bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid analysis for methylglyoxal bis(guanylhydrazone) by reversed-phase ion-pair liquid chromatography.
Authors: Gildersleeve D L , Tobes M C , Natale R B
Published: January 2020

We describe a "high-performance" reversed-phase ion-pair liquid-chromatographic procedure for measuring methylglyoxal bis(guanylhydrazone) (MGBG) in plasma, urine, and bone-marrow leukocytes. Specimens of plasma and bone-marrow leukocytes are deproteinized with perchloric acid, then neutralized with KOH. Urinary MGBG is isolated by liquid-solid extraction in a C18 Sep-Pak. The chromatographic system consists of a 45 X 4.6 mm (i.d.) octadecylsilyl (C18, 5-microns particle) column and a mobile phase consisting of methanol/sodium acetate buffer (200 mmol/L, pH 4.5), 2/3, by vol. The acetate buffer also contains 20 mmol of 1-octanesulfonate and 40 mg of sodium azide per liter. The column effluent is monitored at 283 nm. At a flow rate of 3.0 mL/min, MGBG is eluted in 1.67 min. The detection limit is 20 nmol/L, and peak height varies linearly with concentration from 0.02 to 40 mumol/L. Analytical recovery exceeds 99%. Within-day CVs ranged from 0.9% to 2.9%, between-day CVs from 4.2% to 6.2%.
Plasma apolipoprotein pattern in fish-eye disease examined by high-resolution two-dimensional electrophoresis.
Authors: Marshall T , Williams K M , Holmquist L , Carlson L A , Vesterberg O
Published: January 2020

We compared the plasma protein patterns of the two living patients suffering from fish-eye disease with those of appropriate controls, using high-resolution two-dimensional electrophoresis. Quantitative abnormalities were detected in plasma polypeptides corresponding to the isoforms of apolipoproteins A-I and A-II. The disease was characterized by normal concentrations of proapo A-I but dramatically subnormal concentrations of the other apo A-I isoforms and apo A-II. No significant difference was detected in the concentrations of other plasma proteins. These findings are discussed in relation to other apolipoprotein disorders.
Comparison of improved precipitation methods for quantification of high-density lipoprotein cholesterol.
Authors: Warnick G R , Nguyen T , Albers A A
Published: January 2020

We compared the standard Lipid Research Clinics heparin-Mn2+ (46 mmol/L) method and five improved precipitation methods for quantification of high-density lipoprotein (HDL) cholesterol. Three of these methods--a dextran sulfate-Mg2+ procedure, reported as a Selected Method, a modified heparin-Mn2+ (92 mmol/L) method, and a modified phosphotungstate-Mg2+ procedure--all gave similar results. Three other methods--the standard heparin-Mn2+ (46 mmol/L) method and two polyethylene glycol methods (75 g/L or pH 10 reagent at 100 g/L final concentrations)--gave slightly higher values for HDL cholesterol. Addition of NaCl or glucose to specimens did not significantly change protein precipitation. In terms of sedimentation effectiveness with hypertriglyceridemic specimens, the methods were ranked in the following order: polyethylene glycol (pH 10, 100 g/L) greater than dextran sulfate-Mg2+ greater than heparin-Mn2+ (92 mmol/L) = polyethylene glycol (75 g/L) greater than phosphotungstate-Mg2+ greater than heparin-Mn2+ (46 mmol/L).
A sensitive high-temperature electrothermal atomic absorption analysis for Rb+ in erythrocytes and plasma of normal and hypertensive persons.
Authors: Hallis K F , Boon N A , Perkins C M , Aronson J K , Grahame-Smith D G
Published: January 2020

This method for determination of Rb+ in human plasma and erythrocytes by graphite-furnace atomic absorption spectrophotometry has a sensitivity of 29 nmol/L for plasma, 12 nmol/L for erythrocytes. The detection limit is 24 nmol/L for plasma, 4.8 nmol/L for erythrocytes. This assay is approximately 30-fold more sensitive than previously reported techniques involving atomic absorption spectrophotometry, enabling use of smaller samples. The rubidium signal is linear with concentration up to 1.2 mumol/L, and addition of other cations to the matrix produces only minor alterations in the Rb+ signal. We measured plasma and erythrocytic Rb+ concentrations in healthy subjects and in patients with untreated essential hypertension. In both, our values are similar to those previously reported for healthy individuals.
Collection and Handling of 24-Hour Urine Specimens for Measurement of Analytes Related to Renal Calculi
Authors: Ng Ronald H , Menon Mani , Ladenson Jack H
Published: January 2020

Vol. 30

p 468: In line 7, left column, HCl should be 12 mol/L.

p 536: In Figs. 4 and 5, change the unit on the ordinates to "%." Change the ordinate numbers on Fig. 4 from 0, 20, 40, 60, 80, and 100 to 0, 5, 10, 15, 20, 25. On Fig. 5, change the ordinate number from 120 to 30.

p 725: In line 8, right column, change "logarithmic probability," to "probit." In Figs. 2 and 3, replace the terms "log 10" and "double log" by "probit" and "simple log," respectively.

p 729: Insert "γ" in the empty space on line 23, right-hand column, so the expression reads "100(1 – γ)%."

pp 856–7: Exchange the legends for Figures 2 and 3.

p 1111: Units in the left-hand Table should be "µg/L," not "g/L."

p 1260: In the first column, just under equation 2, delete "β" and insert the words "for a."

p 1294: In paragraph three, line 6, for "blunted" read "blunt."

p 1353: Transpose Figures 2 and 3 over the (unchanged) legends.

0.53 1.02 2.71 5.0

p 1364: The sixth line of data, columns 2–4 should read "176.4 ± 43," "470 ± 198," and "0.5." The final line of data, columns 2–4, should read "1.95 ± 0.47," "5.05 ± 2.12," and "0.4," and, under "Range" the following should be inserted at the bottom of the column:

0.4–0.7

0.2–0.8

0.1–0.7.

p 1365: The data in columns 2 and 4, and some in column 3, of Table 3 should read:

See table in the PDF file

p 1429: The middle column, next to last paragraph, should read: "Hb 27 g/L" and "leukocytes 186 x 109/L."

p 1812: Column 1, fifth line of text: change "decreasing" to "increasing."

p 1813: Column 2, x refers to whole blood rather than plasma, y to plasma.
Pharmacokinetic analysis of a case of isopropanol intoxication.
Authors: Natowicz M , Donahue J , Gorman L , Kane M , McKissick J , Shaw L
Published: January 2020

A comatose 46-year-old woman, admitted to the emergency room, had isopropanol and acetone concentrations of 2000 and 120 mg/L, respectively, in her serum. She had no known history of acute isopropanol intoxication and was otherwise physically healthy. Pharmacokinetic analysis showed that the elimination of both isopropanol and its major metabolite acetone obeyed apparent first-order kinetics with half-lives of 6.4 and 22.4 h, respectively. These data contrast with the commonly held view that isopropanol is slowly metabolized. Concentrations of these analytes in cerebrospinal fluid 6 h after admission were similar to those in serum. This is the first report of the pharmacokinetics of both agents in a nonalcoholic person, and it gives the first data on concentrations of these substances in cerebrospinal fluid.
Fluoroimmunoassays and immunofluorometric assays.
Authors: Hemmilä I
Published: January 2020

Fluorescent probes and fluorometric methods have gained increasing interest in the field of clinical immunology, not only as one additional alternative to radioimmunoassays, but also in producing cheap, stable, and safe reagents and rapid and sensitive assays. One of the main goals has been the development of homogeneous assays: assays based on fluorescence polarization, fluorescence quenching, excitation transfer, or enzymically releasable probes are widely applied, especially in drug monitoring. The development of suitable solid-phase separation techniques has facilitated utilization of fluorescence in heterogeneous assays, which in general have wider applications, from proteins and viruses to small haptens. Lately new alternative fluorescent probes and methods have been introduced. For example, the use of fluorescent phycobiliproteins or porphyrin derivatives with long-wavelength emission and large Stokes shift or, in particular, the rare earth chelates with unique fluorescent properties well suited to time-resolved measurement have opened new possibilities towards more sensitive immunoassays.
Radioimmunoassay for arylsulfatase A in urine.
Authors: Laidler P M , Ryder K W , Glick M R , Oei T O , Van Etten R L
Published: January 2020

Purified arylsulfatase A (EC 3.1.6.1) from human urine was radioiodinated under conditions that caused no significant loss of antigenic activity. We used this labeled arylsulfatase A (specific radioactivity 4-7.5 Ci/g) together with nonlabeled enzyme and rabbit antiserum produced against homogeneous enzyme to develop a radioimmunoassay for arylsulfatase A in urine. A solid-phase, second-antibody technique (Immunobead Second Antibody; Bio-Rad Laboratories) was used to separate free enzyme from antigen-antibody complexes. The working range of the assay was 0.1-4.0 ng of enzyme; within- and between-assay CVs were around 10%, and the analytical recovery was 105.5% (SD 7.7%). The lower limit of detection was 0.08 ng of arysulfatase A per assay, substantially less than that of typical activity-based assays. Over a wide range of urinary arylsulfatase A activities, results by this method agreed well (r = 0.99) with those obtained by activity assays. We measured the enzyme in urines of 59 healthy volunteers and 92 patients with different diseases, including a group of colorectal cancer cases, to determine whether this could serve as a reliable marker for cancer of the colon; however, urinary excretion of arylsulfatase A by most patients with colon cancer was within normal limits.
Rapid radiometric assay used to assess lactoferrin in granulocytes.
Authors: Freeman K L , Anderson D C , Hughes B , Buffone G J
Published: January 2020

We describe a rapid, precise, and sensitive radiometric assay for human lactoferrin. In this typical "sandwich"-type assay, anti-human lactoferrin is adsorbed onto a polystyrene sphere and bound lactoferrin is detected by the subsequent binding of 125I-labeled anti-human lactoferrin. The assay is accurate for lactoferrin concentrations of 5 to 1500 micrograms/L and takes about 2.5 h to complete. The within-assay and interassay variations (CV) are 5% and 13%, respectively. Neither lysozyme nor heparin, substances that form complexes with lactoferrin, interfered with lactoferrin measurement by this method. The assay has been applied to the measurement of lactoferrin in polymorphonuclear leukocytes of both healthy adults and neonates. We found significantly (p less than 0.001) less lactoferrin in the latter, an abnormality that may be related to known functional deficits of polymorphonuclear leukocytes during the newborn period.
Choriogonadotropin measured with the Tandem-E immunoenzymetric assay system.
Authors: Bock J L , Furgiuele J , Segen J C
Published: January 2020

We evaluated the Hybritech Tandem-E procedure for quantifying choriogonadotropin (hCG) in human serum. In this "sandwich"-type assay, two monoclonal antibodies directed against different regions of the hCG molecule are used, one coated on a plastic bead, the second conjugated to alkaline phosphatase. The assay can detect as little as 1.0 int. unit of the hormone per liter, shows a linear response up to at least 200 int. units/L, and has good precision. By prolonging the incubations for formation of the sandwich and for substrate hydrolysis, one can achieve higher sensitivity at the expense of a narrower linear range. Correlation with a conventional radioimmunoassay for the beta subunit of hCG was generally excellent, but in one instance the Tandem-E gave an apparently false positive result.
Molar absorptivity and the blank correction factor.
Authors: Kroll M H , Elin R J
Published: January 2020

In photometry, where both the product formed and one or several reactants absorb light at the same wavelength, the absorbance of the "blank" of the sample at the end of the reaction may be less than that measured at the beginning of the reaction, because of consumption of reactant(s). The blank correction factor for the determined result with one light-absorbing reagent is epsilon P / (epsilon P - epsilon R), where epsilon R and epsilon P are the molar absorptivities of the reagent and the product, respectively. We derived a factor for the case when more than one reagent absorbs light at the same wavelength as the measured product. This factor is independent of the concentration of reagent(s) and can correct the determined result or absorbance for the consumption of light-absorbing reagent(s) during the reaction.
Immunoenzymetric assay for creatine kinase MB with subunit-specific monoclonal antibodies compared with an immunochemical method and electrophoresis.
Authors: Chan D W , Taylor E , Frye R , Blitzer R L
Published: January 2020

Results of an immunoenzymetric assay (TANDEM-E CKMB) for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme, in which subunit-specific monoclonal antibodies are used, were compared with those by an immunochemical method (Isomune-CK) and electrophoresis (Corning agarose gel). The study involved 200 patients; greater than 500 samples were analyzed by all three methods. The analytical performances were acceptable. Between-method correlation coefficients ranged from 0.881 to 0.975. Two reference intervals were established for the immunoassays: 0-4 micrograms/L (TANDEM) and 0-4 U/L (Isomune) for "normal" patients; 0-9 micrograms/L (TANDEM) and 0-14 U/L (Isomune) for noninfarct patients. Agreement with respect to increased CK-MB as defined by the reference intervals for the noninfarct patient was 96% between TANDEM and electrophoresis, 90% between Isomune and electrophoresis. All three methods are acceptable for use in determining CK-MB, but the overall diagnostic efficiencies for the mass or activity concentration of the isoenzyme and for its proportion of total CK activity, based on the predictive value model, are 92% (electrophoresis, 0-7 U/L), 90% (electrophoresis, 0-4%), 92% (TANDEM, 0-9 micrograms/L), 88% (TANDEM, 0-3% index), 88% (Isomune, 0-14 U/L), and 83% (Isomune, 0-4%). All three methods can detect CK-MB in serum, but its presence is not necessarily diagnostic of acute infarct. We recommend using the actual concentration of CK-MB to evaluate patients with suspected acute myocardial infarct, and the percentage of CK-MB when total CK is very high.
Total cholinesterase in plasma: biological variations and reference limits.
Authors: Lepage L , Schiele F , Gueguen R , Siest G
Published: January 2020

Factors affecting biological variation in total cholinesterase activity in plasma were studied in a population of 3372 apparently healthy subjects at least four years old. Determination of the dibucaine number, after inhibition of the activity by dibucaine, made it possible to specify the contributions of genetic and physiological characteristics to variations in the total activities by using a statistical method of segmentation. The main factors modifying plasma cholinesterase activity in males are genetic status (dibucaine number) and degree of overweight (subscapular skinfold). In females, hormonal status (puberty or menopause), genetic status (dibucaine number), and the use of oral contraceptives are the factors mainly responsible for the variations of plasma cholinesterase activity. These data allow us to propose reference limits for total plasma cholinesterase.
Head-space gas-chromatographic determination of 3-hydroxybutyrate in plasma after enzymic reactions, and the relationship among the three ketone bodies.
Authors: Kimura M , Kobayashi K , Matsuoka A , Hayashi K , Kimura Y
Published: January 2020

In this sensitive, reproducible method for determination of D-3-hydroxybutyrate (3-OHB) in plasma, it is converted to acetone by use of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)/lactate dehydrogenase (EC 1.1.1.27) coupled with acetoacetate decarboxylase (EC 4.1.1.4). The resulting acetone is detected by head-space gas chromatography. The lowest concentration of 3-OHB detectable in plasma was 2 mumol/L. The calibration curve showed a linear relationship for 3-OHB concentration from 0 to 5 mmol/L (r = 0.999). Analytical recovery of 3-OHB (50 mumol/L) was 97.9 (SD 3.8)%. The method was developed for determination of the three ketone bodies in plasma. The ratio of acetone to acetoacetate was not significantly different (p greater than 0.2) between normals (n = 31) and diabetics (n = 86). In normal subjects, the ratio of 3-OHB to acetoacetate was 1.20 (SD 0.44). In diabetic patients, the ratio correlated with the logarithm of the total ketone body concentration (r = 0.828).
K, Ca, Mg, and Zn in platelets, as determined by atomic absorption spectrometry with use of a sealed decomposition bomb and discrete nebulization.
Authors: Makino T
Published: January 2020

In this new method for precise and accurate determination of K, Ca, Mg, and Zn in platelets, small amounts of platelets, prepared by differential centrifugation and cell washing, are decomposed in a homemade mini-vessel, a sealed Teflon bomb. The metal ions in the decomposed sample are measured by atomic absorption spectrometry with discrete nebulization. Overall, CVs ranged from 0.8 to 4.8%. We investigated sex-related differences (none were found) and intra-individual variations.
Factors affecting the binding of disopyramide to serum proteins.
Authors: Shaw L M , Altman R , Thompson B C , Fields L
Published: January 2020

We investigated the influence of the following factors on the binding of disopyramide to serum proteins: method of drug quantification [enzyme immunoassay (EMIT) compared with liquid chromatography (HPLC)], separation technique (ultrafiltration vs equilibrium dialysis), temperature, pH, and total concentration of disopyramide. EMIT and HPLC measurements of disopyramide in ultrafiltrates prepared from 50 sera agreed well: EMIT = 1.046 HPLC + 0.042, (r = 0.928, SEE = 0.04 mg/L). Free disopyramide concentrations in ultrafiltrates of dialyzed sera were similar to those measured in the corresponding dialysates by the EMIT method for 30 patients' sera: ultrafiltrate of serum retentate = 1.053 dialysate + 0.042. Concentrations of free [14C]disopyramide were little affected by temperature. The concentration of free disopyramide decreased as the pH was increased from 7.0 to 7.8. The concentration of free disopyramide, as determined by ultrafiltration, is strongly and directly related to total drug concentration. In the sera of 50 cardiac patients receiving chronic therapy with disopyramide and with total disopyramide concentrations of 0.5 to 5.8 mg/L, the proportion of free disopyramide ranged from 16 to 54%.
Rapid assessment of albumin concentration by immunoturbidimetry.
Authors: Biou D , Thérond P , Israël A , Demelier J F
Published: January 2020

The dye-binding methods involving bromcresol green and bromcresol purple may seriously overestimate albumin in serum that has a below-normal albumin/globulin ratio, particularly in nephrotic syndrome hypoalbuminemia. To overcome this problem, we developed a new immunoturbidimetric method for albumin with use of a centrifugal analyzer. Results by this method (y) and by radial immunodiffusion (x) agreed well and yielded the regression equation: y = 0.91x + 1.33 (n = 40, r = 0.96). Compared with laser immunonephelometry, our method is easier and faster (15 min for the overall determination). Thus, the proposed immunoturbidimetric method is particularly suitable for the rapid indirect assessment of oncotic pressure and indirectly for monitoring intravascular body fluids.
Improved column-chromatographic isolation of glycosylated hemoglobin S.
Authors: Cirotto C , Allegrucci M , Cartechini M G , Compagnucci P , Santeusanio F , Bolli G
Published: January 2020

We investigated glycosylated hemoglobin S by means of chromatography on Bio-Rex 70. The selected elution conditions were similar to those described by Trivelli et al. (N Engl J Med 284: 353-357, 1971), except for modified ionic strength and accurate temperature control. This enabled us to isolate a minor hemoglobin fraction whose properties, as determined by chromatography, electrophoresis, and two-dimensional maps of its tryptic peptides, were typical of a hemoglobin S tetramer with blocked N-terminal residues of the beta subunits. The colorimetric test indicated this to be a glycosylated hemoglobin. This procedure is notably improved over previous chromatographic techniques for isolating glycosylated hemoglobin S. Moreover, it can be easily used in any laboratories where Trivelli's method is routinely in use.
Quality control of reflectometric determinations of glucose in dried blood spots on filter paper.
Authors: von Schenck H , Lönnström L , Engström M
Published: January 2020

We evaluated the effect of sampling of capillary blood on filter paper on the later analysis for glucose. We found the method simple and reliable. Determination at the central laboratory of glucose in blood collected onto filter paper and comparison of the results with those obtained with test strips read in reflectometers at outpatient units is easy. Collecting duplicate samples on filter paper facilitates quality control by avoiding the complications that arise from using quality-control solutions that are not directly comparable with fresh blood, and it avoids the disturbances of test strip chemistry attributable to the glycolysis inhibitors added to blood samples intended for quality control.
Thyroid-function assessment by use of enzyme immunoassays.
Authors: Proença M T , de Moura L S
Published: January 2020

We used an enzyme immunoassay to determine the normal reference intervals for thyroxin, triiodothyronine, and thyrotropin in groups of children, ranging from newborns to 15-year-olds. Results compared well with those by radioimmunoassay. There were no substantial differences between plasma and serum samples for thyroxin and triiodothyronine, but thyrotropin concentrations differed significantly (p less than 0.05, n = 20 each).
Applications of recombinant DNA to pathologic diagnosis.
Authors: Sauls C D , Caskey C T
Published: January 2020

Recombinant DNA techniques are contributing to the understanding of the pathogeneses of genetic, neoplastic, and viral diseases, and are used in the diagnosis of certain genetic and viral diseases. Such techniques will have wider application in the future and will play an increasing role in the clinical laboratory. The technology of this field rests upon the cleavage of DNA by certain enzymes, restriction endonucleases, and upon the ability to locate specific sequences of nucleotides in a cleaved DNA sample by using known fragments of DNA ("probes") labeled with radioisotopes or biotin. To produce useful probes, one "clones" multiple copies of the same DNA fragment in bacteria. The use of DNA probes in the clinical laboratory is valuable in antenatal diagnosis, genetic counseling, and post-natal diagnosis of genetic diseases, especially hematologic diseases and inborn errors of metabolism. DNA probes can also be used to detect viral genetic material in clinical specimens.
Radioimmunoassay for detecting hepatitis B core antigen in serum from patients with chronic hepatitis B infection.
Authors: Quiroga J A , González O , Carreño V , Mora I , Porres J C , Gutiez J , Hernández-Guío C
Published: January 2020

The core antigen component of hepatitis B virus (HBV), HBcAg, may be used as a marker of viral replication. We describe a radioimmunoassay for HBcAg in serum, and compare results with those by other similar methods. Samples are centrifuged over a sucrose gradient containing 2-mercaptoethanol, and the resulting pellets are resuspended in a nonionic detergent solution. After a bead coated with anti-HBcAg is incubated with this suspension, 125I-labeled anti-HBcAg is added, and is also bound by the antigen. The specificity of the method was verified by blocking with purified IgG antibodies to HBcAg. When we tested by this method for HBcAg in sera from 60 patients with chronic HBV infection, all those with circulating HBV-DNA polymerase tested positive for HBcAg. All sera from HBV-negative controls showed no detectable HBcAg. The correlation between the presence of HBcAg and HBV-DNA polymerase was significant (r = 0.715, p less than 0.001). Samples can be tested more quickly and easily with this method, and its sensitivity compares well with that of other similar methods.
Immunoenzymometric assay and radioimmunoassay measure different populations of antibody against human acetylcholine receptor.
Authors: Hinman C L , Kellogg C M , Ernstoff R M , Rauch H C , Hudson R A
Published: January 2020

We used a "sandwich"-type immunoenzymometric assay (IEMA) and a radioimmunoassay (RIA) to measure antibody against the human nicotinic acetylcholine receptor in serum from individuals with myasthenia gravis, with markedly different results for certain specimens, as measured by the two techniques. In some cases, antibody concentrations were high by RIA but low by IEMA; in others, the reverse was found. Such differences persisted through 30 months after thymectomy. An investigation of potential causes of this disparity suggests that high IEMA measurements reflect specific anti-receptor antibody and are not artifactual. The IEMA is recommended as an adjunct to the RIA because some patients with myasthenia gravis who have low concentrations of anti-receptor antibodies as measured by RIA have significantly above-normal concentrations of anti-receptor antibodies as measured by IEMA.
A monoclonal-antibody-based immunoradiometric assay for apoprotein B in low-density lipoprotein.
Authors: Slater H R , Tindle R W , Packard C J , Shepherd J
Published: January 2020

Five different mouse monoclonal antibodies were raised against apoprotein B, the major protein in human low-density lipoproteins (LDL). Competitive binding studies suggested that these antibodies recognized four different epitopes on the lipoprotein particle. All antibodies bound to very-low-density and intermediate-density lipoproteins, which are the metabolic precursors of LDL and also contain apoprotein B. One antibody (P1C1) displayed a relative specificity for apoprotein B in LDL as compared with that in very-low-density lipoprotein and was used as a radiolabeled first antibody in an immunoradiometric assay for this lipoprotein. Measurements, either on isolated LDL or directly on plasma samples, demonstrated that this assay provides a rapid and precise method of quantification.
Benzethonium chloride method for proteins adapted to centrifugal analysis.
Authors: McDowell T L
Published: January 2020

A benzethonium chloride method for measuring protein concentrations in several types of body fluids has been adapted to the Cobas Bio centrifugal analyzer. The ability of this analyzer to add two reagents stepwise, measuring absorbances after each addition, is essential for proper blanking of urine specimens. The method has excellent curve stability and precision and yields results that correlate well with those of four other protein methods.
On the natural history of hypergastrinemia.
Authors: Lindstedt G , Runsteen D , Lundberg P A , Bengtsson C , Lapidus L , Nyström E , Kilander A F
Published: January 2020

We determined total gastrin and pepsinogen I in frozen serum samples from 175 overnight-fasted women 54 years old, and from 81 overnight-fasted women 60 years old, who took part in a population study in 1968-69. We also assayed samples from some of these women, who participated in clinical follow-up studies in 1974-75 and 1980-81: all of the women in the initial group whose serum gastrin concentration exceeded the 85th centile value and, as a reference group, a randomized subsample of women whose initial serum gastrin concentration was less than the 80th centile. Samples with total gastrin concentration greater than 400 ng/L were also assayed for gastrin-17 and gastrin-34. We found that: a pronounced increase of serum gastrin persisted throughout the study period for most of these postmenopausal women, indicating that conversion of type A gastritis (antrum-sparing) to pan-gastritis is uncommon; unexplained high concentrations of pepsinogen I in relation to the reference interval for young and middle-aged adults, as well as in relation to serum gastrin, were common; and the gastrin-17/gastrin-34 ratio is not correlated with the outcome of pronounced hypergastrinemia.
Rapid procedure for fecal porphyrin assay.
Authors: Lockwood W H , Poulos V , Rossi E , Curnow D H
Published: January 2020

Hydrochloric acid extraction of feces in the presence of ether yields an extract suitable for spectrophotometric estimation of total porphyrin and for further separation by "high-performance" liquid chromatography (HPLC) or thin-layer chromatography. A total porphyrin reference interval of less than 200 nmol/g dry weight of feces was established from data on 106 normal subjects on an unrestricted diet. Total fecal porphyrin values in 11 porphyria cutanea tarda patients were considerably higher than given by the widely used Rimington method (respective means, 652 and 239 nmol/g dry weight). Our HPLC method for separation of porphyrin methyl esters on a silica column, with quantification by fluorescence, is described. HPLC separations performed on 23 porphyria cutanea tarda patients gave the following mean proportions of total fecal porphyrins: dicarboxylics 21%, coproporphyrin 9%, isocoproporphyrins 28%, pentacarboxylporphyrin 9%, hexacarboxylporphyrin 11%, heptacarboxylporphyrin 18%, and uroporphyrin 4%.
Rate-nephelometric determination of fibronectin in plasma.
Authors: van Helden W C , Kok-Verspuy A , Harff G A , van Kamp G J
Published: January 2020

We describe a kinetic immunonephelometric method for the determination of fibronectin in human plasma, used with the Beckman ICS rate nephelometer. The method is rapid and cost-effective. Two commercially available controls stated by the manufacturer to contain 200 and 295 mg/L were found to contain 198 and 290 mg/L, respectively. Mean analytical recovery was 104%. Within-run precision (CV) for normal samples was 3.8%, between-day precision 5.1%. For samples containing subnormal concentrations of fibronectin, these figures were 3.8% and 6.7%, respectively. Results by the method described here agreed and correlated well with those by a commercially available turbidimetric assay. With appropriately diluted samples, the range of measurement is 40 to 1000 mg/L. Normal values for women and men were 286 (SD 84) and 340 (SD 55) mg/L, respectively, in good agreement with values published by others.
Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma.
Authors: Rosalki S B , Foo A Y
Published: January 2020

Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.
Improved preparation of cholesterol calibration and control sera.
Authors: Terlingen J B , van Dreumel H J , van Heiningen A , Boerma G J , Koedam J C
Published: January 2020

We prepared several serum batches with various cholesterol concentrations, to be used as calibrators and controls in a proficiency testing program of an organization in The Netherlands that is in charge of the standardization of cholesterol determinations for epidemiological purposes. The sera were of human origin, to avoid abnormal matrix effects. To decrease the cholesterol content in some samples, we adsorbed them onto colloidal silicic acid. To increase it, we added lipoproteins that had been precipitated from human serum with heparin and calcium ions. The precipitation method we used allowed us to dilute the serum as little as possible and to keep the final concentrations of calcium and heparin as low as possible, while maintaining a high cholesterol content. By mixing these sera having high and low cholesterol concentrations, we could prepare batches with any desired concentration. The stabilities of these sera were excellent. We used the sera to calibrate enzymic determinations of cholesterol.
Determination of serum triglycerides by an accurate enzymatic method not affected by free glycerol.
Authors: Sullivan D R , Kruijswijk Z , West C E , Kohlmeier M , Katan M B
Published: January 2020

In this automated single-run enzymatic procedure for specific determination of triglycerides in serum, free glycerol is removed from the reaction mixture by pre-incubation with glycerol phosphate oxidase and peroxidase. The subsequent addition of lipase and the chromogen, 4-aminoantipyrine, results in the formation of color proportional to the amount of triglycerides in serum. Standards containing triolein in aqueous detergent are used to calibrate the method. For serum pools from the Centers for Disease Control with target values of 0.74, 1.41, and 2.63 mmol/L, the method produced biases of +0.01, -0.05, and 0.00 mmol/L, respectively (mean: -0.01 mmol/L or -0.4%). The mean coefficient of variation was 1.4% within and 2.5% between days; the combined CV, 2.9%. Ninety 6-microL serum samples can be analyzed per hour. The method is more accurate and precise than one based on an NADH-coupled enzyme system with separate addition of lipase.
Urinary creatine: biochemical indicator for evaluation of sickle cell crises.
Authors: Beyer C , Statius van Eps L W , Kastelein J J , Brandjes D P , Mairuhu W M , van den Ende A
Published: January 2020

In a patient with known sickle cell beta 0-thalassemia we measured serum lactate dehydrogenase (LD) activity and 24-h urinary creatine excretion rate as markers to evaluate sickle cell crises. We believe that a distinction based on biochemical findings can be made between hemolytic and painful vaso-occlusive sickle cell crises with muscular involvement. To assess hemolytic crises by objective biochemical measures, we have used assay of LD activity, and to assess painful crises with muscular involvement objectively, the 24-h urinary creatine excretion rate. We conclude that hemolytic crises are characterized by high serum LD activities. Furthermore, we conclude that--at least in this patient--painful crises are accompanied by high 24-h urinary creatine excretion rates. Our findings suggest that muscle involvement may play an important role in painful vaso-occlusive sickle cell crises.
Combined liquid chromatography/radioimmunoassay with improved specificity for serum digoxin.
Authors: Gault M H , Longerich L , Dawe M , Vasdev S C
Published: January 2020

This method for assaying digoxin in serum with improved specificity combines small-column extraction of serum, "high-performance" liquid chromatography, and RIA of the eluted fractions. Analytical recoveries of 1.0, 0.5, and 0.1 microgram/L standards were 95%, 93%, and 84%, respectively. The CVs for duplicates and replicates of sera with values of 0.5 to 1 microgram/L were 4 to 6%. Fifty-nine sera from 50 patients receiving digoxin were so studied. All digoxin metabolites appear to cross react with antibody to digoxin to various degrees. The most polar metabolites were quantitatively the most important, their average cross reactivity being 33%. For eight patients the value for digoxin by the present method was less than 60% of the RIA value. Sera from nine patients not taking digoxin but with falsely high digoxin values were also studied by the present method. The digoxin peak was well resolved from those for (a) digoxin metabolites (except dihydrodigoxin), (b) digitalis-like factors in neonates and in patients with renal failure or combined hepatic and renal failure, and (c) two cross reacting drugs and their metabolites.
Binding of Al by protein in plasma of patients on maintenance hemodialysis.
Authors: Cochran M , Patterson D , Neoh S , Stevens B , Mazzachi R
Published: January 2020

Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.
Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored enzyme immunoassays.
Authors: Thorpe G H , Kricka L J , Moseley S B , Whitehead T P
Published: January 2020

Certain phenol derivatives, including p-iodophenol and p-phenylphenol, enhance light emission from the horseradish peroxidase-catalyzed oxidation of cyclic diacyl hydrazides such as luminol. The light emission decays slowly (glowing for several minutes) and its intensity may be greater than 1000-fold that of the unenhanced reaction. The enhanced system enables rapid, sensitive assay of peroxidase conjugates. We describe its application in immunoassays for human choriogonadotropin, digoxin, and factor VIII-related antigen. Luminescent quantification of peroxidase labels has been directly incorporated into immunoassays based on beads, tubes, or microtiter plates, used in conjunction with photodetectors such as photomultiplier tubes or instant photographic film. Enhancement with phenol derivatives exceeds that achieved with 6-hydroxybenzothiazole derivatives and depends on pH and enhancer concentration. Emission spectra of phenol-enhanced and unenhanced reactions are remarkably similar, suggesting that the enhancers do not act as more efficient emitters but exert their action earlier in the complex reaction between peroxidase, oxidant, and luminol.
Three methods compared for determining phosphatidylglycerol in amniotic fluid.
Authors: Coapman-Hankin R A , Kiechle F L , Epstein E , Artiss J D , Zak B
Published: January 2020

Phosphatidylglycerol is one component of amniotic fluid that is unaffected by several of the reported interferences with conventional thin-layer chromatographic measurement of phospholipid. To compare a rapid immunological agglutination test, "Amniostat-FLM", with two-dimensional thin-layer chromatography, we determined phosphatidylglycerol by both methods in 41 amniotic fluid specimens obtained at 31 to 40 weeks of gestation. We also assayed phosphatidylglycerol in 14 of these specimens by an enzymic, colorimetric procedure. The agglutination test is rapid and simple but relatively insensitive; it yielded positive results for only seven of 23 specimens in which phosphatidylglycerol was detected by thin-layer chromatography. In 18 specimens in which no phosphatidylglycerol was detected by thin-layer chromatography, results by Amniostat-FLM were also negative; however, eight of these specimens, assayed by the enzymic method, had phosphatidylglycerol present. Apparently, the Amniostat-FLM detects phosphatidylglycerol only at concentrations exceeding 25 mumol/L, or more than 15% of the total phospholipid composition.
N-[(carbamoylmethyl)amino]ethanesulfonic acid improves phenotyping of alpha 1-antitrypsin by isoelectric focusing on agarose gel.
Authors: Chappell D J
Published: January 2020

The hereditary deficiency variants of alpha 1-antitrypsin that are associated with diseases such as emphysema are usually identified by use of isoelectric focusing on polyacrylamide gels. Agarose is a simpler, faster, safer, and more reliable medium for this, but resolution often is not as good. I describe a method in which the ultrathin agarose gel contains N-[(carbamoylmethyl)amino]ethanesulfonic acid as a "separator," to flatten the pH gradient and improve separation of the alpha 1-antitrypsin isoforms. The resolution obtained equals or surpasses that of conventional methods based on use of either polyacrylamide or agarose. Haptoglobin, which interferes with isoelectric focusing on polyacrylamide, does not interfere with this method; other advantages are also discussed.
Use of DNA immobilized on plastic and agarose supports to detect DNA by sandwich hybridization.
Authors: Polsky-Cynkin R , Parsons G H , Allerdt L , Landes G , Davis G , Rashtchian A
Published: January 2020

Cloned Salmonella DNA, which has been immobilized irreversibly on plastic and agarose solid supports, can form hybrids in both single-layer and "sandwich" hybridization protocols. In single-layer hybridization, 3 micrograms of immobilized DNA bound at least 30 fmol of a specific 800-base DNA sequence (equivalent to 8.5 ng, or the amount of that sequence present in 4 X 10(10) organisms). In a 4-h sandwich hybridization protocol, as little as 14 amol (equivalent to 8 pg, or the amount of that sequence present in 1 X 10(7) organisms) of a 1600-base sequence of DNA could be detected. The methods described should be applicable to use with any set of probes--not just from Salmonella--that fulfill the criteria specified. The ability to perform DNA hybridizations on solid-phase matrices such as those used for immunoassay should bring DNA hybridization into the realm of routine clinical laboratory procedures.
Fluorescence depolarization assay for quantifying alpha-amylase in serum and urine.
Authors: Hofman M , Shaffar M
Published: January 2020

We have developed a new method for quantifying alpha-amylase (EC 3.2.1.1) in serum and urine by fluorescence depolarization. Amylase in the sample catalyzes the hydrolysis of the substrate, a fluorescein-labeled amylose. This results in decreased fluorescence polarization, owing to the increased rate of rotation of the amylose fragment relative to the intact substrate. The TDx amylase assay is calibrated with six human-serum-based pancreatic amylase calibrators. Amylase activities are determined by interpolation from the calibration curve, which is stored in the TDx analyzer's memory. Results correlate well with those by the Du Pont aca assay and the Beckman "DRI-STAT" assay. Endogenous glucose does not interfere. CVs are less than 6%, and the reagents are stable in liquid form.
Particle concentration fluorescence immunoassay: a new immunoassay technique for quantification of human immunoglobulins in serum.
Authors: MacCrindle C , Schwenzer K , Jolley M E
Published: January 2020

A new fluorescence immunoassay technique, particle concentration fluorescence immunoassay (PCFIA), has been developed for quantifying the human immunoglobulins (IgA, IgM, and IgG). In these "two-site sandwich assays," the capture antibody is immobilized on small polystyrene spheres and the tracer is fluorescein-labeled antibody. Polystyrene particles less than 1 microm in diameter make up the solid phase, to which goat anti-human antibody for each respective assay is attached. Serum specimens are diluted (5000-fold for IgA or IgM, 20 000-fold for IgG) placed on the 96-well Pandex assay plate; and mixed with the solid phase and tracer (fluorescein-labeled goat anti-human IgA, IgM, or IgG), which are added automatically by the Pandex Screen Machine. This instrument incubates the reaction mixture for 17 min at ambient temperature, separates the bound and free label by filtration, washes the solid phase, and determines the total particle-bound fluorescence by front-surface fluorimetry or epifluorescence, calculates results, and generates detailed reports. Ninety-six specimens may be analyzed in 29 min or 960 specimens in 136 min. Results by PCFIA for IgA, IgM, and IgG in serum correlated well with those by rate nephelometry.
Simultaneous determination of total IgE and allergen-specific IgE in serum by the MAST chemiluminescent assay system.
Authors: Brown C R , Higgins K W , Frazer K , Schoelz L K , Dyminski J W , Marinkovich V A , Miller S P , Burd J F
Published: January 2020

We have developed a chemiluminescent immunoenzymometric system. The first commercial application of this chemiluminescent assay (CLA) is the measurement of total IgE and allergen-specific IgE in human serum. The CLA system is a second-generation adaptation of the MAST RIA allergy profiling system. The MAST CLA system assay protocol consists of three steps: overnight incubation of serum, a 4-h incubation with enzyme-labeled antibody, and a 30-min chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with an inexpensive microprocessor-controlled infrared transmittance densitometer. The novel luminogenic substrates used yield a constant light output for over 2 h with an intensity at least 10-fold greater than that of commercial chemiluminescent reagents. The MAST CLA system exhibits sensitivity, specificity, and precision equal to that of the MAST RIA system (r = 0.96 for 40 serum samples analyzed with 25 allergens). As many as 35 different allergens per sample can be quantified in a single assay. The MAST CLA system requires no standard curve or volume-dependent pipetting steps, incorporates both positive and negative controls for each sample, and quantifies allergen-specific IgE at picomolar concentrations.
The use of DNA probes in studies of human cytomegalovirus.
Authors: Spector S A , Spector D H
Published: January 2020

Hybridization assays provide a sensitive and rapid means for studying the molecular biology of viral replication and for identifying viral nucleic acid in biological specimens. Such assays are attractive because the detection of virus does not require intact virions or concomitant viral protein synthesis, both of which may be absent in a latently infected cell or in a virus-associated tumor. For molecular and clinical studies on human cytomegalovirus (HCMV), we have cloned and characterized subgenomic EcoRI fragments representative of the entire genome of HCMV strain AD169. To study the epidemiology of HCMV infections and to identify the presence of HCMV nucleic acid in urine, blood, Kaposi's sarcoma, and other tissues, we have used various hybridization techniques, including DNA dot/slot-blot hybridization, Southern blot hybridization, and in situ cytohybridization. These studies demonstrate how cloned molecular probes can be used to study the molecular biology, pathogenesis, and treatment of viral infections.