bluuebunny/crossref_metadata_2025 huggingface dataset:
Alanine aminopeptidase in serum: automated optimized assay, and effects of age, sex, smoking, and alcohol consumption in a selected population.
Authors: Mueller P W , Phillips D L , Steinberg K K
Published: January 2020

In the diagnosis of chronic (as opposed to acute) liver diseases, combinations of indicators are needed to improve specificity. Alanine aminopeptidase (AAP; microsomal aminopeptidase, EC 3.4.11.2) activity in serum reportedly is a very sensitive indicator of intrahepatic cholestasis and biliary obstruction; it is also particularly useful in diagnosing chronic liver disease when combined with an indicator of hepatocyte damage such as aspartate aminotransferase or alanine aminotransferase. We optimized the assay of AAP in serum, automated the assay by using a centrifugal analyzer, then used this automated assay to determine activity in 202 individuals, ages one to 73 years. The preliminary results were analyzed in terms of the effects of age, sex, smoking, and alcohol consumption on AAP activity in serum. Striking sex-related differences were observed: AAP activity in males declined 2.5 times more rapidly with age than did that in females; indeed, activity in adult females remained essentially constant. Moreover, AAP values were higher in men who smoked than in those who did not, the difference being of borderline significance by analysis of covariance (p = 0.0865) but significant by partial correlations (p = 0.02). No similar differences were seen for women smokers and non-smokers. When the effects of other variables were held constant, alcohol consumption alone did not significantly correlate with AAP activity in men or women.
A two-step radioimmunoassay for free triiodothyronine in serum.
Authors: Rajan M G , Samuel A M
Published: January 2020

Using a high-affinity solid-phase-bound antibody (Ka = 1.2 X 10(11) L/mol), we have standardized a two-step radioimmunoassay for free triiodothyronine (FT3) in serum, based on immunoextraction. The method was validated by comparison with an equilibrium-dialysis procedure (r = 0.96) involving RIA of T3 in the dialysate standardized with the same antibody and by a commercial (Liso-Phase, International-CIS) method. The two-step RIA could detect as little as 0.2 pg per milliliter. The mean CVs within and between assays were 9% and 12%, respectively. FT3 values measured in 30 normal adults ranged from 1.77 to 4.77 ng/L. Comparison with ratios of total T3 to thyroxin-binding globulin showed good agreement in normal subjects, pregnant women, and hypothyroid and hyperthyroid patients.
A new enzymic determination of guanidinoacetic acid in urine.
Authors: Shirokane Y , Utsushikawa M , Nakajima M
Published: January 2020

We developed and evaluated a colorimetric method for enzymic determination of guanidinoacetic acid (GAA) in urine. Endogenous urinary urea was first eliminated by urease (EC 3.5.1.5), and the added urease was then removed from the sample by centrifugal ultrafiltration. GAA in the ultrafiltrate was subsequently hydrolyzed by guanidinoacetate amidinohydrolase (EC 3.5.3.2) to glycine and urea. The latter substance produced an orange chromogen reacting with o-phthalaldehyde and N-(1-naphthyl)-N'-diethylethylenediamine, the absorbance of which at 465 nm was linearly related to concentrations as high as 200 mg/L for standard solutions of GAA. Analytical recovery of GAA added to urine ranged from 94 to 112% (mean 101%) and the within-run and between-run precision (CVs) of the method for the urinary GAA determination averaged 2.2 and 3.5%, respectively. Results correlated well (r = 0.983) between the present method and a high-performance liquid chromatographic method. The proposed method is accurate and simple. We saw a great decrease in urinary GAA of patients with suspected or proven renal insufficiency as compared with that of healthy volunteers.
A patent law primer for clinical chemists and other scientists.
Authors: Blecher M
Published: January 2020

Patenting and commercialization by academic scientists, despite potential drawbacks, are on balance highly desirable if technology is to be transferred from the laboratory to the public use, and if the scientist and his institution are to be encouraged to participate in this transfer. If that premise is accepted, there is much that academic institutions can do to foster utilization of their biotechnological discoveries. Such institutions should have a patent policy that is known to all and that includes a professional patent administrator and clear administrative procedures for carrying out such policy. Scientists should be trained to recognize and protect their inventions and to appropriately disclose their inventions to their patent officers. Ideally, scientists should know the rudiments of the patent statutes of their own country and should be aware of what constitutes trade secrets. Scientists should be given guidance in working with patent attorneys in the preparation and prosecution of patent applications. Finally, given human nature, institutions should see to it that their scientists are provided with a suitable environment in which to invent, and appropriate incentives to do so.
Presence of nonesterified and acylcarnitine in human polymorphonuclear leukocytes and mononuclear cells.
Authors: Katrib K , Adlouni H A , Férard G
Published: January 2020

We demonstrate the presence of nonesterified carnitine and acylcarnitine in leukocytes, but not in erythrocytes, from 16 healthy adults. After carefully separating the different kinds of blood cells we measured significant amounts of nonesterified carnitine and acylcarnitine in polymorphonuclear leukocytes (28.5 +/- 6.1 and 18.5 +/- 6.3 mumol/10(9) cells) and mononuclear cells (25.4 +/- 5.2 and 14.8 +/- 4.5 mumol/10(9) cells). We also measured nonesterified carnitine, long-chain acylcarnitine, and short-chain acylcarnitine in plasma after fractionation with perchloric acid and obtained the following values (mean +/- SD): 41.4 +/- 2.6, 3.9 +/- 1.2, and 6.0 +/- 1.6 mumol/L, respectively. The mean percentages of total carnitine (n = 6) in polymorphonuclear leukocytes, mononuclear cells, and plasma were approximately 62%, 27%, and 13% of whole-blood carnitine, respectively (mean recovery was 102%). The percentage of acylated carnitine was 37% in leukocytes, as compared with 19% in plasma.
Rapid detection of leukemia cells by use of a complement-mediated cytolytic reaction and an imaging sensor system.
Authors: Suzuki M , Tamiya E , Kataoka T , Tokunaga T , Karube I
Published: January 2020

We describe a system for detection of leukemia cells involving complement-mediated cytotoxic reaction and an image processing system, consisting of a charge-coupled-device image sensor, an image memory board, a personal computer, and a phase-contrast microscope. Then added to a cell suspension, monoclonal antibody specific to the fetal thymus antigen-1 of the mouse leukemia GRSL cell produced cytolysis of only GRSL cells. This cytolysis decreased the brightness of the cells observed by phase-contrast microscopy. The remaining brightness was subtracted from that of the phase-contrast image of the cells before cytolysis, which had been converted to a digital signal and stored in computer memory. Measurement time is 2 s. The time course for complete GRSL cytolysis, as measured with this system, is 12 min; overall measurement time, including reaction time, is approximately 15 min. GRSL cells in a suspension of mixed cells were determined specifically by the system.
Lactate dehydrogenase isoenzyme 1 as determined by inhibition with 1,6-hexanediol and by two other methods in patients with myocardial infarction or cardiac-bypass surgery.
Authors: Shamberger R J
Published: January 2020

In this assay for lactate dehydrogenase (EC 1.1.1.27) isoenzyme 1 (LD-1), 1,6-hexanediol is added to serum after total LD has been measured. After incubation for 5 min the total LD remaining is determined. Samples from patients who had a myocardial infarction or who had undergone bypass surgery were assayed simultaneously by the 1,6-hexanediol method, the Roche "Isomune LD," and by electrophoresis on agarose gel. For 101 analyses of sera from bypass patients, correlations were high for results by the inhibitor method and electrophoresis (r = 0.96), by Roche Isomune and the inhibitor method (r = 0.96), and by the Roche method and electrophoresis (r = 0.97). In general, values for total LD were quite comparable by the three methods, but results by the inhibitor method seemed slightly higher and showed greater variability (CV) than those by the electrophoretic method. The assay is simple--requiring one reagent addition and a short incubation--and inexpensive: LD-1 can currently be determined for less than 1 over the cost of determining total LD.
Interference by goat-derived anti-lymphocyte globulin in a double-antibody radioimmunoassay of digoxin.
Authors: Coon M J , England B G , Annesley T M , Giacherio D A
Published: January 2020

Therapy with anti-lymphocyte and anti-thymocyte globulins, two animal-derived preparations used as immunosuppressive agents, results in high concentrations of foreign immunoglobulin circulating in plasma. We describe a case that illustrates how the presence of these immunoglobulins can interfere with double-antibody radioimmunoassay procedures.
Cellular oncogenes and human carcinogenesis.
Authors: Der C J
Published: January 2020

Experimental studies over the past decade have identified 30 or so cellular genes as potential oncogenes. The genetic events that lead to cellular oncogene activation may result in the excessive or inappropriate expression of the gene, or the expression of an aberrant gene product. Although the involvement of these putative cellular oncogenes in human oncogenesis has not been proven, the accumulation of considerable experimental evidence strongly implicates some role of these genes in the malignant process. The inactivation of certain genetic loci (suppressor genes) may also contribute to tumor progression.
Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.
Authors: Sorroche P , Bianchi-Bosisio A , Sinha P K , Gelfi C , Righetti P G
Published: January 2020

This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.
Radioimmunoassay of atrial natriuretic polypeptide in heat-treated human plasma.
Authors: Iinuma K , Ikeda I , Ogihara T , Hara H , Shima J , Kurata K , Kumahara Y
Published: January 2020

In this simple, sensitive radioimmunoassay (RIA) of atrial natriuretic polypeptide (hANP) in human plasma, nonspecific interference is minimized by deproteinizing the plasma by heat treatment at 85 degrees C for 10 min. We directly measure alpha-hANP in the supernates by RIA, with use of antiserum that recognizes the N-terminal region of alpha-hANP. The minimal detectable value was 0.4 pg per tube. The intra-assay CV was 6.6% (n = 8). The mean concentration of hANP in plasma of 54 healthy volunteers was 41 (SD 29) ng/L. Concentrations of hANP in plasma increased after saline infusion and high salt intake for one week in patients with essential hypertension. High concentrations were also measured in patients with renal failure and congestive heart failure. This method, which requires no extraction or purification with column chromatography, is especially useful for simultaneous measurement of several samples.
Identification and quantification of a protein-bound ligand in uremic serum.
Authors: Takeda N , Niwa T , Tatematsu A , Suzuki M
Published: January 2020

A major protein-bound ligand in the serum of chronic hemodialysis patients was isolated from heat-deproteinized uremic serum by "high-performance" liquid chromatography (HPLC). The isolated compound was identified as 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid by use of liquid secondary-ion mass spectrometry and gas chromatography-mass spectrometry. On HPLC, an authentic sample of this compound showed a retention time identical to that of the protein-bound ligand peak. The concentration of the furancarboxylic acid in serum, as estimated by HPLC, is markedly greater in chronic hemodialysis patients than in normal subjects.
Mathematical treatment of data for calculating activities of alpha-amylase isoenzymes.
Authors: Redondo F L
Published: January 2020

Methods for determining alpha-amylase isoenzymes by selective inhibition with a wheat-germ protein are practical and easy, and give accurate, precise results. The incomplete specificity of the inhibitory action is not a major drawback but does necessitate mathematical treatment of the data (i.e., enzymic activities measured before and after preincubation with the inhibitor) to ascertain the amount of the different isoamylases. Such an algorithm is quite simple and straightforward, because the isoenzymes can be calculated either arithmetically or geometrically, by using a linear standard curve, empirically obtained, that relates the fraction of activity remaining after inhibition (R/T) to the pancreatic isoenzyme fraction or to the percentage of total alpha-amylase (P/T or 100 X P/T). An alternative method, plotting R/T against the ratio of pancreatic to salivary isoenzyme (P/S), is inconvenient, necessarily yields a nonlinear curve, needlessly complicates the calculations, and has been a persistent source of confusion in many articles dealing with the differentiation of isoamylases.
Nonenzymatic glycation of immunoglobulins does not impair antigen-antibody binding.
Authors: Morin L G , Austin G E , Burkhalter A
Published: January 2020

We glycated immunoglobulins from commercial kits designed to measure human ferritin, thyrotropin, and transferrin, and compared the calibration curves for assays utilizing glycated antibodies with those of assays utilizing non-glycated antibodies. Glycation was verified by borate affinity chromatography and assay with thiobarbituric acid reagent. We found no evidence that antigen-antibody binding is impaired by nonenzymatic glycation of antibodies. Our results provide no evidence in support of the supposition that glycation may be a contributory factor in the decreased resistance of diabetics to infection.
Enzyme-linked immunosorbent assay for 17 alpha-hydroxyprogesterone in dried blood spotted on filter paper.
Authors: Maeda M , Arakawa H , Tsuji A , Yamagami Y , Isozaki A , Takahashi T , Haruki E
Published: January 2020

In this rapid, cost-effective, enzyme-linked immunosorbent assay for 17 alpha-hydroxyprogesterone (17-OHP) eluted from dried blood spotted on filter paper, second antibody is coated onto the microwell plate and horseradish peroxidase (EC 1.11.1.7) is the label enzyme. Antiserum to 17-OHP was prepared by using 4-(2-carboxymethylthio)-17-OHP-bovine serum albumin conjugate as immunogen. Enzyme conjugate was prepared from 4-(2-carboxymethylthio)-17-OHP and peroxidase. The blood spots are assayed in the microwells without extraction or centrifugation steps. The detection limit of the assay is 1 microgram/L, equivalent to 3.5 pg (10.6 fmol) per disc. Intra- and interassay CVs at two steroid concentrations (7.38 and 22.79 micrograms/L) ranged from 3.74 to 11.90% (n = 5), and 9.49 and 9.83% (n = 5), respectively. Results correlated well (r = 0.91) with those of a fluorescence enzyme immunoassay. The sensitivity, specificity, and precision of this method make it potentially useful in the mass screening of neonates for congenital adrenal hyperplasia.
Delta bilirubin: absorption spectra, molar absorptivity, and reactivity in the diazo reaction.
Authors: Doumas B T , Wu T W , Jendrzejczak B
Published: January 2020

Delta bilirubin (B delta), isolated from serum, has an absorption maximum near 440 nm and a molar absorptivity of 72,000 L mol-1cm-1 in either Tris HCl (0.1 mol/L, pH 8.5) or phosphate (0.13 mol/L, pH 7.4) buffer. This absorptivity exceeds by approximately 50% and 59%, respectively, that of unconjugated bilirubin in the same buffers. This finding suggests that substantial errors can be incurred in direct spectrophotometry of bilirubins in serum. In the total diazo (TBIL) assay (Clin Chem 1985;31:1779-89), the color yield from B delta increases by 10% as the final diazo concentration is increased from 0.27 to 0.81 mmol/L. In the direct (DBIL) assay, if done in HCl (50 mmol/L), B delta yields approximately 15% more color as the diazo concentration is increased from 0.51 to 1.53 mmol/L, whereas in acetate buffer (0.4 mol/L, pH 4.7) the corresponding color yield is 25% greater. However, the absolute color yield for the reaction in HCl exceeds that in acetate buffer. In both the TBIL and the DBIL assay, B delta reacts slowly, nearly complete reaction requiring 10 min. Thus, B delta may be seriously underestimated in diazo (especially DBIL) methods in which short reaction times (20 s to 1 min) are used.
Improved assay for etoposide in plasma by radial-compression liquid chromatography with electrochemical detection.
Authors: el-Yazigi A , Martin C R
Published: January 2020

We describe a rapid, sensitive, and expedient radial-compression liquid-chromatographic method for routine quantification of etoposide (VP-16-213) in plasma of cancer patients undergoing chemotherapy with this agent. Teniposide was used as the internal standard. The mobile phase was a mixture of 10 mmol/L phosphate solution and methanol (50.7/49.3, by vol), adjusted to pH 3 with phosphoric acid. The flow rate was 2 mL/min. We used a 5-micron particle size C18 cartridge with a radial-compression separation system. The effluent was monitored with an electrochemical detector. Plasma samples are extracted with chloroform and the chloroform is evaporated. The residue, reconstituted in mobile phase, is injected onto the cartridge. Assay results were linearly related to concentration (r greater than 0.9944) over the range examined, 0.010-28.0 mg/L. The intra-run CV was less than 4.4%, and we encountered no interference from anticancer drugs or other compounds. We present data for plasma sampled at different intervals from two cancer patients being infused with etoposide.
Improved accuracy of cholinesterase phenotyping after participation in a proficiency survey.
Authors: Evans R T , Walker A , Bowness K M
Published: January 2020

We report results of an investigation into the proficiency of cholinesterase (EC 3.1.1.8) phenotying, assessed in 13 laboratories between 1983 and 1986. Thirty-two specimens of serum were distributed for analysis: two, each in duplicate, from the eight genotypes that can be recognized by differential enzyme inhibitor numbers. The accuracy of genotype ascription was markedly improved over that observed in an earlier study in which 12 of these laboratories took part, although the proportion of clinically significant errors did not change. Consequently, although participation in a proficiency testing program can lead to a considerable enhancement of performance, we still recommend the use of reference centers for detailed cholinesterase studies.
Isoelectric focusing of neuraminidase-treated alkaline phosphatase isoenzymes on agarose gel.
Authors: Root C F , Fine J S , Clayson K J
Published: January 2020

We attempted to separate bone and liver alkaline phosphatase (EC 3.1.3.1) isoenzymes in human serum by isoelectric focusing on agarose gel. We found that in a pH 3-10 gradient the liver and bone isoenzymes focused into so many bands over a narrow pH range such that the information could not be quantified. However, when the bone isoenzyme in serum was first desialylated at 37 degrees C for a minimum of 6 h, catalyzed by neuraminidase (EC 3.2.1.18) at pH 5.8-6.0, we could detect four distinct bands with pls of 6.7, 6.8, 6.9, and 7.0. Under the same conditions, the liver isoenzyme in human serum focused into one band at pH 7.0. The multiple banding we observed for the desialylated bone isoenzyme has not been previously reported. The method is suited as a qualitative technique for detecting the bone alkaline phosphatase isoenzyme in serum.
Generation 04 sodium slides and generation 04 reference fluid evaluated for use with the Ektachem 400 Analyzer.
Authors: Jack R M , Noble W
Published: January 2020

Evaluation of a new generation of sodium slides and reference fluid for use with the Kodak Ektachem 400 Analyzer demonstrated improved performance in generation 04 over the previous generation 03 slide for sodium determinations. A decrease in between-replicate imprecision and a correction of the "serum" assigned values of the calibrators used to calibrate the slide used with the reference fluid account for the improvement. When generation 04/Ektachem results (y) were compared with results obtained with a Beckman ion-selective electrode (x), linear regression statistics were: slope = 0.88, y-intercept = 15.4 mmol/L, and Syx = 2.1 mmol/L. The average fixed bias in comparison with the Beckman electrode decreased from +1.9 mmol/L with generation 03 to -0.6 mmol/L with generation 04 materials. Random error is indicated by a decrease in Syx from 2.8 mmol/L for generation 03 to 2.1 mmol/L for generation 04. Application of Deming's regression, a more accurate portrayal of the generation 04 sodium data set, produces a slope of 1.0 and a y-intercept of -0.6 mmol/L.
Preparation and stability of low-cost liquid quality-control serum stabilized with ethanediol.
Authors: Premachandra P , Wood P L , Hill P G , Browning D M , Olazabal D A
Published: January 2020

The use of quality-control serum to monitor laboratory performance is an essential part of any health laboratory. However, laboratories in many countries are disadvantaged because of the non-availability or high cost of commercial quality-control sera. Here we describe the stability of bovine serum stabilized with ethanediol, prepared at low cost by simple techniques that are available in most laboratories.
Initial assessment of tumor-associated antigen CA-125 in patients with ovarian, cervical, and testicular tumors.
Authors: Buamah P K , Cornell C , Skillen A W , Cantwell B M , Harris A L
Published: January 2020

Serum CA-125, a glycoprotein antigen, has been measured in serum of patients with ovarian cancer, cervical cancer, or nonseminomatous germ-cell tumor of testis. Increased concentrations were found in 21 of 27 patients with epithelial ovarian cancer and two of three patients with ovarian teratoma. Changes in CA-125 concentrations in serum during chemotherapy mirrored the progress of the disease as assessed by clinical and radiological evidence. Although CA-125 provides no real asset for diagnosis, it should have value as a marker for monitoring response to chemotherapy.
Effect of albumin and lamellar bodies on fluorescence polarization of amniotic fluid.
Authors: Ivie W M , Swanson J R
Published: January 2020

Using a probe and procedure developed for the Abbott TDx analyzer, we investigated the influence of albumin on fluorescence polarization of amniotic fluid. Polarization readings increased when albumin was added to either "mature" or "immature" amniotic fluids. Albumin added to a fraction rich in lamellar bodies (10,000 X g pellet) greatly increased polarization readings, but albumin added to the lamellar-body-free fraction resulted in only small increases. Adding increasing amounts of the lamellar-body-rich fraction from mature and immature pools to a constant amount of albumin decreased the polarization readings. We saw no correlation between amniotic fluid polarization and albumin concentration but found a significant correlation between polarization values and either total extractable phospholipids (r = 0.725, P less than 0.001) or the ratio albumin/total extractable phospholipids (r = 0.784, P less than 0.001). The concentration of albumin in amniotic fluid was variable and correlated poorly with gestational age.
Absorbance errors from using pieced-together rotors in the Roche Cobas-Bio centrifugal analyzer.
Authors: Miller W G , Armitage E K
Published: January 2020

Two Cobas-Bio analyzers were found to have significantly increased imprecision, owing to absorbance errors, when we used rotors pieced together from segments of four to 10 cuvets. The difference in CV between whole/unbroken and four-cuvet or 10-cuvet segment rotors was significant by Student's unpaired t-test (P = 0.038 and less than 0.0001, respectively) and by the Mann-Whitney U test (P less than 0.0001 for either). Pieced-together rotors were found to have plastic fragments in some cuvets, both adjacent to the break points and in other positions within the segment. There was no significant difference in delta A between adjacent cuvets at break positions or in between break positions by unpaired t-test (P = 0.214) or by the Mann-Whitney U test (P = 0.235). There was a significant difference by either test (P less than 0.0001) for the delta A between adjacent cuvets in broken rotors and those in whole/unbroken rotors. We conclude that random errors in absorbance are observed both in cuvets adjacent to break positions and in cuvets removed from the break point when pieced-together rotors are used.
Cefoxitin interferes with the "Clini-Skreen" column method for urinary 17-hydroxycorticosteroids.
Authors: Kroll M H , Jackson A J , Elin R J
Published: January 2020

Cefoxitin interferes with determination of urinary 17-hydroxycorticosteroids. The apparent concentration of hormone is increased from three- to 10-fold in samples from patients receiving cefoxitin when the Amberlite XAD-2 "Clini-Skreen" column is used. To determine the mechanism of interference, we reacted aqueous solutions of cefoxitin, cortisol, cortisone, and 11-deoxycortisol with phenylhydrazine; recorded the adsorption spectra; and determined the molar absorptivities and the equilibrium and rate constants. Also, we recorded the absorption spectra of phenylhydrazine with eight other cepha antibiotics and benzylpenicillin. Cortisol, cortisone, 11-deoxycortisol, and cefoxitin react with phenylhydrazine and absorb light with superimposable spectra and absorption maxima of 410 nm. The other antibiotics react with phenylhydrazine but absorbance maxima of the products vary, none being at 410 nm. Cortisol, cortisone, and 11-deoxycortisol react with phenylhydrazine 35-fold faster, have equilibrium constants ninefold greater, and have molar absorptivities 1.6 times that of cefoxitin. Thus, cefoxitin interferes with determination of urinary 17-hydroxycorticosteroids by forming a chromophore with the same absorbance maximum and with a molar absorptivity similar to cortisol, but much more slowly.
Automated determination of angiotensin-converting enzyme in serum.
Authors: Peters R H , Golbach A J , van den Bergh F A
Published: January 2020

This is an adaptation of the Fujirebio "ACEcolor" kit for automated measurement of angiotensin-converting enzyme (EC 3.4.15.1) in serum with the Cobas Fara centrifugal analyzer. The linear range extends to an activity of 110 U/L. Results obtained by the present method and by the manual method were identical, and correlated closely (r = 0.983) with those by Cushman's modified method. The reference interval for 77 adult blood-bank donors was 9-25 U/L (mean 17, SD 4 U/L). Within-run and between-run CVs are 1.7 and 4.0%, respectively. The present method permits rapid, precise, and economical measurement of the enzyme and allows users of a Cobas Fara centrifugal analyzer to introduce a fully automated assay for angiotensin-converting enzyme into their clinical laboratory.
Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma.
Authors: Peters T
Published: January 2020

An attempt is made to review the steps in the biosynthesis of plasma proteins by the liver and to point out intermediate precursor forms that might escape to the plasma. With the use of immunochemical techniques such as immunofixation electrophoresis it is possible to identify these precursors when they present an unusual bands on protein electrophoresis. The discovery of the role of vitamin K in the formation of the blood-clotting proteins, for example, followed the immunochemical detection of an abnormal form of inactive prothrombin in plasma. The chief types of precursors likely to occur in blood are forms that are incompletely glycosylated, phosphorylated, or sulfated, or which are single peptide chains of multichain proteins. Precursors containing basic propeptides such as proinsulin or proalbumin may also appear. By being alert to the possible appearance of intracellular forms in plasma, the clinical chemist may be able to relate their presence to the nature of the disease process responsible.
Nonisotopic M13 probes for detecting the beta-globin gene: application to diagnosis of sickle cell anemia.
Authors: Sheldon E , Kellogg D E , Levenson C , Bloch W , Aldwin L , Birch D , Goodson R , Sheridan P , Horn G , Watson R
Published: January 2020

M13 DNA probes labeled with biotinylated psoralen and a streptavidin-horseradish peroxidase conjugate provide nonradioactive detection of the sickle cell and normal alleles of the beta-globin locus. The two biotinylated probes contain single-stranded sequences complementary to two different Sau3AI restriction fragments from the 5' region of the beta-globin gene and double-stranded M13 vector sequences. These probes are labeled with biotinylated psoralen photochemically linked to DNA. After hybridization, the presence of biotinylated probe bound to target DNA is detected in 3 h by using a streptavidin-horseradish peroxidase conjugate and the substrate, 3,3',5,5'-tetramethylbenzidine. Digestion of the normal (beta A) allele of the beta-globin gene with MstII (or isoschizomers) yields a 1.14-kb restriction fragment, while digestion of the mutant beta S allele yields a 1.34-kb fragment. These fragments can be resolved by gel electrophoresis and detected by Southern blot hybridization. The nonradioisotopic probe system can detect the beta-globin restriction fragment in as little as 0.5 microgram of human DNA and can distinguish heterozygotes (beta A beta S) from homozygotes (beta A beta A or beta S beta S) in 2.0 micrograms of human DNA.
Measurement of intact human parathyrin by an extracting two-site immunoradiometric assay.
Authors: Blind E , Schmidt-Gayk H , Armbruster F P , Stadler A
Published: January 2020

This is an immunoradiometric assay of intact human parathyrin, hPTH(1-84). One antibody, directed against the N-terminal part of the hormone, was produced in goats and conjugated covalently to cellulose particles. hPTH(1-84) and the N-terminal fragments were extracted from EDTA-treated plasma by these particles and thus concentrated. Another antibody, against synthetic hPTH(53-84), was raised in rabbits; this bound to the C-terminal part of the hormone. The final step was labeling the second free binding site of this antibody with 125I-labeled Tyr52-hPTH(53-84) and measuring the bound radioactivity. This assay can detect intact PTH in concentrations as low as 0.6 pmol/L (1.2 X 10(-16) mol per tube). The assay did not cross react with hPTH(1-34), hPTH(1-44), hPTH(28-48), hPTH(39-84), hPTH(44-68), or hPTH(53-84) in concentrations up to 6400 pmol/L. In 60 normal subjects, hPTH(1-84) concentrations ranged from 1.9 to 6.8 pmol/L; in 32 patients with primary hyperparathyroidism, from 7.0 to 80 pmol/L. The hormone was not detected in four patients with hypoparathyroidism.
"Delfia" and "Amerlite": two sensitive nonisotopic immunoassay systems for assay of thyrotropin compared.
Authors: Parnham A J , Tarbit I F
Published: January 2020

We assessed the LKB "Delfia" (time-resolved dissociation-enhanced lanthanide fluoroimmunoassay) and the Amersham "Amerlite" (enhanced luminescent immunometry) assays of thyrotropin in serum. Both assays are sensitive (respective detection limits: 0.02 and 0.04 milli-int. unit/L) and have very good within- and between-batch precision over a wide range of thyrotropin concentrations. Results by the two methods correlate well (r = 0.992); the regression equation is: Amerlite = 0.915 Delfia - 0.33 milli-int. unit/L. The standard curve for the Delfia assay was linear, but that for the Amerlite assay showed some deviation from linearity below 0.5 milli-int. unit/L. Both assays have a negative bias in comparison with radiolabeled immunoradiometric assays, as judged by results for samples from the Quality Assurance Scheme. Both assays discriminate well between hyper-, hypo-, and euthyroid subjects, and results for thyrotropin for most patients with nonthyroidal illness were within the euthyroid reference interval. Both assays are convenient to perform and are based on systems that provide a viable alternative to radioimmunoassay.
Rapid typing of serum paraproteins by immunoblotting without antigen-excess artifacts.
Authors: Norden A G , Fulcher L M , Heys A D
Published: January 2020

In this new immunoblotting procedure for determining the heavy-chain class and light-chain type of monoclonal serum immunoglobulins, proteins are transferred from agarose electrophoretic gels to nitrocellulose by brief capillary blotting. Paraproteins transferred are detected with appropriate horseradish peroxidase-conjugated antisera to light chain and heavy chain. Examination of 121 serum specimens probably containing a paraprotein (as detected by protein staining) by immunoblotting and by immunofixation gave the same results for 116 specimens: paraproteins were typed in 103 specimens and their presence was excluded in 13. Immunoblotting required one repeat analysis (as compared with 22 for immunofixation). In only 100 min we could type as many as 10 paraproteins, and the procedure did not show antigen-excess artifacts. These results suggest that immunoblotting may be preferable to immunofixation for routine typing of paraproteins.
Deciding the optimum interval between specimen collections: theory and nomograms.
Authors: Fraser C G , Browning M C
Published: January 2020

The time interval between collection of specimens from an individual patient is usually determined empirically. For analytes whose values decline according to first-order kinetics--for example, enzyme activities in serum after an acute myocardial infarction, tumor markers in serum after excision of the tumor, and drugs in serum after an overdose--the minimum time between collections (delta T) depends on the elimination half-life (t) and the analytical precision (CVA), according to the equation: delta T = (1/log2) X t X log (2.33 CVA/100 + 1). Nomograms showing this relationship graphically have been generated.
Analytical systems ranked by freedom from interferences.
Authors: Glick M R , Ryder K W
Published: January 2020

We determined the effect of hemolysis, lipemia, and bilirubinemia on clinical-chemical analytical results under standardized conditions, for serum specimens prepared by us. Our purpose was to assess results obtained with 22 commonly available analytical systems. The quantitative rating scheme described is derived from the observed interference(s) divided by the number of analytical methods evaluated. The combined ranking reveals which chemistry analyzers are least affected and which are most affected by the added substances. Generally, systems that incorporate physical barriers or protein-separation steps perform better than those without thin-film layers, glass-fiber barriers, or dialysis membranes. Among the "direct" analyzers, fewer interferences are seen if appropriate "blanking" wavelengths are used, especially if a "specimen blank" absorbance is used appropriately in the analytical system. Centrifugal analyzers tended to perform poorly, according to the criteria presented here.
Cholinesterase assay automated in the Cobas-Bio centrifugal analyzer.
Authors: Ratnaike S , Gray F , Deam D
Published: January 2020

We describe a method for measuring plasma cholinesterase (EC 3.1.1.8) and dibucaine and fluoride numbers by using the Cobas-Bio. Benzoyl choline chloride is used as substrate. Reaction conditions are the same as in the corresponding manual method. The reaction is stopped with physostigmine. Choline oxidase (EC 1.1.3.17) is coupled with 4-aminoantipyrene. In the presence of peroxidase (EC 1.11.1.7), the 2-hydroxy-3,5-dichlorobenzenesulfonate indicator reaction gives a red product, measured at 505 nm. The analyzer is used in the "Multi Run" mode, with plasma cholinesterase and dibucaine and fluoride inhibition concurrently measured for eight samples. Coefficients of correlation between the manual and present method for plasma cholinesterase and dibucaine and fluoride numbers in 40 patients of various phenotypes were 0.843, 0.923, and 0.717, respectively. The inter-batch CV for each of the three assays was 2% to 3%.
Automated quantification of thyrotropin by radial partition immunoassay.
Authors: Rugg J A , Flaa C W , Dawson S R , Rigl C T , Leung K S , Evans S A
Published: January 2020

We describe a radial partition enzyme immunoassay in which fully automated quantification of human thyrotropin (hTSH) takes less than 11 min. This "sandwich"-type assay involves two monoclonal antibodies, both specific for the intact hTSH molecule. The solid phase consists of tabs of glass-fiber filter paper containing a pre-immobilized monoclonal anti-hTSH antibody complexed with a goat antibody specific for the Fc region of mouse IgG. The patient's sample is first applied to the central "reaction zone" of the tab, wherein hTSH binds to the immobilized antibody. Application of a buffered solution containing enzyme-labeled Fab' fragments of the second monoclonal anti-hTSH antibody initiates "sandwich" formation. A wash buffer containing a fluorogenic substrate elutes unbound conjugate to the tab periphery. The bound enzyme conjugate is quantified by measuring the rate of increase in fluorescence in the reaction zone of the tab, then converting the rate to clinical units by comparison with a stored calibration curve. The clinical utility and performance of the present assay compare favorably with those of other sensitive assays for hTSH.
Bias between enzymatic methods and the reference method for cholesterol.
Authors: Kroll M H , Lindsey H , Greene J , Sliva C , Hainline A , Elin R J
Published: January 2020

Assaying 312 serum samples, we compared four enzymatic methods for serum cholesterol with the Reference Method (modified Abell-Kendall) of the Centers for Disease Control (CDC). The means for the aca, TDx, and SMAC methods (2.27, 2.27, and 2.24 g/L, respectively) were significantly higher (P less than 0.05) than those of the Reference Method and the RA-1000 method (2.19 and 2.18 g/L, respectively). The biased methods had positive proportional and (or) systematic biases. Results with these methods were 2.6% to 4.9% higher than with the Reference Method. The assigned concentrations of cholesterol in the calibration materials for the SMAC and aca agreed with those obtained by the Reference Method, but were lower for the TDx and higher for the RA-1000. These findings document positive biases for cholesterol with three enzymatic methods and suggest that misassignment of calibrators is not primarily responsible for the biases found with the aca and SMAC. It may, however, to be a significant factor for the TDx.
Variations in the composition of low- and high-density lipoproteins during the acute phase of myocardial infarction.
Authors: Mainard F , Madec Y , Robinet N
Published: January 2020

We analyzed correlations between apolipoprotein B (apo B), cholesterol and phospholipids (preponderant lipids) in low-density lipoproteins (LDL) as well as between apolipoprotein A1 (apo A1) and these same lipids in high-density lipoproteins (HDL), during the acute phase of myocardial infarction. In LDL, a very elevated and stable correlation (r) was observed between these parameters, and the coefficients of regression (b) did not differ significantly during the period studied. In HDL, there was a decrease in r and b values from day 1 to day 2, then an increase after day 2. We hypothesize that these disturbances in HDL composition may be due to a greater endocytosis of LDL at day 2, leading to intracellular increase in cholesterol and phospholipids. Part of these lipids could be taken up by HDL molecules, causing a transient overload.
Four assay methods for glycated hemoglobin compared as screening tests for diabetes mellitus: the Islington Diabetes Survey.
Authors: Forrest R D , Jackson C A , Gould B J , Casburn-Budd M , Taylor J E , Yudkin J S
Published: January 2020

We assessed the utility of four methods of glycated hemoglobin assay (agar gel electrophoresis with Schiff base, agar gel electrophoresis with prior removal of Schiff base, boronate affinity chromatography, and isoelectric focusing) as screening tests for diabetes mellitus, studying 223 subjects undergoing an oral glucose-tolerance test after fasting and 2 h after ingestion of 75 g of glucose. Assessment of glucose tolerance status according to the 1980 World Health Organization diagnostic criteria indicated that 13 subjects (5.8%) had diabetes, 48 (21.5%) had impaired glucose tolerance, and nine (4.0%) could not be classified (six of these because of missing values). Use of receiver-operating characteristic curves to compare the assays as screening tests showed the affinity chromatography assay to be superior. Assays for glycated hemoglobin after fasting had better precision than post-glucose-load assays as screening tests, and test characteristics of glycated hemoglobin assays on fasting subjects were similar to those of the blood-glucose estimation after fasting. We conclude that measurement of glycated hemoglobin may be a useful screening test for diabetes, and that such measurement of stable glycated hemoglobin is as accurate as measurement of fasting blood-glucose in screening for diabetes. For this use, we found methods that measure stable glycated hemoglobin superior to that measuring both stable and labile Schiff-base fractions.
Galactosemia detection from phenylketonuria screening.
Authors: Henderson M J , Shapiro L , McCowan C
Published: January 2020

We describe a case of classical galactosemia in which the diagnosis was first suggested by the finding of a moderately increased blood-spot phenylalanine concentration. The child was clinically unaffected at six days when the initial sample was collected. Prompt institution of dietary management averted a serious metabolic crisis.
Gas-chromatographic measurement of carboxyhemoglobin in blood from mothers and newborns.
Authors: Fogh-Andersen N , Eriksen P S , Grinsted J , Siggaard-Andersen O
Published: January 2020

HbCO in blood sampled from 20 mothers and newborns immediately after birth was measured with a new, simple gas-chromatographic method for CO. The mean ratio of HbCO to total hemoglobin for 13 non-smoking mothers did not differ significantly from that for their infants (mean 0.38%, SD 0.26% vs 0.38%, SD 0.13%), but the HbCO ratio varied more in the mothers than in the infants (P less than 0.05). The infants of seven cigarette-smoking mothers, tobacco-abstinent for 7 h during labor, had higher HbCO ratios than their mothers (mean 1.88% vs 1.28%, P less than 0.05). The results are in harmony with the concept of equal affinities of fetal and adult hemoglobin for CO and a long time for passage of CO across the placenta.
Liquid-chromatographic measurement of cyclosporin A and its metabolites in blood, bile, and urine.
Authors: Christians U , Zimmer K O , Wonigeit K , Maurer G , Sewing K F
Published: January 2020

Using solid-phase extraction columns and "high-performance" liquid-chromatographic (HPLC) analysis, we could determine cyclosporin A and nine of its metabolites in blood, bile, and urine. To facilitate calculations of concentrations of cyclosporin A and its metabolites from the chromatograms, we used cyclosporin D as internal standard. For the HPLC analysis we used two sequential 250-mm analytical columns filled with reversed-phase octyl (C8) sorbent, eluting with a concave gradient of water, adjusted to pH 3.0 with phosphoric acid, and acetonitrile. Peaks were detected at 205 nm. For characterization of the chromatographic peaks, we isolated, by semi-preparative HPLC, 32 fractions representing peaks potentially related to cyclosporin A metabolites and re-injected them into the HPLC system under the same conditions as authentic cyclosporin A metabolites. Analytical recovery was 70-80%. The inter-assay CV for bile was 7.2%, for urine 12.3%. The method was used for routine monitoring of cyclosporin A and its metabolites.
Direct spectrophotometry of bilirubin in serum of the newborn, with use of caffeine reagent.
Authors: Vink K L , Schuurman W , van Gansewinkel R
Published: January 2020

We recently described a direct spectrophotometric method for unconjugated bilirubin, with caffeine reagent (Clin Chem 1986;32:1389-93). Because this method is independent of the protein matrix we used it for the preparation of bilirubin standards (Clin Chem 1987;33:1817-21). Now, in this paper, we utilize the caffeine reagent in setting up a bilirubin method for serum from neonates. This resulted in a two-wavelength (465 and 528 nm) equation, which fully corrects for HbO2 interferences. In combination with a bilirubin standard, this equation may be transformed into a simple relative formula for use with this simple dilution method. We studied this two-wavelength method with 55 neonates' sera, comparing results with those by both the diazo method of Doumas et al. (Clin Chem 1985;31:1779-89) and the borate method of Hertz et al. (Scand J Clin Lab Invest 1974;33:215-30). We found that this new method is independent of hemolysis and of the matrix of the sera. Therefore, it is very suitable for use in neonatology.
Highly specific enzyme immunoassay for the beta-subunit of human thyrotropin with use of monoclonal antibodies.
Authors: Iijima Y , Endo Y , Hata N , Fujita H , Unoki M , Miyal K
Published: January 2020

A highly specific enzyme-linked "sandwich" immunoassay is described for determining free human thyrotropin (hTSH) beta-subunit in serum by using a anti-hTSH beta-subunit monoclonal antibody conjugated with beta-D-galactosidase (EC 3.2.1.23) and a solid phase consisting of silicone rods coated with another monoclonal antibody. We could detect as little as 0.04 ng of beta-subunit per assay. The measurable range of hTSH beta-subunit concentrations in serum was 0.4 to 50 micrograms/L. The assay demonstrated little or no cross reactivity with intact hTSH, hTSH alpha-subunit, or human choriogonadotropin. The mean CVs were 12.2% within assay, 13.9% between assay. The hTSH beta-subunit was not detectable in sera from healthy subjects, patients with hyperthyroidism, or two patients with pituitary tumors producing TSH. It was measurable (at concentrations of 0.65 to 2.70 micrograms/L) in sera from eight of 23 hypothyroid patients. In five of the hypothyroid patients examined, the concentration of hTSH beta-subunit in serum increased after administration of thyroliberin. This method may be useful in elucidating the physiological and pathological significance of the hTSH beta-subunit and in examining the function of the hypothalamus-pituitary-thyroid axis.