic disorders can also produce these IgG abnormalities, but they can usually be distinguished from multiple sclerosis by other clinical and laboratory data.
Evaluation of a new free-thyroxin assay.
Authors: Wellby M L , Guthrie L , Reilly C P
Published: January 2020

The Amerlex Free Thyroxin (T4) Radioimmunoassay Kit (Amersham International Ltd.) is a new direct equilibrium radioimmunoassay for free T4 based on an antiserum with very high affinity for T4, and a unique 125I-labeled T4 analog as tracer. It is a very simple single-tube radioimmunoassay, making use of Amerlex particles to separate antibody-bound from free species. Interassay precision (CV) is 3.7% at 13 pmol/L and 2.3% at 30 pmol/L; within-assay precision is 4.2% at 21 pmol/L. The reference interval is 11-22 pmol/L. The assay did not misclassify any patients tested who had untreated myxedema or untreated thyrotoxicosis. The free T4 assay excelled both the free T4 index and the T4/T4-binding globulin ratio in correcting for increased thyroxin-binding globulin from pregnancy, and it was better than the index but not better than the ratio in correcting for increased thyroxin-binding globulin in users of oral contraceptives.
Improved simultaneous gas-chromatographic analysis for homovanillic acid and vanillylmandelic acid in urine.
Authors: Leiendecker-Foster C , Freier E F
Published: January 2020

We describe an improved gas-chromatographic method for the simultaneous quantitation of the catecholamine metabolites, homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid) and vanillylmandelic acid (3-methoxy-4-hydroxymandelic acid). Our improvements in the method of Muskiet et al. (Clin. Chem. 23: 863, 1977) include a shorter program time and a longer silylation interval. Recovery and precision data obtained by this improved technique are similar to those of Muskiet et al. Vanillylmandelic acid results (y) were compared with those by the method of Pisano et al. (Clin. Chim. Acta 7: 285, 1962). The relation is expressed by the equation y = 0.52 + 1.05x (Sy . x = 2.33 mg/24 h and r = 0.997). Results for homovanillic acid (y) were compared with those by the method of Knight and Haymond (Clin. Chem. 23: 2007, 1977); the equation was y = 0.84 + 0.90x (Sy . x = 2.04 and r = 0.97). Retention times are also reported for several phenolic acids and other related compounds found in urine.
Liquid-chromatographic monitoring of 5-methyltetrahydrofolate in plasma.
Authors: Giulidori P , Galli-Kienle M , Stramentinoli G
Published: January 2020

The liquid-chromatographic measurement of 5-methyltetrahydrofolate in biological fluids is described. The sensitivity of the spectrophotofluorometric detector used allows direct evaluation of basal concentrations of the compound in plasma. Because it is resolved from the other common folates and from methotrexate, the procedure is suitable for monitoring it in plasma of patients receiving high-dose therapy with methotrexate.
Optimized assay for serum angiotensin-converting enzyme activity.
Authors: Hurst P L , Lovell-Smith C J
Published: January 2020

This optimized two-point kinetic assay for serum angiotensin-converting enzyme is based on the colorimetric determination of hippurate with cyanuric chloride/dioxan reagent. The hippurate is released from hippuryl-L-histidyl-L-leucine by angiotensin-converting enzyme in the presence of chloride ion. CVs for the method (within-run and between-run) ranged from 2.1 to 3.2%. Linearity extends up to 200 U/L. Results are unaffected by lipemia and icterus. Hemoglobin in concentrations greater than 1.5 g/L causes a slight negative interference. Reference intervals for men and women are 22-82 U/L and 25-69 U/L, respectively.
Separation and detection of lipoproteins in human serum by use of size-exclusion liquid chromatography: a preliminary report.
Authors: Busbee D L , Payne D M , Jasheway D W , Carlisle S , Lacko A G
Published: January 2020

Components of human serum can be separated on the basis of differences in relative molecular mass by using size-exclusion "high-performance" liquid chromatography. Lipoproteins in fractions of the eluate can be quantitated by conventional chemical and enzymatic methods. Alternatively, if lipoproteins in the serum are selectively prestained with diformazan dye, the column effluent can be monitored spectrophotometrically at 580 nm, so that only the lipoprotein components of serum are detected. Samples of purified low-density lipoproteins, so stained and analyzed, provide peak-area values that are proportional to their concentration as evaluated by chemical methods. With this technique, the various lipoprotein classes can be quickly separated and their concentration estimated. These techniques should prove useful in clinical and research laboratories.
Improved thin-layer chromatography of disaturated phosphatidylcholine in amniotic fluid.
Authors: Tsai M Y , Cain M , Josephson M W
Published: January 2020

We describe an indirect test of fetal lung maturity: the quantitation of disaturated phosphatidylcholine in amniotic fluid. The lipids in samples of amniotic fluid from 172 patients were reacted with osmium tetroxide, and disaturated phosphatidylcholine was then isolated by thin-layer chromatography. Interfering substances were retained by a pre-adsorbent layer. The charred disaturated phosphatidylcholine, quantitated by densitometry, was compared to standard dipalmitoyl phosphatidylcholine. Both within-run and between-run coefficients of variation were about 10%. Blood and meconium do not interfere. Six infants developed respiratory distress when disaturated phosphatidylcholine concentrations of amniotic fluid drawn within 72 h of delivery were less than 5.5 mg/L. A concurrently determined lecithin/sphingomyelin ratio falsely predicted lung maturity in one of these. In seven other samples for which lecithin/sphingomyelin ratios suggested lung immaturity but disaturated phosphatidyl-choline predicted maturity, none of the infants developed respiratory distress. In normal pregnancies, measurement of disaturated phosphatidylcholine in amniotic fluid appears to be a better predictor of fetal lung maturity than is measurement of the lecithin/sphingomyelin ratio. Further studies are needed to determine if this analysis is a better predictor in diabetic pregnancies.
Automated enzyme assays by use of a centrifugal analyzer with fluorescence detection.
Authors: Pearson K W , Smith R E , Mitchell A R , Bissel E R
Published: January 2020

The first commercially available centrifugal analyzer having fluorescence detection capability was used to develop kinetic fluorometric assays for several proteinases. The substrates were all synthetic oligopeptides incorporating 7-amino-4-trifluoromethylcoumarin, a molecule that can be detected either spectrophotometrically or fluorometrically. We thus compared the centrifugal analyzer spectrophotometric and fluorometric detection systems, finding fluorescence detection to be 50-fold more sensitive. We also compared the sensitivity of the fluorescence detector to that of a conventional spectrofluorometer by determining the minimum detection limit for each enzyme on both instruments; we found them to be similar in sensitivity. As an illustrative application, we measured the cathepsin B-like activity in serum samples from 55 women. The median enzyme activity of women taking oral contraceptives and pregnant women was increased two- and threefold, respectively, over the control group (about 5% CV within run, and 10% CV between runs).
Liquid-chromatographic determination of cimetidine, its known metabolites, and creatinine in serum and urine.
Authors: Ziemniak J A , Chiarmonte D A , Schentag J J
Published: January 2020

We describe a liquid-chromatographic procedure for determination of cimetidine, its hydroxymethyl-, sulfoxide-, and guanyl urea metabolites, and creatinine in patients serum and urine. SKF 92374 is used as the internal standard. Protein in 0.5 mL of serum or diluted urine is precipitated with 2 mL of acetonitrile, the organic and aqueous phases are separated by adding 0.3-0.5 g of anhydrous K2HPO4. The organic phase is evaporated, and 0.5 mL of 50 mmol/L HCl is added. This solution is washed with 3 mL of water-saturated isoamyl alcohol, the aqueous phase is extracted with 3 mL of methylene chloride and enough K2HPO4 to saturate the solution. The methylene chloride is evaporated, the residue reconstituted with 100 microL of mobile phase, and a 25-microL aliquot injected onto the chromatographic column (Dupont Sil). The mobile phase is acetonitrile/methanol/water/ammonium hydroxide (1000/50/50/2, by vol). The column effluent is monitored at 228 nm. Lower limits of detection ranged from 0.05 mg/L for cimetidine to 0.2 mg/L for guanyl urea. We determined cimetidine and its principal metabolites in the serum of a patient receiving cimetidine for the treatment of Zollinger-Ellison syndrome, and have assessed use of the assay in a clinical setting.
Analysis for paraquat by second- and fourth-derivative spectroscopy.
Authors: Fell A F , Jarvie D R , Stewart M J
Published: January 2020

We describe simple, precise methods, based on second and fourth-derivative spectroscopy, of analysis for paraquat in serum, plasma, or dialysis fluid. Plasma paraquat, after ion-pair extraction and dithionite reduction, yields a sharp, stable absorption peak at 396 nm. Derivative spectroscopy confers enhanced sensitivity (detection limit, 5 microgram/L) and improved rejection of spectroscopic interference by diquat, hemolysis, bilirubin, or lipemia. Derivative peak amplitudes are linearly related to paraquat dichloride concentration from 10 to 1000 microgram/L in plasma, and the methods are precise (CV 2.7-5.2% for 50 microgram/L concentration) and accurate. Results of derivative assays for 18 cases of paraquat poisoning correlate well with those by radioimmunoassay (r = 0.999). Derivative spectroscopy enables monitoring of paraquat concentrations in the plasma of poisoned patients during treatment by hemoperfusion or hemodialysis. A rapid assay developed for dialysis fluid (detection limit, 2 microgram/L) permits the efficacy of hemodialysis to be routinely assessed.
High-affinity binding of folate to a serum protein in chronic myelogenous leukemia: effect of binder concentration, pH, and temperature.
Authors: Holm J , Hansen S I , Lyngbye J
Published: January 2020

Serum from a patient with chronic myelogenous leukemia was subjected to anion-exchange chromatography on DEAE-Sepharose CL-6B. High-affinity binding of [3H]folate to front effluent, eluted at a low salt gradient, was studied in equilibrium-dialysis experiments (37 degrees C, pH 7.4). As suggested by the data, folate binding displayed positive cooperativity. Dilution of the binder solution resulted in a shift to a simple non-cooperative binding type and increased binding affinity. Furthermore, binding was inhibited at pH 5.0 and at low temperature (7 degrees C). This study demonstrates important similarities between high-affinity folate binding in milk and serum: positive cooperativity and dependence of binding affinity on concentration of binder. Identical mechanisms may underly these phenomena in milk and serum. The apparent relationship between binding type and concentration of binder shown herein seems to agree fairly well with recent observations on sera from groups of healthy persons.
Enzyme immunoassay and enzyme inhibition assay of methotrexate, with use of the centrifugal analyzer.
Authors: Pesce M A , Bodourian S H
Published: January 2020

Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mumol/L and there is no interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.
Laser Doppler velocimetry vs heater power as indicators of skin perfusion during transcutaneous O2 monitoring.
Authors: Enkema L , Holloway G A , Piraino D W , Harry D , Zick G L , Kenny M A
Published: January 2020

Laser Doppler velocimetry (LDV) and heater power (HP) were compared as indicators of skin perfusion during transcutaneous oxygen tension (ptcO2) monitoring in adults. The LDV probe was fitted to a Radiometer ptcO2 attachment ring, which was fixed to the forearm. ptcO2, electrode temperature, HP, and LDV measurements were recorded continuously and simultaneously. LDV was more specific and sensitive to changes in cutaneous perfusion. Moreover, changes in perfusion were signalled four to 24 times faster by LDV than by HP. Changes in ptcO2 corresponded to changes in LDV more closely than to changes in HP in all experiments. HP was more specific and sensitive to changes in electrode and air temperatures. Once hyperemia was established at 44 degrees C and the electrode heater was turned off (HP = 0), LDV measurements frequently showed persistence of hyperemia (up to 25 min). Under these circumstances ptcO2 decreased linearly with decreasing electrode temperature in the range of 44-32 degrees C. LDV performance was referenced by monitoring velocity measurements taken on a cylinder revolving at known velocities. We conclude that LDV is a useful adjunct to ptcO2 monitoring and better than HP in assessing skin blood flow. It should enhance quality control of transcutaneous O2 monitoring.
Continuous-flow serum albumin determination by reaction with picrate ions, with use of a flow-through picrate ion electrode.
Authors: Diamandis E P , Papastathopoulos D S , Hadjiioannou T P
Published: January 2020

An automated potentiometric method for serum albumin determination by use of the picrate/albumin reaction is described. A continuous-flow system and a specially designed flow-through picrate ion electrode were used in making the measurements. Various factors affecting the reaction, such as pH, picrate ion concentration, and reaction time, were studied. Peak height in millivolts and albumin concentration were linearly related in the range 10-70 g/L. Both within-run and day-to-day, the CV for the method was about 2%. Analytical recovery of albumin added to serum samples ranged from 97.0 to 110.3%, averaging 102.2%. Results compare favorably with those by the established bromcresol green method. The proposed method is suitable for routine use and for screening tests.
Relation of age to isoenzyme pattern and total activity of amylase in serum.
Authors: Bossuyt P J , Van den Bogaert R , Scharpé S L , Van Maercke Y
Published: January 2020

Pancreatic and salivary isoenzymes of amylase were determined in serum from 70 subjects. Thin-layer gel/isoelectric focusing was used to separate the isoenzymes. Because other studies (J. Lab. Clin. Med. 90: 141-151, 1977) show that the major isoamylases have isoelectric points between 5.8 and 7.2, we focused the sera on polyacrylamide gel plates with a pH gradient from 5.5 to 8.5. The separated amylase fractions were made visible by direct incubation with a commercially available dye-starch polymer. Isoelectric focusing proved to be convenient, precise, and reproducible, and it can be used as a routine analysis to detect even slight changes in serum amylase distributions. We found that the isoamylase distribution is age dependent, whereas total amylase activity shows no correlation with age.
The Worthington "ultrafree" device evaluated for determination of ultrafiltrable calcium in serum.
Authors: Toffaletti J , Tompkins D , Hoff G
Published: January 2020

We evaluated a commercially-available disposable device ("Ultrafree," Worthington Diagnostics) for the anaerobic preparation of protein-free ultrafiltrates from serum for measurement of ultrafiltrable calcium. Sufficient filtrate for the analysis is obtained with 10 min from 0.2 to 0.4 mL of serum at room temperature. We assessed these ultrafilters with regard to permeability of calcium citrate, exclusion of proteins, frequency of leakage, and effect of temperature on results. Within-run and day-to-day coefficients of variation for human serum pools were 1.2 and 1.5%, respectively. Reference intervals (in mmol/L) for total (2.16-2.58), ultrafiltrable (1.44-1.67), dialyzable (1.25-1.41), and ionized (1.04-1.25) calcium have been determined for a healthy population of 69 women and 81 men, ages to 18 to 65 years. The device appears to be the most practicable yet available for use in making this measurement.
An optically clear hypercholesterolemic hypertriglyceridemic quality-control material prepared from animal lipid sources.
Authors: Proksch G J , Bonderman D P
Published: January 2020

A hyperlipidemic control serum can be simple prepared from animal lipid sources. Beta- and pre-beta-lipoproteins containing cholesterol and triglyceride are removed from porcine serum by treatment with dextran sulfate and calcium ions. A triglyceride-rich fraction containing only trace amounts of cholesterol is isolated from chicken egg-yolks. The two fractions are then combined in 40 mmol/L sodium bicarbonate to give the desired values for cholesterol and triglyceride. The preparation is stabilized against surface denaturation during long-term storage at 5 degrees C perhaps for as long as two years, by adding 0.25 g of Triton X-100 surfactant per liter, and against an accidental exposure to short-term freezing by adding 10 g of sucrose per liter. We used this solution as a diluent to reconstitute lyophilized bovine serum. The resulting product, having been prepared from only animal sources, is free of hepatitis-associated constituents, and is remarkably clear, homogeneous, and stable. Results obtained with it are precise.