bluuebunny/crossref_metadata_2025 huggingface dataset:
Assay of cerebrospinal fluid protein: a rate biuret method evaluated.
Authors: Finley P R , Williams R J
Published: January 2020

We evaluated a rate colorimetric method (Beckman) for measuring total protein in cerebrospinal fluid. The automated instrument we used was Beckman's ASTRA TM. A 100-microL sample of spinal fluid is introduced into the biuret reagent in the reaction cell and the increase in absorbance at 545 nm is monitored for 20.5 s. Solid-state circuits determine the rate of alkaline biuret-protein chelate formation, which is directly proportional to the total protein concentration in the sample. The linear range of measurement is 120 to 7500 mg/L. Day-to-day precision (CV) over the range of 150 to 1200 mg/L ranged from 15.2 to 2.3%. The method was unaffected by radical alteration of the albumin/globulin ratio, but there is a positive interference in the presence of hemoglobin, a suppression in the presence of bilirubin, and no effect by xanthochromia. The method is precise, accurate, rapid, and convenient. The method was compared with the trichloroacetic acid method as performed on the Du Pont aca III, giving a correlation coefficient (r2) of 0.9693. The method is precise, accurate, rapid, and convenient.
Column enzyme immunoassay for secretory immunoglobulin A in serum.
Authors: Yamamoto R , Kimura S , Hattori S , Ishiguro Y , Kato K
Published: January 2020

This enzyme immunoassay for specific measurement of secretory immunoglobulin A concentrations in human serum involves use of a small chromatographic column as a solid-phase. Serum samples are incubated for 2 h with beta-D-galactosidase-labeled antibody to secretory component, then passed through a 0.1-mL Sepharose 4B column containing antibodies to human immunoglobulin A. After the column is washed to remove the unbound label, the buffer in the column is replaced by a solution of o-nitrophenyl-beta-D-galactoside (a beta-D-galactosidase substrate) and incubated at 25 degrees C overnight. The enzyme reaction is stopped by washing the column with sodium carbonate solution, and the absorbance of the eluate is measured at 420 nm. The concentration of secretory immunoglobulin A can be determined with a minimum detectable sensitivity of 3 mg/L, without interference from free immunoglobulin A and secretory component in the same samples.
Clinical and laboratory evaluation of a commercial kit for measurement of serum free thyroxin ("Amerlex Free T4").
Authors: Nasralla M , Hassan M , Al-Ansari A A , Kutob M , Al-Abdul Razak A , Al-Awqati M A
Published: January 2020

Different indexes of thyroid function were determined in conjunction with values obtained with a new commercial radioimmunoassay kit for serum free thyroxin, in 49 apparently healthy subjects, 87 pregnant women, and 142 outpatients attending the thyroid clinic. The results indicate a diagnostic success rate of 88% when free thyroxin was measured instead of estimating the so-called free thyroxin index. Furthermore, in three cases of papillary carcinoma the concentration of free thyroxin was increased, although all the other laboratory tests indicated a euthyroid state. Technically, the method is simple, rapid, and precise, and it would be of most value in the small hospital laboratory lacking the facilities of a comprehensive thyroid-function test service.
Remazol Brilliant Blue as a pre-stain for the immediate visualization of human serum proteins on polyacrylamide gel disc electrophoresis.
Authors: Saoji A M , Jad C Y , Kelkar S S
Published: January 2020

We describe the pre-staining of proteins in normal human serum with Remazol Brilliant Blue before separation by disc electrophoresis. Critical to reproducibility are: dye concentration of 0.16 mol/L in a Tris-glycine buffer (pH 8.3), use of equal volumes of serum and dye solution, a tagging period of 2 h at room temperature, and electrophoresis of 0.1 mL of the mixture at 2.5 mA/gel for about 2 h. Advantages include speed, quality of resolution, and low background. This method was compared with Amido Black post-electrophoresis staining in 35 sera. Of these, 16 showed identical results with respect to the number of bands. In the other 19, Amido Black showed more bands in the post-transferrin region and, sometimes, in the post-albumin region. The pre-stained gels showed slower electrophoretic mobilities of the components. Protein bands eluted from pre-stained gels retained immunological reactivity.
Heterozygote detection in congenital adrenal hyperplasia.
Authors: Handelsman D J , Howe C J , Conway A J , Turtle J R
Published: January 2020

Detection of heterozygote carriers for congenital adrenal hyperplasia by use of a modified tetracosactide (a synthetic corticotropin) stimulation test with prior overnight dexamethasone suppression proved to have a diagnostic accuracy of 95%. Discrimination of heterozygotes from normals was best when we used a criterion based on the ratios of 17 alpha-hydroxyprogesterone to cortisol at baseline and at 30 min after intravenous administration of 250 micrograms of tetracosactide.
A challenge of several concepts of free thyroxin index for assessing thyroid status in patients with altered thyroid-binding protein capacity.
Authors: Wilke T J
Published: January 2020

I compared values for the free thyroxin index derived from data obtained by four triiodothyronine-uptake kits for 93 euthyroid subjects with normal or altered thyroxin-binding globulin binding capacity. In addition, I devised a mathematical model of an "ideal" free thyroxin index. Only one uptake kit satisfactorily normalized values for the free thyroxin index in the increased thyroglobulin groups, as compared with the control. The other three demonstrated contrasting patterns of free thyroxin index variation as compared with normal, when thyroglobulin concentrations were increased. By all four uptake tests, values for the free thyroxin index in thyroglobulin-deficient patients were significantly lower than normal. Values derived from one uptake kit behaved similarly to those of the "ideal" model. The failure of the free thyroxin index to compensate for increases in thyroglobulin concentration is therefore a function of specific uptake methods rather than a characteristic of free thyroxin index per se. However, the free thyroxin index is not a true reflection of thyroid status in patients with thyroglobulin deficiency. The model suggests that a curvilinear relationship between triiodothyronine uptake and the concentration of thyroxin-binding globulin can result in a free thyroxin index that corrects for increases in thyroglobulin binding capacity.
Free thyroid hormone index, thyroid hormone/thyroxin-binding globulin ratio, triiodothyronine uptake, and thyroxin-binding globulin compared for diagnostic value regarding thyroid function.
Authors: Wilke T J
Published: January 2020

The thyroid hormone/thyroxin-binding globulin (TBG) ratio and the free thyroid hormone index (FTI) were compared in 372 subjects classified according to age, sex, and biochemical and clinical findings. Age-related variations in thyroid function tests were investigated, as was the relationship between triiodothyronine uptake and TBG. Men, but not women, showed significant age-dependent changes in concentrations of thyroid hormones. FTI was as good as the thyroid hormone/TBG ratio in hyperthyroidism and was a better index of thyroid status in pregnancy, TBG deficiency, and hypothyroidism. In addition, the triiodothyronine uptake correlated extremely well with TBG (r = -0.95, p less than 0.001) and was very efficient in detecting decreased and significantly increased concentrations of TBG. I conclude that FTI is a better discriminator of functional status of the thyroid over a wider range of TBG values than is the thyroid hormone/TBG ratio. Further, the triiodothyronine uptake test produced diagnostic information equivalent to that of TBG estimation and thus should not be replaced in routine use.
Estrone sulfate concentrations in plasma of normal individuals, postmenopausal women with breast cancer, and men with cirrhosis.
Authors: Rémy-Martin A , Prost O , Nicollier M , Burnod J , Adessi G L
Published: January 2020

Estrone sulfate is quantitatively the most important estrogen in plasma. A method for its determination in human plasma is described, and the precision, accuracy, sensitivity, and specificity are defined. Free steroids were extracted from plasma with diethyl ether and steroid sulfates were isolated with use of Vlitos' reagent (methylene blue in dilute H2SO4/Na2SO4 solution). After enzymic hydrolysis, estrone was isolated by chromatography on Celite and measured by radioimmunoassay. The mean concentrations (nmol/L +/- 1 SD) of estrone sulfate were 2.51 +/- 0.90 for plasma from 13 women in follicular phase, 5.33 +/- 1.55 for 17 women in luteal phase, 0.89 +/- 0.60 for 44 postmenopausal women, and 0.96 +/- 0.43 for 24 postmenopausal women with breast cancer. Results for postmenopausal women with or without breast cancer did not differ significantly. For 13 normal men, estrone sulfate concentrations were 2.62 +/- 0.79 nmol/L, and for a group of 19 cirrhotic men the mean value was 1.43 +/- 0.95 nmol/L, significantly lower than normal.
Characterization and elimination of a factor in serum that interferes with turbidimetry and nephelometry of lipase.
Authors: Kannisto H , Lalla M , Lukkari E
Published: January 2020

We studied a serum component ("pseudolipase") that interferes with both turbidimetric and nephelometric lipase measurements. Pseudolipase activity in serum was found in the presence of rheumatoid factor and in other disorders of active immune processes. The incidence of this interferent in sera seems to be greater than reported previously. We describe a method for eliminating its effect without affecting pancreatic lipase activity, by precipitating some of the proteins, including IgM, with polyethylene glycol.
Clinical acceptance of a multi-test reference region for biochemical-panel results.
Authors: Durbridge T C
Published: January 2020

Results of a panel of six biochemical tests on a patient's specimen were mathematically combined into a "six-test signal strength" (STSS) value. This value indicated the overall extent of change from physiological results, and it was calculated in a way that ensured that a STSS value less than or equal to 1 occurred in 95% of apparently healthy people. STSS was reported with the test results for hospital inpatients during a four-month trial period. Doctors requested a repeat of the panel less often when a low STSS was reported, even if some test results were outside their separate reference intervals. Clinicians expressed differing opinions about its usefulness, some finding that a high STSS value had saved them from overlooking abnormal results, others not finding the value to be any practical advantage. Using a multi-test normal region resolves a statistical dilemma, while compounding the problem of knowing what results really mean.
Protocol for the study of drug interferences in laboratory tests: cefotaxime interference in 24 clinical tests.
Authors: Baer D M , Jones R N , Mullooly J P , Horner W
Published: January 2020

We developed a protocol to study interference by cefotaxime and one of its major metabolites with 24 common chemical tests. Serum pools simulating specimens from healthy adults of both sexes, pregnant women, patients with liver disease, cardiac disease, or renal disease, and patients receiving gentamicin and tobramycin were supplemented with high and low concentrations of cefotaxime and desacetylcefotaxime. Using a discrete analyzer (the American Monitor Parallel), we tested 12 replicate samples from each condition for 24 analytes. Although statistically significant changes were found in many tests, 85% of the differences were less than 15% of the control value and more than half were less than 5%. The apparent concentration of creatinine was not significantly changed. Test results for phosphorus were increased in patients who were receiving gentamicin and tobramycin. No other changes were considered clinically significant.
Response curves for radioimmunoassay.
Authors: Finney D J
Published: January 2020

In quantitative estimates from radioimmunoassay, one of four types of response curves is usually used: a freehand curve, a spline function, an equation based upon mass-action considerations, or a logistic equation. This paper comments briefly on the subjectivity and labor of the first and on the overparametrization of the second. It is chiefly concerned to compare the single binding-site equation with a simple or modified logistic. Whatever the theoretical merits of the binding-site approach (these are not under discussion), estimation of parameters is difficult. The paper shows that under many but not all circumstances a four- or five-parameter logistic will fit data at least as well over a wide range of doses. This is particularly so when both the binding-site concentration and the equilibrium constant are small.
Semi-automated continuous-flow enzyme immunoassay for antiepileptic drugs in serum.
Authors: Hayashi S , Kurooka S , Arisue K , Kohda K , Hayashi C
Published: January 2020

We have developed a semi-automated method for measuring five kinds of antiepileptic drugs in serum by successfully adapting commercial competitive-binding enzyme immunoassay kits (MARKIT; Dainippon) for use with a continuous-flow analyzer (Technicon AutoAnalyzer II equipped with a dialyzer). The free enzyme-labeled drug is automatically separated by a microfilter from the competitive immunoreaction mixture between labeled and unlabeled drug for anti-drug immunoglobulin coupled to bacterial cell walls. The concentrations of the antiepileptic drugs in serum samples can be determined by automated measurement of enzyme activity of the enzyme-labeled drugs. Results of the semi-automated method correlated well with those obtained by manual enzyme immunoassay, gas-liquid chromatography, and "high-pressure" liquid chromatography. The correlation coefficients were all greater than 0.95, showing the practicality of this method for therapeutic monitoring of antiepileptic drugs.
Determination of bethanidine in plasma by liquid-chromatography with a microbore reversed-phase column.
Authors: Shipe J R , Arlinghaus A F , Savory J , Wills M R , DiMarco J P
Published: January 2020

In this novel method for quantifying bethanidine in plasma, after a multi-step extraction of bethanidine and internal standard from 2.0 mL of plasma, the drugs are separated on a "microbore" C18 reversed-phase column and quantified by their ultraviolet absorbance at 210 nm. The isocratic chromatographic separation takes about 15 min with use of an ion-pairing regent in the mobile phase (acetate buffer/acetonitrile, 9/1 by vol) and a flow rate of 0.25 mL/min. Sensitivity is increased relative to conventional columns, and solvent consumption is reduced by 90%. The standard curve is linear to at least 5 mg/L, and the detection limit is 0.02 mg/L. The within-run precision of the method is excellent (CV 4%) at a midrange concentration of 1.25 mg/L.
Polarization fluoroimmunoassay of 11-deoxycortisol in serum and saliva.
Authors: Al-Ansari A A , Massoud M , Perry L A , Smith D S
Published: January 2020

We developed polarization fluoroimmunoassays for 11-deoxycortisol in serum and saliva. To avoid interfering factors, the steroid was initially extracted from the biological fluids with dichloromethane. Assays could then be completed without any further separation procedures or need to correct for blank signals. The serum assay was suitable for following the response to the metyrapone test and results correlated acceptably with those by an established, specific, direct 125I-radioimmunoassay. The method is not sufficiently sensitive to detect 11-deoxycortisol in normal saliva, but greatly increased concentrations were found in post-metyrapone saliva and results agreed well with those by the radioimmunoassay as modified for salivary assay. Salivary 11-deoxycortisol assay would provide a convenient means of monitoring results of the metyrapone test.
Solid-state extraction of cortisol from plasma or serum for liquid chromatography.
Authors: Hofreiter B T , Mizera A C , Allen J P , Masi A M , Hicok W C
Published: January 2020

Solid-state extraction of plasma or serum offers significant advantages over traditional liquid-liquid methods in cortisol determinations by liquid chromatography (HPLC). Sample preparation is facilitated and HPLC column contamination is minimized. Extract yield from the solid-state columns is enhanced by sample pretreatment with methanol. Cortisol values by solid-state extraction/HPLC correlate well (r = 0.92) with those from a commercial radioimmunoassay procedure.
Abnormal regulation of pyridoxal 5'-phosphate in Reye's syndrome.
Authors: Faraj B A , Camp V M , Caplan D B , Ahmann P A , Kutner M
Published: January 2020

Seeking to determine the effect of liver disease associated with Reye's syndrome on the regulation of plasma pyridoxal 5'-phosphate, we measured this compound in plasma from 11 patients with biopsy-proven Reye's syndrome. Its concentrations in plasma are significantly higher [37.5 (SD 6.13) micrograms/L] at the onset of the disease than after treatment [8.50 (SD 2.9) micrograms/L] or in a group of hospitalized patients with no evidence of liver disease [8.4 (SD 1.5) micrograms/L]. The concentration of pyridoxal 5'-phosphate in plasma at the time the patients entered the hospital correlated significantly with their activities of serum alanine aminotransferase.
Profiles of urinary volatiles from metabolic disorders characterized by unusual odors.
Authors: Burke D G , Halpern B , Malegan D , McCairns E , Danks D , Schlesinger P , Wilken B
Published: January 2020

The profiles of urinary volatiles from patients with phenylketonuria, maple syrup urine disease, isovaleric acidemia, or trimethylaminuria (fish-odor syndrome) were in each case vastly different from the normal urinary volatiles profile. In the maple syrup urine disease case, metabolites that occur distal to the block were found and a mechanism for their formation is suggested. A new major metabolite in isovaleric acidemia was also found. As well as providing a reliable diagnostic tool for diseases characterized by odors, the analysis of urinary volatiles may provide information to help our understanding of still unexplained aspects of the diseases.
Optimizing reference values for the measurement of alpha 1-antitrypsin in serum: comparison of three methods.
Authors: Gaidulis L , Muensch H A , Maslow W C , Borer W Z
Published: January 2020

We studied three methods (rate nephelometry, radial immunodiffusion, and trypsin-inhibitory capacity) for their ability to detect those individuals with a deficiency of alpha1-antitrypsin. The phenotype represented in 170 serum samples was determined by isoelectric focusing as the reference method. All three methods correctly identified Pi Z, Pi S, and Pi SZ phenotypes but varied in their ability to detect Pi MZ and Pi MS phenotypes. The rate-nephelometric method was the least sensitive in detecting Pi MZ and Pi MS variants because of the inappropriately low reference interval suggested by the manufacturer. We found that the three screening methods are comparable when the limiting values are properly selected. We suggest that the reference value for the rate-nephelometric method be increased from 0.85 g/L to 1.40 g/L to improve the sensitivity of the test.
Two macromolecular complexes between alkaline phosphatase and immunoglobulin A in a patient's serum.
Authors: Wenham P R , Chapman B , Smith A F
Published: January 2020

We report the presence of two separate macromolecular complexes between an immunoglobulin and alkaline phosphatase (EC 3.1.3.1) in the serum of a patient with cholestatic liver disease. The complexes were seen as slow-moving bands on electrophoresis on polyacrylamide gel (70 g/L). Estimates of relative molecular mass for the two bands were 785 000 and 490 000 by gel chromatography on Sephacryl S300, and 640 000 and 420 000 by gradient gel-electrophoresis. Inhibitor and heat-inactivation studies showed that the two bands had similar properties, resembling the liver isoenzyme but more heat stable. Immunological studies suggested that both bands consisted of a complex of liver alkaline phosphatase with kappa type immunoglobulin A.
Hepatic infarction: biochemical study of a case.
Authors: Hoag G N , Orr T P , Amies D R
Published: January 2020

Hepatic infarction was observed post mortem in a 27-year-old man who died of aortic dissection. Blood had been sampled at admission and 12 and 19 hours later. Values for aspartate aminotransferase and alanine aminotransferase in serum were markedly above normal, whereas those for alkaline phosphatase and gamma-glutamyltransferase were only marginally increased. A threefold-increased creatine kinase was ascribable solely to isoenzyme CK-3, suggesting muscle breakdown. Moreover, total lactate dehydrogenase activity was increased threefold, accounted for by a ninefold increase in LD-5 isoenzyme. Those enzyme activities in serum that evidently are associated with acute hepatocellular necrosis increase quickly in hepatic infarction, and CK isoenzyme assay is a useful adjunct if LD-5 increases are significant.
Isolation and characterization of electrophoretic variants of human prostatic acid phosphatase.
Authors: Hibbard M D , McCarthy R C , Markowitz H
Published: January 2020

Prostatic acid phosphatase (EC 3.1.3.2) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.
Prognostic value of combined data on enzymes and inflammation markers in plasma in cases of severe head injury.
Authors: Bourguignat A , Albert A , Férard G , Tulasne P A , Kempf I , Métais P
Published: January 2020

We measured certain enzyme activities (aldolase, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase) and inflammation markers (alpha 1-antitrypsin, C-reactive protein, fibrinogen, and leukocytes) each day for four days in plasma of patients with severe head injury. The univariate prognostic efficiency of each biochemical parameter was assessed 24, 48, 72, and 96 h after trauma. By stepwise multivariate analysis applied every day, we found that (a) four variables, two enzymes (lactate dehydrogenase and aspartate aminotransferase) and two inflammation markers (C-reactive protein and leukocytes), sufficed to reliably predict the patient's outcome and (b) data recorded at 72 h best discriminated between survivors and nonsurvivors. A risk index based on the four selected variables and validated on a large control sample allowed the correct allocation of, respectively, 90% of survivors and 88% of nonsurvivors at 72 h. We discuss why results obtained at 72 h are more predictive than those obtained at any other of the times considered.
Clinical evaluation of two direct procedures for free thyroxin, and of free thyroxin index determined nonisotopically and by measuring thyroxin-binding globulin.
Authors: Waud J M , Chan D W , Drew H M , Oropeza M J , Sucupira M S , Scheinin B , Garrison G M , Mayo M , Taylor E , Stem J
Published: January 2020

We have investigated the clinical utility of two direct radioimmunoassays for free thyroxin, an enzyme-inhibition immunoassay, and a direct measurement of thyroxin-binding globulin (TBG) by radioassay. All assay methods correctly identified greater than or equal to 90% of euthyroid, hyperthyroid, and hypothyroid patients who had normal TBG concentrations. In patients with altered TBG concentrations, none of the assays correctly classified all categories of patients. However, the direct assays of free thyroxin concentrations were able to classify correctly more patients with altered TBG concentrations than did the free thyroxin index methods. The free thyroxin index methods evaluated may be acceptable for routine use, if the concentration of thyroxin and the measurement of TBG capacity are reported along with the index value. Patients with altered TBG concentrations included a group of euthyroid pregnant patients. Significant decreases in free thyroxin in the third trimester were detected by all the assays studied. For patients in the first and second trimester, the mean free thyroxin concentration measured varied with the assay method.
DNA interference with radioimmunoassay procedures.
Authors: Matsuo N , Koide S S
Published: January 2020

A bacterial factor preventing precipitation of immune complexes was purified from a culture of Streptococcus faecalis (AK strain) by gel filtration on Sephadex G-100 and Bio-Gel A-150m columns. The factor was identified as DNA on the basis of its ultraviolet absorption maximum at 260 nm, hydrolysis by deoxyribonuclease, Mr exceeding 1.5 X 10(8), and positive reaction with diphenylamine. DNA interferes with the immunoassay by preventing precipitation of immune complexes. Only double-stranded DNA and polynucleotides showed this property. Evidently, double-stranded DNA will give false-positive interference with the usual RIA procedures.
Radioimmunoassay for pancreatic polypeptide, and its age-related changes in concentration.
Authors: O'Hare M M , Daly J G , Buchanan K D
Published: January 2020

Pancreatic polypeptide (PP), a recently discovered pancreatic hormone, is potentially a marker for endocrine tumors. Consequently, we devised a radioimmunoassay for it, using antisera (raised in rabbits) to bovine PP, 125I-labeled bovine PP (purified by anion-exchange chromatography), and human PP standards. Concentrations circulating in fasting, normal subjects were measured. Statistical analysis of the results revealed a skewed distribution. An age-related increase was also observed. Evaluating PP concentrations in sera from 23 patients with endocrine tumors, we found increased values in a few cases of Zollinger-Ellison syndrome, medullary carcinoma of the thyroid, carcinoid syndrome, and one tumor producing vasoactive intestinal polypeptide (VIPoma). In contrast, values from five insulinomas and one glucagonoma were within the normal reference interval. Thus, an increased value for PP in a fasting individual may suggest the diagnosis of an endocrine tumor but is not a diagnostic prerequisite.
Concurrent measurement of theophylline and caffeine in neonates by an interference-free liquid-chromatographic method.
Authors: Ou C N , Frawley V L
Published: January 2020

A sensitive liquid-chromatographic method was developed for the simultaneous measurement of theophylline and caffeine within 6 min. The excellent resolution of the method allows the complete separation of theophylline, internal standard, and caffeine from frequently encountered interfering drugs including acetaminophen, acetazolamide, acetylsalicylate, ampicillin, 8-chlorotheophylline, cefazolin, cephalosporin C, cephalothin, cephapirin, chloramphenicol, dyphylline, metronidazole, 3-methylxanthine, salicylate, sulfadiazine, sulfamerizine, sulfamethazine, sulfamethoxazole, sulfisoxazole, and theobromine. The chromatographic system involves a Waters' Radial-Pak C18 reversed-phase column and acetonitrile in 0.1 mol/L potassium phosphate buffer, pH 4.0, (9.5/90.5 by vol) as the mobile phase. The method can detect theophylline or caffeine concentrations as low as 0.5 mg/L in 50 microL of serum. Precision and accuracy are excellent.
Separation of bound and free ligand by ethacridine (Rivanol) in thyroxin radioimmunoassay.
Authors: Bhupal V , Mani R S
Published: January 2020

In this simple and low-cost radioimmunoassay (RIA) procedure for thyroxin (T4), ethacridine (6,9-diamino-2-ethoxyacridine lactate; Rivanol) is used to separate antibody-bound ligand and free ligand. This acridine dye precipitates free T4 not bound to antibody. At pH 8.6, it precipitates more than 90% of 125I-labeled T4. Precipitation is rapid and is constant over the concentration range of 1 to 3 g of ethacridine per liter; time of addition and temperature are not critical. The inherent yellow color of the dye gives it the advantage of a color-coded reagent. Analysis of 65 serum samples by the proposed method and by a procedure involving polyethylene glycol gave similar results: correlation coefficient, 0.988; slope, 0.981; y-intercept, -1.42. Within- and between-assay variations (CV) were less than 5 and 6%, respectively.
A radioisotopic method for fructose-1-phosphate aldolase assay that facilitates diagnosis of hereditary fructose intolerance.
Authors: Shin Y S , Moro V , Doliwa H , Endres W
Published: January 2020

A sensitive new method in which D-[U-14C]fructose-1-phosphate is used for fructose-bisphosphate aldolase (EC 2.1.2.13) assay is described. The radioactive fructose-1-phosphate compound was prepared from [U-14C]fructose by use of partly purified fructokinase (EC 2.7.1.4). With this method we measured normal values for aldolase in human liver (2.4-10.0 nmol/min per mg of protein), kidney (3.6-3.8), and intestine (4.2-10.0) as well as Km values for fructose-1-phosphate (approximately 1.0-2.2 mmol/L). In patients with hereditary fructose intolerance the aldolase activity in liver and intestine was less than 10% of normal values. The Lineweaver-Burk plots for data from patients with hereditary fructose intolerance were hyperbolic, indicating a structural alteration in the enzyme.
Quality-control sera for routine determination of aluminum by electrothermal atomic absorption spectroscopy.
Authors: Leung F Y , Henderson A R
Published: January 2020

Several commercially available quality-control sera were analyzed for aluminum content by atomic absorption spectroscopy with a stabilized-temperature graphite furnace. The values obtained ranged between 4 and 1250 micrograms/L (0.148 to 46.235 mumol/L). No significant difference was detected for between-vial variation for four lots of quality-control sera (p greater than 0.05). Control sera stored in 1-mL polypropylene vials and frozen at -20 degrees C for up to six months showed no significant variation in aluminum content (p greater than 0.05), but those stored in their original glass containers had significantly increased aluminum content (p less than 0.001) over a four-week period.
Preparation of F(ab')2 fragments of immunoglobulin G.
Authors: Killion J J , Holtgrewe E M
Published: January 2020

We describe a simple protocol for the preparation of F(ab')2 fragments of immunoglobulin G, based upon the known Fc- binding properties of protein A-Sepharose. The fragment preparations of xenogeneic and allogeneic anti-IgG were noncytotoxic to intact target cells, and were able to block the cytotoxicity of intact antibody. This method should therefore be useful for functional studies not requiring biochemical homogeneity.
Coproporphyrin in urine of newborns with meconium aspiration syndrome.
Authors: Francoual J , Lindenbaum A , Dehan M , de Verneuil H , Nordmann Y , Leluc R
Published: January 2020

We evaluated coproporphyrin in the first urine passed by newborn infants with and without meconium aspiration, by spectrophotometric analysis and thin-layer and "high-performance" liquid chromatography. Urines of newborn infants without meconium aspiration contained only very small quantities of coproporphyrin, detected, after partial purification, by "high-performance" liquid chromatography. Urines of newborn infants with meconium aspiration contained large quantities of coproporphyrin, identified by all three techniques. Urinary coproporphyrin as measured spectrophotometrically correlates well with the "urinary meconium index," and the method is simple, rapid, and reliable, even for samples containing hemoglobin.
Clinical value of free-thyroxin measurements.
Authors: Braun B E , Müller M J , Seitz H J , Krieg M
Published: January 2020

Although discrepancies have been reported between free thyroxin (fT4) in serum and actual thyrometabolic status, the concentration of free thyroid hormones in blood is thought to be an important determinant of the thyrometabolic status of patients. Therefore, the diagnostic value of fT4 measurement has been emphasized. This prompted us to evaluate the diagnostic significance of direct fT4 measurement. We conclude that: (a) The different fT4 radioimmunoassays we examined did not excel the total thyroxin, fT4 index, or thyroxin/thyroxin-binding globulin ratio in the laboratory assessment of thyroid status and alterations in thyroid hormone-thyroxin-binding globulin relationships. (b) The fT4 values obtained by direct or indirect methods always shifted in parallel to total thyroxin with respect to borderline increased or decreased total thyroxin, subclinical hypothyroidism, isolated hypertriiodothyroninemia, and thyroxin substitution.
Solid-phase chemiluminescence immunoassay for plasma estradiol-17 beta during gonadotropin therapy compared with two radioimmunoassays.
Authors: De Boever J , Kohen F , Vandekerckhove D
Published: January 2020

Estradiol in plasma was determined with a simple solid-phase immunoassay based on chemiluminescence in patients undergoing ovulation induction. In the assay, we use a purified monoclonal antibody to estradiol-17 beta, with estradiol-6-carboxymethyl oxime-aminobutylethyl isoluminol as the chemiluminescent marker. The antibody is covalently coupled to polymer beads. Bound and free ligand are separated by simple centrifugation. Values so determined were compared with those determined with two different radioimmunoassay (RIA) methods, one involving the same monoclonal solid-phase antibody plus iodinated estradiol, 125I-RIA, the other a polyclonal antibody and tritiated estradiol, 3H-RIA. In the latter RIA, dextran-coated charcoal was used to separate bound and free ligand. Values determined by the three methods agreed well: for chemiluminescence vs 125I-RIA, r = 0.93; for chemiluminescence vs 3H-RIA, r = 0.94; for 125I-RIA vs 3H-RIA, r = 0.94. The three methods are similar in sensitivity, specificity, and precision. The present method is simple and reliable, taking advantage of the specificity and sensitivity intrinsic to the use of an antibody without the inconveniences associated with use of an isotopically labeled ligand.
Quality control of acid phosphatase assay improved by direct reconstitution of lyophilized control materials with citric acid.
Authors: Blick K E , Commens M J , Passey R B
Published: January 2020

Current quality control of assays of acid phosphatase (EC 3.1.3.2) with thymolphthalein substrate is inadequate when certain control-suppliers' instructions are followed for reconstituting and stabilizing human lyophilized control material. If such materials are reconstituted with a citric acid solution, then stored frozen, control vial-to-vial variability is minimized, as are losses of activity in freshly reconstituted controls and losses during room-temperature storage of the control material. Effects of pH of the reconstituting solution on the acid phosphatase activity of human lyophilized controls are also discussed.
Multilayer film analysis for glucose in 1-microL samples of plasma.
Authors: Neeley W E , Zettner A
Published: January 2020

With the ultramicroanalytical system described here we can measure glucose in 1 microL of plasma or serum. The sample is placed on a dry, multilayer film element (Eastman Kodak), where a colored spot about 3.5 mm in diameter develops. The reflectance of these spots is measured with a reflectance digital matrix photometer that was conceived, designed, and constructed in our laboratory. The spot is illuminated with monochromatic light and its image is projected by a camera lens onto the photosensitive surface of a linear photodiode array containing 512 individual photodetectors. The photodetector signals are processed by a computer to obtain the reflectance and diameter of the spot. The latter is proportional to sample volume. Because the reflectance of the spot does not depend greatly on sample volume, accurate pipetting is not required. The coefficients of variation of repeatable glucose analyses were, for 400, 3000, and 5120 mg/L, 1.7, 2.3, and 2.8%, respectively. The correlation coefficient (r) between glucose analyses by our method (y) and with the Ektachem 400 (x) was 0.9918; the regression equation was y = 1.07x - 94.3 mg/L.
Advancing in tandem: clinical endocrinology and clinical chemistry.
Authors: Hershman J M
Published: January 2020

Here I review some of the recent accomplishments in clinical endocrinology and clinical chemistry, point out the direction of future work, and try to predict some of the advances of the next decade. Endocrinology, perhaps more than any other branch of clinical medicine, is rooted in biochemistry and physiology. Endocrinologists have been leaders in the development of methods, which have been adapted by clinical chemists for wider applications than those conceived originally. For the sake of brevity, I shall not recount the early history of endocrinology in this century--or the enormous progress since Starling coined the term "hormone" and stated the physiological role of these chemical messengers in 1905. Progress in endocrinology in recent years has been astounding.
Sensitive fluorometry of heat-stable alkaline phosphatase (Regan enzyme) activity in serum from smokers and nonsmokers.
Authors: Maslow W C , Muensch H A , Azama F , Schneider A S
Published: January 2020

We developed a simple, sensitive enzymatic assay involving the fluorogenic substrate naphthol AS-MX phosphate [(3-hydroxy-2-naphthoic acid 2,4-dimethylanilide) phosphate] to measure heat-stable alkaline phosphatase (EC 3.1.3.1), the Regan isoenzyme, in human serum. The day-to-day CV was 5.7% for a serum activity of 0.080 arbitrary units/L. Measurable amounts of enzyme were detected in most normal individuals. The mean for 51 nonsmokers was 0.068 (SD 0.037) arb. units/L; for 25 smokers it was 0.440 (SD 0.360) arb. units/L. Activity of this isoenzyme in smokers was as much as 10-fold the upper normal limit for nonsmokers. Activation of this tumor marker by smoking has not received attention hitherto. We conclude that a truly normal range can only be established among nonsmokers. The isoenzymes in smokers, nonsmokers, and pregnant women were similar in their heat stability, immunologic cross reactivity, and inhibition by L-phenylalanine.
Amniotic fluid fluorescence polarization value at physiological temperature: a marked improvement in assessing fetal lung maturity.
Authors: Barkai G , Mashiach S , Modan M , Serr D M , Lanir D , Lusky A , Goldman B , Shinitzky M
Published: January 2020

Determination of fetal lung maturity by measurement of the fluorescence polarization (P) value of the amniotic fluid at room temperature has become the method of choice in an increasing number of perinatal units because of its simplicity and relatively high predictive value. Nevertheless, its power to discriminate between cases with and without hyaline membrane disease (HMD) needs improvement. To this end, we assessed the discriminative power of the P value at the physiological temperature of 37 degrees C (P37) as compared with the power at 25 degrees C (P25). The study group consisted of 288 consecutive cases at risk for preterm delivery. Samples from all 288 cases were measured at 25 degrees C and samples from 112 of these were measured concurrently at 37 degrees C as well. HMD occurred in 27 infants of the total group, nine of whom belonged to the subgroup tested at both temperatures. When sensitivity was fixed at 100% the specificity of P37 was 97% as compared to 79% for P25 (p less than 0.001). The percentage of cases with infants free of HMD who had borderline P values was also significantly smaller: 1% vs 21%, respectively (p less than 0.001). Although data on more HMD cases are needed to establish the precise threshold of lung maturity for P37, we conclude that P37 is a considerably better discriminator for fetal lung maturity determination than P25.
Centrifugal analyzer used for enzyme immunoassay of progesterone and choriomammotropin.
Authors: Terouanne B , Marchand J , Calzolari C , Monnier J , Nicolas J C , Pau B , Descomps B
Published: January 2020

Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.
Effect of albumin concentration on the assay of serum free thyroxin by equilibrium radioimmunoassay with labeled thyroxin analog (Amerlex Free T4).
Authors: Amino N , Nishi K , Nakatani K , Mizuta H , Ichihara K , Tanizawa O , Miyai K
Published: January 2020

Serum free thyroxin (FT4) in normal nonpregnant and pregnant subjects was measured by radioimmunoassay (RIA) with Amerlex FT4 RIA (Amersham International) and LiquiSol FT4 RIA (Damon Diagnostics) kits. Amerlex FT4 values in serum from pregnant women were lower than those in serum from nonpregnant women, but LiquiSol FT4 values were similar in serum from both groups. Amerlex FT4 values were directly correlated with the concentrations of albumin in serum and inversely correlated with those of thyroxin-binding globulin, but not with prealbumin concentrations. No significant correlations were observed between LiquiSol FT4 values and serum concentrations of thyroxin-binding proteins. Amerlex FT4 values were normal in patients with excess, deficient, or decreased thyroxin-binding globulin. Albumin added to serum samples increased Amerlex FT4 values but not LiquiSol FT4 values. Albumin inhibited the binding of labeled thyroxin analog to the solid-phase thyroxin antibody. These data indicate that the albumin concentration influences FT4 values as measured by an RIA involving a thyroxin analog and that Amerlex FT4 values should be carefully interpreted when the patient has an abnormal concentration of serum albumin.
Proportion of hemoglobin S in blood, as determined from solubility measurements.
Authors: Bowie L J , Dohnal J C
Published: January 2020

We exploited the concentration dependence of hemoglobin S on its solubility in concentrated phosphate buffer to determine the percentage of hemoglobin S or hemoglobin A in whole blood or hemolysates. A sample is incubated for 1 h at 37 degrees C in "SickleQuik" (General Diagnostics) tubes, and the absorbance of the aqueous phase is then measured at 555 nm and 650 nm, the latter wavelength to correct for turbidity. The fractional hemoglobin S or hemoglobin A content is read from standard-curve data on mixtures of hemoglobin A and hemoglobin S. Such curves can be prepared with the use of hemoglobin A alone, because under our conditions all hemoglobin S is precipitated. The curve is reproducible for up to three weeks. Day-to-day CVs ranged from 1.8% at 10% hemoglobin S to 8.1% at 68% hemoglobin S. For similar concentration ranges, day-to-day CVs for electrophoresis, with densitometry, ranged from 4.9% to 18%. Results on 29 patients are presented. We recommend the method for rapid quantification of hemoglobin S percentage for patients with sickle cell disease in acute-care situations.
Measurement of glycosylated hemoglobin on cellulose acetate membranes by mobile affinity electrophoresis.
Authors: Ambler J , Janik B , Walker G
Published: January 2020

In this method for separating glycosylated from nonglycosylated hemoglobin in blood by electrophoresis on cellulose acetate membranes, we exploit the affinity of low-molecular-mass dextran sulfate for the nonglycosylated fraction, which increases the mobility of the latter relative to that of glycosylated hemoglobin. After the membrane strips are cleared and stained, the two fractions are quantified densitometrically. As evaluated by use with blood from diabetics, results compare well with those by chromatography on short columns and by electrophoresis in commercial agar gel films.
Improved Ellman procedure for erythrocyte cholinesterase.
Authors: George P M , Abernethy M H
Published: January 2020

The procedure of Dietz et al. (Clin. Chem. 19: 1309-1313, 1973) for plasma cholinesterase (EC 3.1.1.7) gives a background absorbance of 1.4 A when extended to erythrocyte cholinesterase (EC 3.1.1.8) measurement, because the peak absorbance of the reaction product, 5-thionitrobenzoate, coincides with the hemoglobin Soret band at 410 nm. Consequently, the precision of erythrocyte cholinesterase measurements is poor, and the test is restricted to laboratories with a spectrophotometer having a high signal-to-noise ratio. Use of the detergent benzethonium chloride (Hyamine 1622) instead of quinidine sulfate to stop enzyme action allows readings to be made at 440 nm because the hemoglobin band is shifted to 405 nm and its peak intensity is decreased. Moreover, detergent micelle interactions shift the peak absorbance of the 5-thionitrobenzoate from 410 to 435 nm. Overall, the blank absorbance is decreased to about 0.4 A. This results in an assay that is twice as precise as the previous version and is suited for use in a routine laboratory with a moderate-quality spectrophotometer. Thus erythrocyte cholinesterase measurements can readily be made, to complement plasma cholinesterase in the investigation of exposure to organophosphates.
Direct determination of inorganic phosphorus in serum with a single reagent.
Authors: Muñoz M A , Balón M , Fernandez C
Published: January 2020

Estimation of inorganic phosphate in serum based on the formation of "molybdenum blue" may be simplified by eliminating the reduction stage. The yellow complex formed by the reaction of phosphate with molybdate in an acid medium is measured at 390 nm. The yellow complex behaves in accordance with Beer's law over a wide concentration range (to at least 80 mg of P per liter in the initial sample); its molar absorptivity at 390 nm is 2.51 X 10(3) L mol-1 cm-1. Sensitivity is increased in the presence of a detergent (triethanolamine lauryl sulfate), which is also used to dissolve the proteins. Because only one reagent is used (a stable combination of, per liter, 20 mmol of sodium molybdate, 82 mmol of nitric acid, and 100 mg of the detergent), the method is simple and rapid. We describe a manual procedure and an automated one. Within-run precision was 1.9%, and day-to-day precision less than 7%. Results by the automated method compare favorably (r = 0.96) with those obtained by Drewes's method (Clin Chim Acta 39: 81-88, 1972).