bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-site immunofluorometric assay for pregnancy-specific beta 1-glycoprotein (SP1).
Authors: Sykes A , Chard T
Published: January 2020

We describe an immunoassay for human pregnancy-specific beta 1-glycoprotein by use of fluorescein-labeled antibodies to this protein. The technique is generally similar to that of a "two-site" immunoradiometric assay but has the advantage of stability of tracer and avoidance of radiation hazard. However, the procedure requires relatively large quantities of antibody. Furthermore, the within-assay coefficient of variation (11.8%) is greater than that for radioimmunoassay (6.5%). This results from imprecision of the end-point detection, and some inherent difficulties in optimizing a "two-site" immunometric system. Concentrations of endogenous material as measured by the fluorometric assay are 1.9-fold higher than those obtained with radioimmunoassay, probably because the former detects multiple forms of this heterogeneous material.
Separation of serum prednisolone and prednisolone-21-hemisuccinate by extraction and their concurrent determination by radioimmunoassay.
Authors: Yanagibashi K , Mizuchi A , Yotsumoto H , Miyachi Y
Published: January 2020

We describe a simple, sensitive, and reliable radioimmunoassay for prednisolone and prednisolone-21-hemisuccinate in serum. The antiserum produced in rabbits to prednisolone-21-hemisuccinate/bovine serum albumin was specific for prednisolone and prednisolone-21-hemisuccinate. A simple dichloromethane extraction permitted the separation of prednisolone from prednisolone-21-hemisuccinate in the serum samples. Interference by cortisol, although not insignificant, is minimized in this assay. We used the method to measure prednisolone and prednisolone-21-hemisuccinate concentrations after a bolus injection of prednisolone-21-hemisuccinate into human beings and mice.
Measuring percent oxygen saturation of hemoglobin, percent carboxyhemoglobin and methemoglobin, and concentrations of total hemoglobin and oxygen in blood of man, dog, and baboon.
Authors: Dennis R C , Valeri C R
Published: January 2020

We used an automated four-wavelength spectrometer to measure the concentration of total hemoglobin, percent oxyhemoglobin, carboxyhemoglobin, and methemoglobin, and concentration of oxygen bound to hemoglobin in the blood of humans, dogs, and baboons under clinical and various experimental conditions. Measurements of total hemoglobin and methemoglobin with this simple method were comparable to those with standard spectrometric procedures. Carboxyhemoglobin measurements were comparable to those made with gas chromatography, and measurements of oxygen content were comparable to those made with the galvanic cell method. The new instrument is as accurate as the comparison methods used to evaluate it in all parameters, is reliable, and measurements take only 63 s per sample. In addition, it requires minimal operator training, infrequent need for calibration, and no sample preparation.
Nitrogen estimation in biological samples by use of chemiluminescence.
Authors: Ward M W , Owens C W , Rennie M J
Published: January 2020

We describe an apparatus modified for the chemiluminescent estimation of nitrogen in biological and clinical samples. Analytical characteristics have been assessed and results compared with those by the traditional Kjeldahl method. The chemiluminescence method, much faster and more sensitive than the traditional method, is at least as accurate, precise, and reproducible. Costs are low, and the method should find a place in laboratories needing, for example, rapid assessments of nitrogen balance in surgical patients and renal function in patients with renal failure, or estimations of small amounts of specific nitrogen-containing chemical substances in biological samples.
(University of Pennsylvania Case Conference): oligoclonal immunoglobulins in cerebrospinal fluid in multiple sclerosis.
Authors: Keshgegian A A , Coblentz J , Lisak R P
Published: January 2020

In the normal state, immunoglobulins are polyclonal. They migrate as a broad band on electrophoresis, reflecting the diversity of immunoglobulin species secreted by the many clones of immunoglobulin-producing cells. In lymphoproliferative disorders, monoclonal immunoglobulins are often found, migrating as electrophoretically homogeneous bands, reflecting the large amount of a single immunoglobulin being secreted by the single proliferating neoplastic clone. In certain neurological diseases, such as multiple sclerosis, the cerebrospinal fluid (CSF)2 often exhibits an oligoclonal immunoglobulin pattern consisting of several electrophoretically homogeneous bands in the gamma-globulin regin (Figure 1). We present two cases of neurological disease with an oligoclonal immunoglobulin pattern in the CSF electropherogram. In the discussion we consider the definition, etiology, and pathogenesis of multiple sclerosis, and laboratory tests that are useful in multiple sclerosis, emphasizing methods for the demonstration and interpretation of an oligoclonal immunoglobulin pattern.
Improved liquid-chromatographic determination of caffeine in plasma.
Authors: Blanchard J , Mohammadi J D , Conrad K A
Published: January 2020

We describe a rapid, specific, and sensitive liquid-chromatographic micro-method for caffeine in plasma. Each plasma sample can be assayed within about 15 min of its receipt. Samples are denatured with acetonitrile, centrifuged, and the supernate is chromatographed on a reversed-phase column. Only 100 microL of plasma is required, and concentrations as low as 0.3 mg/L can be measured accurately. Other xanthines and their metabolites do not interfere. The small sample required makes the procedure ideally suited for measuring caffeine in the plasma of infants and small animals as well as adults.
Evaluation of quality-control materials for high-density-lipoprotein cholesterol quantitation.
Authors: Warnick G R , Mayfield C , Albers J J
Published: January 2020

Commercial quality-control materials and frozen plasma and serum pools were evaluated for high-density lipoprotein (HDL) cholesterol quantitation by the heparin-Mn2+ and phosphotungstate-Mg2+ precipitation methods and by ultracentrifugal and electrophoretic methods. We evaluated among-day precision; inter-method differences in HDL cholesterol quantitation; lipid composition and electrophoretic mobility of th lipoprotein density fractions; and the effects of modifications in reagent concentration and temperature. Frozen plasma pools (in-house) and frozen serum pools (prepared by the Center for Disease Control) were determined to be suitable HDL controls. Also, a commercial lyophilized serum free of very-low-density lipoprotein (VLDL) to which VLDL is added in a diluent solution (Analytical Control) also appeared suitable as an HDL control material. Conventional lyophilized pools (Lyphochek Elevated, Monitrol I, Ortho Normal, Precilip) were less suitable as control materials because of poor between-day precision for one or both of the precipitation methods and, in some cases, because of large absolute differences observed between the HDL methods. A serum pool stabilized with ethylene glycol (Beckman Level I) was the least as appropriate as an HDL control material. Other lyophilized pools--Lipid Fraction Control (Hyland) prepared by a modified lyophilization process and Lipidophor (Immuno Diagnostika) prepared by lyophilization with a stabilizing agent--appear promising.
Relation between maternal weight and serum alpha-fetoprotein concentration during the second trimester.
Authors: Haddow J E , Kloza E M , Knight G J , Smith D E
Published: January 2020

Maternal serum alpha-fetoprotein concentrations are influenced by maternal weight during the second trimester. Heavier pregnant women have lower median values, apparently as a result of a diluting effect of larger blood volume. This phenomenon is of clinical interest because alpha-fetoprotein concentration in a pregnant woman's serum is one of the factors considered in assessing risk of poor outcome. A revision of the reference interval for alpha-fetoprotein to take body weight into account might improve its use as a diagnostic aid, especially in heavier women.
Liquid-chromatographic quantification of plasma phenylalanine, tyrosine, and tryptophan.
Authors: Neckers L M , Delisi L E , Wyatt R J
Published: January 2020

Phenylalanine, tyrosine, and tryptophan are isolated and quantified by "high-pressure" liquid chromatography, with fluorescence detection. An isocratic mobile phase and reversed-phase column are used to provide rapid and reproducible measurement of these amino acids in as little as 1 to 2 microL of human plasma.
Measurement of near-normal concentrations of erythrocyte protoporphyrin with the hematofluorometer: influence of plasma on "front-surface illumination" assay.
Authors: Schifman R B , Finley P R
Published: January 2020

Zinc protoporphyrin in erythrocytes increases in iron depletion. Because the hematofluorometer directly measures zinc protoporphyrin in whole blood, it may therefore be a useful screening instrument for detecting iron deficiency. We evaluated its performance with normal to slightly above-normal zinc protoporphyrin concentrations (0.2 to 2.0 mg/L) in erythrocytes, because this is a critical range for differentiating normal and iron-deficient individuals. There was excellent correlation (r2 = 0.900) between erythrocyte protoporphyrin as measured by an extraction procedure and as measured directly with the hematofluorometer. However, at these concentrations, plasma caused hematofluorometer readings to be spuriously high, by 2.4 to 89.4%, an effect due to the instrument's optical design. The effect can be eliminated by washing the cells free of plasma or by allowing erythrocytes to displace plasma by settling to the illuminated surface before the measurement. The interference does not affect the hematofluorometer's sensitivity, but abnormal results obtained for whole blood should be confirmed with more-specific tests, and interlaboratory precision may be influenced if whole-blood samples are used. A 1-min delay from sample placement to measurement decreased zinc protoporphyrin values by 2.6 to 36.9%. We also describe the use of cryopreserved control erythrocytes, for which the CV is 2.3% for a concentration of 0.96 mg per liter of erythrocytes, which are not affected by time of measurement, and which are stable for three months.
Evaluation of the IFCC-recommended procedure for serum aspartate aminotransferase as modified for use with the centrifugal analyzer.
Authors: Bruns D E , Savory J , Titheradge A C , Cross R E , Wills M R
Published: January 2020

The IFCC-recommended procedure for aspartate aminotransferase (EC 2.6.1.1) was adapted to the centrifugal analyzer and evaluated during five years. The main hindrance to widespread use of the recommended method is the need for pre-incubation with pyridoxal phosphate. The present method includes a 10-min pre-incubation with pyridoxal phosphate. Extensive evaluation of the method with and without this pre-incubation confirms earlier reports that it eliminates some significant errors, especially for samples from patients with cardiac disease. However, the pre-incubation can be briefer than the recommended 10 min and still provide acceptable results.
Extended clinical trial and evaluation of glucose determination with the Eastman Kodak Ektachem GLU/BUN Analyzer.
Authors: Bandi Z L , Fuller J B , Bee D E , James G P
Published: January 2020

We followed the "abbreviated precision protocol" of the National Committee for Clinical Laboratory Standards for the evaluation of precision, accuracy, and carryover in analyses for glucose with the "Ektachem." We analyzed 760 clinical samples by this technique, by the FDA Proposed Class Standard glucose reference method, and with the Beckman System I GLU/BUN Analyzer. Precision and accuracy were estimated for 500, 1000, 1200, 1500, and 3000 mg/L glucose concentrations in 100, 30, or 20 microL of serum or plasma. Potential interference of 19 compounds was evaluated. Random error (1.965 X SD) was 22, 30, 34, 40, and 88 mg/L. Systematic error was 8, 1.5, -2, -5, and -27 mg/L. Total analytical error was 30, 32, 36, 46, and 110 mg/L for analysis of 100 microL of serum at the above-stated glucose concentrations. The greatest interference (-39 mg/L) in the glucose (at 1200 mg/L) determination was caused by L-ascorbate (40 mg/L). Glucose concentrations as determined with the Ektachem were found to be linearly related to the expected concentration up to at least 5660 mg/L. Carryover was not statistically significant.
Urinary metanephrine radioimmunoassay: comparison with the colorimetric assay.
Authors: Raum W J , Swerdloff R S
Published: January 2020

A radioimmunoassay involving an 125I ligand has been developed and applied to the measurement of urinary metanephrine. To validate the clinical usefulness of this assay, we compared measurement of metanephrine by radioimmunoassay and of total urinary metanephrines by the Pisano colorimetric method. The radioimmunoassay is specific for metanephrine, whereas the colorimetric method measures both metanephrine and normetanephrine. We used both methods to determine urinary metanephrine or total metanephrines in subjects with essential hypertension, pheochromocytoma, the syndrome of multiple endocrine adenomatosis type 2, and normotensive volunteers. The mean and upper limit of normal (3 SD) for metanephrine by radioimmunoassay in our normotensive volunteers was 94.2 microgram/24 h and 229 microgram/24 h, respectively, which compares well with reported values of 87.6 microgram/24 h and 319 microgram/24 h by non-radioimmunoassay methods. Both radioimmunoassay and colorimetry accurately identified five patients with known pheochromocytoma. Good correlation (r = 0.993) was demonstrated between the two assays in a comparison of patients with essential hypertension and pheochromocytoma. We conclude that the radioimmunoassay is at least equivalent to the colorimetric methods in distinguishing pathological and normal catecholamine secretion, and is faster, more precise, and 1000-fold more sensitive.
A clinical evaluation of various delta check methods.
Authors: Wheeler L A , Sheiner L B
Published: January 2020

To evaluate the performance of delta check techniques, we analyzed 707 unselected pairs of continuous-flow test results, using three different delta check methods. If any of the test results (plus the urea nitrogen/creatinine ratio and the anion gap) failed one of the checks, the reason for the failure was sought by examining subsequent test results, retesting specimens, and (or) reviewing te patient's chart. Each delta check failure was accordingly classified as a true or false positive. The percentage of positives we judged to be true positives ranged from 5 to 29%. Each of the three methods had test types with low and high percentages of true positives. We conclude that with the delta check methods one can detect errors otherwise overlooked, but at the cost of investigating many false positives, because, in the population we studied, disease processes or therapy often caused large changes in a series of test results for a patient.
Day-to-day variation in serum ferritin concentration in healthy subjects.
Authors: Pilon V A , Howanitz P J , Howanitz J H , Domres N
Published: January 2020

We report our observations on day-to-day variation in serum ferritin, serum iron, total iron-binding capacity, and percent saturation of binding proteins with iron in 13 ostensibly healthy subjects during five weeks. The average intrasubject coefficients of variation were 14.5, 28.5, 4.8, and 28.0%, respectively. Precision studies on control samples showed greater within-assay and between-assay analytical variation for serum ferritin than for serum iron or total iron-binding capacity. Evidently, serum ferritin varies less in a given individual from day to day than do serum iron and percent iron saturation. Thus, a single measurement of serum ferritin may be a more reliable index of iron stores than an isolated determination of either serum iron or percent iron saturation.
Measurement of amniotic fluid surfactant.
Authors: Freer D E , Statland B E
Published: January 2020

Measurement of the production of surfactant is the most direct means of prenatally assessing fetal pulmonary maturity. We review assays which have evolved for measuring surfactant, classifying them into two general categories: biochemical quantitation and biophysical measurements. Biochemical quantitation assays include the amniotic fluid lecithin/sphingomyelin ratio and quantitation of lecithin and other surfactant phospholipids. Biophysical measurements include measurement of surface-tension-lowering ability of extracts of amniotic fluid lipid and evaluation of surface-tension-related properties such as foam stability and microviscosity. Assays of surfactant are subject to certain pre-analysis sources of variation over which the analyst has no control, such as variability in total in vivo amniotic fluid volume, incomplete in vivo mixing of surfactant with amniotic fluid, and presence of contaminating blood or meconium. We also examine other factors such as centrifugation speed and time, and storage of the amniotic specimen before analysis. These factors can dramatically affect analyses, and must be carefully controlled by the analyst. In general, both biochemical and biophysical approaches to surfactant analysis are useful diagnostically. When properly performed, both approaches give results that correlate well, both with each other and with clinical outcome. Because "mature" and "immature" values overlap, none of the assays can completely eliminate false predictions, whether of fetal maturity or fetal immaturity.
Concanavalin A binding of alpha-fetoprotein in amniotic fluid as an aid in the diagnosis of neural tube defects.
Authors: Buamah P K , Taylor P , Ward A M
Published: January 2020

Concanavalin A nonreactive alpha-fetoprotein was determined in samples of amniotic fluid from 16 abnormal pregnancies complicated by anencephaly (7), open spina bifida (6), intra-uterine death (1), anencephaly with exomphalos (1), or open spina bifida with exomphalos (1), and in amniotic fluid from 50 normal pregnancies with gestational age between 13 and 24 weeks. In all 16 cases with fetal malformations, the proportion of nonreactive alpha-fetoprotein was significantly decreased (median 5.3%) as compared with amniotic fluid from pregnancies with a normal outcome (median 39.7%). The results confirm that this measurement is useful in the diagnosis of neural tube defects, especially when the concentration of alpha-fetoprotein in amniotic fluid is normal or only slightly above normal and gestational age is uncertain.
Liquid-chromatographic determination of urinary riboflavin.
Authors: Gatautis V J , Naito H K
Published: January 2020

We describe a method for determining urinary riboflavin by "high-pressure" liquid chromatography, with fluorometric detection. An aliquot of a 24-h urine specimen is injected directly into the chromatograph and the natural fluorescence of riboflavin is measured as the compound is eluted. Interference from other fluorophores is obviated because of differing retention times. Urinary components other than riboflavin and its analogs or degradation products exhibit no significant fluorescence at the wavelengths used (450 nm excitation, 530 nm emission). Analytical recovery of added riboflavin was 96.5 (SD 1.1)%. The CV within-run was 0.7%, between-run it was 4.3%. Concentrations of riboflavin as low as 10 micrograms/L are readily detected, with a linear relation of response to concentration to at least 2000 micrograms/L.
Colorimetry of diaphorase in commercial preparations and clinical chemical reagents by use of tetrazolium salts.
Authors: Gella F J , Olivella M T , Pegueroles F , Gener J
Published: January 2020

We describe a procedure for assay of diaphorase activity in commercial purified preparations and in clinical chemical reagents by use of iodonitrotetrazolium chloride or other tetrazolium salts. The method is based on measurement of the formazan produced by enzymic reduction of tetrazolium salts in the presence of NADH. The assay procedure has been optimized for linear kinetics, simplicity of operation, nondetectable blank rates, and extended activity/enzyme concentration proportionality. The proposed method has several advantages over the older assay by use of dichlorophenolindophenol.
Glucagon radioimmunoassay with use of antiserum to glucagon C-terminal fragment.
Authors: Nishino T , Kodaira T , Shin S , Imagawa K , Shima K , Kumahara Y , Yanaihara C , Yanaihara N
Published: January 2020

In this pancreatic-glucagon-specific radioimmunoassay we used C-terminal-region-specific antiserum. OAL-123, produced against a 19-29 C-terminal fragment of porcine glucagon. On measurement of pooled plasma the ranges for intra- and inter-assay coefficients of variation were 4.8-8.1% and 7.5-10.7%, respectively. The concentration of immunoreactive glucagon in plasma of healthy subjects, as measured with the OAL-123 assay system, was 87.9 (SD 23.8) ng/L. Measurement of the same plasma samples with the 30K assay system (30K being an antiserum highly specific for pancreatic glucagon) showed a comparable value, 86.2 (SD 26.3) ng/L. We followed changes in human and dog plasma immunoreactive glucagon concentrations on arginine infusion and after glucose load, using the OAL-123 and the 30K assay systems, with identical results. Combining other results of comparative immunochemical characterization of the OAL-123 and 30K assay systems, we confirmed that the antisera raised against the C-terminal fragment of glucagon can be used in radioimmunoassay of pancreatic glucagon.
Specific 125I-radioimmunoassay for cholyglycine, a bile acid, in serum.
Authors: Miller P , Weiss S , Cornell M , Dockery J
Published: January 2020

The concentration of the conjugated bile acid, cholylglycine, in serum is a sensitive and specific indicator of hepatic function. We describe a convenient, specific, and precise radioimmunoassay for cholylglycine, in which 125I-labeled cholylglycyltyrosine is used as tracer. In addition, a blocking agent in the buffer system eliminates binding of bile acids to serum albumin. Therefore no extraction is required. We found no interference by (a) abnormal concentrations of albumin or gamma-globulin, (b) lipemic sera, (c) hemolyzed sera, (d) anticoagulants, or (e) various commonly used drugs. The reference interval for fasting subjects is estimated to be 0.0 to 0.6 mg/L. Our clinical studies show that serum cholylglycine concentrations are usually abnormal in most hepatobiliary diseases, such as viral hepatitis, alcoholic liver disease, cirrhosis, and pediatric liver diseases.
Liquid-chromatographic determination of bilirubin and its conjugates in rat serum and human amniotic fluid.
Authors: Rosenthal P , Blanckaert N , Kabra P M , Thaler M M
Published: January 2020

This newly developed and highly specific and sensitive procedure was applied to the determination of unconjugated bilirubin and its ester conjugates in rat serum and human amniotic fluid. Bilirubin conjugates in biological samples are converted to methyl esters by alkaline methanolysis, extracted into chloroform, and the unconjugated bilirubin and esterified pigment derivatives are fractionated by "high-performance" liquid chromatography. The separated pigments are measured spectrophotometrically. Bilirubin and its mono- and di-conjugates are readily quantitated, even in previously undetectable concentrations. Linearity was established from 0.07 to 121.2 mumol/L for unconjugated bilirubin, 0.07 to 34.6 mumol/L for the C-8 monoconjugate, 0.06 to 69.3 mumol/L for the C-12 monoconjugate, and 0.17 to 43.7 mumol/L for the di-conjugate fraction. The detection limit was 0.03 mumol/L for unconjugated bilirubin and for each monoconjugate, and 0.1 mumol/L for the di-conjugated pigment.
Effect of feedback to clinical staff of information on clinical biochemistry requesting patterns.
Authors: Grivell A R , Forgie H J , Fraser C G , Berry M N
Published: January 2020

Attempting to reduce the number of unnecessary tests requested, we initiated a program providing continuous feedback to clinicians concerning their requesting patterns of clinical biochemistry laboratory tests. At four-week intervals each medical specialist received data showing the number and costs of tests requested by all members of his team, the number of patients who had tests, and various related information. Comparative summaries, in frequency-histogram format, of the number of specimens submitted and the costs involved allowed each medical specialist to rate his own performance against that of his peers. The program appeared to have no effect on laboratory use, although reliable information concerning workload patterns was accrued. The degree of education of medical students in clinical biochemistry did not appear to influence the use they made of the laboratory after qualification. The specimens per bed-day ratio is suggested as a useful parameter in the identification of inappropriate laboratory use and as a standard parameter for comparing laboratory use in different hospitals.
Clinical applicability of acid phosphatase isoenzyme assay.
Authors: Sun T , Lam K W , Narukar L
Published: January 2020

We compared electrophoretic evaluation of acid phosphatase isoenzymes with spectrophotometric determination of prostatic acid phosphatase in terms of clinical utility. In all of 33 cases of prostatic carcinoma, an increased prostatic fraction was detected; in nine prostatectomized patients, this fraction returned to normal as measured by either technique. Abnormal spectrophotometric results were also seen in 10 cases of benign prostatic hypertrophy and seven cases of non-prostatic disorders, but only two benign prostatic hypertrophy and one non-prostatic case showed a prostatic band (band 2) in an electrophoretogram. Band 2 was not demonstrated in 463 patients affected by a great variety of diseases but without prostatic disorders. A weak band 5 was seen in patterns for most patients, except for cases with metastatic bone tumor and Gaucher's disease, whose serum showed a strong band 5. The specificity of bands 2 and 5 seems to be confirmed by this large series of patients. Measurement of acid phosphatase isoenzymes is recommended as a routine screening test for patients whose serum acid phosphatase is abnormally high, because the isoenzyme study not only indicates the presence or absence of prostatic cancer but also whether or not there is bony metastasis. Other disorders such as Gaucher's disease, different kinds of leukemias, and thrombocythemia may also be detected and distinguished by this screening technique.
Total surface-active phospholipid/sphingomyelin ratio as an index of fetal lung maturity.
Authors: Dohnal J C , Bowie L J
Published: January 2020

As an index of fetal lung maturity, the ratio of total surface-active phospholipid (phosphatidylethanolamine + phosphatidylinositol + phosphatidyglycerol + lecithin) to sphingomyelin performs as well as or better than any other indicator that we tested, showing 92% sensitivity and 98% specificity. The sensitivity and specificity, respectively, of the other methods were: (a) lecithin/sphingomyelin ratio, 92% and 95%; (b) alkaline phosphatase/glutamyltransferase ratio, 100% and 49%; (c) microviscosity, 75% and 97%; and (d) adsorbance at 650 nm, 50% and 73%. The procedure requires 2 h or less and its simplicity allows it to be offered on a 24 h/day basis without necessitating special training.
Method for testing antiserum titer and avidity in nephelometric systems.
Authors: Hudson G A , Ritchie R F , Haddow J E
Published: January 2020

Antiserum performance in a nephelometric system can be characterized by parameters derived from measuring reaction rates. The characterization process is derived from a series of dose-response curves (elicited nephelometric response vs antigen concentration) generated from various dilutions of the antiserum being tested. Antiserum titer can then be calculated by plotting the antigen concentration found at one-half the maximum nephelometric response (Hmax) of each dose-response curve (C50) vs the corresponding antiserum dilution. Antiserum avidity can be calculated by plotting Hmax against its corresponding antiserum concentration. After general expressions are determined for C50 and Hmax vs antiserum concentration, a single dose-response curve suffices for characterizing antisera with respect to titer and avidity. Direct evidence is provided for the validity of C50 and Hmax as measures of titer and avidity by correlating these parameters with antiserum binding strength and with the number of antibodies eluted from immobilized antigen. This method can be applied to evaluate and compare different antiserum lots having the same specificity, to identify reagent inadequacies by comparing antisera of different specificity, and to predict the optimal antiserum dilution to use in performing an assay.
Distribution of enzymes in dog heart and liver; significance for assessment of tissue damage from data on plasma enzyme activities.
Authors: Visser M P , Krill M T , Muijtjens A M , Willems G M , Hermens W T
Published: January 2020

When organ damage is assessed from activities of tissue enzymes in plasma, it is assumed that variations in tissue enzyme content, both among individuals and between different sites within an organ, are small. We checked these assumptions, using canine heart and liver. We determined creatine kinase (CK; EC 2.7.3.2), lactate dehydrogenase (LD; EC 1.1.1.27), aspartate aminotransferase (AST; EC 2.6.1.1), and glucosephosphate isomerase (GPI; EC 5.3.1.9) in different sites of different hearts; the results showed CVs of 9.3, 9.1, 13.5, and 8.2%, respectively. A small transmural gradient in CK is found in the left ventricle. Determination of AST, GPI, and alanine aminotransferase (ALT; EC 2.6.1.2) in different sites of different livers gave CVs of 12.5, 17.0, and 11.6%, respectively. Most of the total variation is interindividual. The unreliability of early data and conflicting reports on transmural myocardial enzyme gradients are discussed. We conclude that by use of proper enzymes, such as LD for the heart and ALT for the liver, organ damage can be estimated, although there are inherent problems in relating enzyme release to loss of tissue mass.
Equations and nomogram for the relationship of human blood p50 to 2,3-diphosphoglycerate, CO2, and H+.
Authors: Samaja M , Mosca A , Luzzana M , Rossi-Bernardi L , Winslow R M
Published: January 2020

We describe a new method for tonometry of small amounts of blood (up to 0.25 mL) at known pO2, PCO2, and temperature, in small, reusable, closed Pyrex flasks. Equilibrated blood is analyzed for oxygen saturation, pH, and organic phosphate concentration with standard techniques, and its p50 (the pO2 at which hemoglobin is half-saturated with oxygen) is determined with full control of all the variables known to affect it. The SD in the measurement of p50 is 0.044 kPa (0.33 mmHg). We made 63 determinations of p50 on normal human blood under different conditions of pH and pCO2, and with different concentrations of 2,3-diphosphoglycerate and ATP. Empirical equations and a nomogram were derived, which allow the calculation of p50 from known values of pCO2, pH, and [2,3-DPG]/[Hb4] molar ratio with a SD of 97 and 114 Pa (0.73 and 0.86 mmHg), respectively.
Continuous-flow enzymic determination of creatine in urine.
Authors: Yasuhara M , Fujita S , Furukawa I , Arisue K , Kohda K , Hayashi C
Published: January 2020

A new enzymic method is described for the determination of creatine in urine by continuous-flow analysis. The measurement is accomplished by transforming creatine to formaldehyde in reactions catalyzed by creatinase (creatine amidinohydrolase) and sarcosine dehydrogenase. The formaldehyde is reacted with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole to form a purple product, which is measured colorimetrically. The method gives a linear standard curve for creatine concentrations up to 100 mg/L. Precision and analytical recovery are excellent, and results correlate well with those by the more-difficult Folin method, which currently is most often used for this analysis.
Bratton-Marshall and liquid-chromatographic methods compared for determination of sulfamethazine acetylator status.
Authors: Whelpton R , Watkins G , Curry S H
Published: January 2020

Synthesis of acetylsulfamethazine has made possible an evaluation of the Bratton-Marshall assay for this compound. Hydrolysis is an essential step in the assay, which depends on color development with the sulfamethazine formed in the hydrolysis. Sulfamethazine decomposes under the conditions needed for hydrolysis of acetylsulfamethazine, and this leads to depressed values for acetylsulfamethazine and to low figures for clinical "percent acetylation." Because of this, a simple "high-pressure" liquid-chromatographic procedure was developed for the assay; it requires no hydrolysis, is rapid in application, and can be applied to as little as 10 microL of capillary blood obtained by finger-to-puncture. We found that the cutoff point between "slow" and "fast" acetylators, when this method was used on samples from 75 subjects collected about 6 h after dosage, was 40% "acetylation" in plasma.
Automated analysis for plasma epinephrine and norepinephrine by liquid chromatography, including a sample cleanup procedure.
Authors: Yamatodani A , Wada H
Published: January 2020

We describe a fully automated method of analysis for plasma epinephrine and norepinephrine by use of a two-column system of "high-performance" liquid chromatography. Catecholamines in deproteinized plasma are purified on the first (preparation) column, then transferred automatically to the second (analytical) column in which epinephrine and norepinephrine are resolved. These compounds are then determined fluorometrically with a continuous-flow reaction system by the trihydroxyindole method. The minimum sensitivity of the method is 0.02 and 0.04 pmol for epinephrine and norepinephrine, respectively. One assay can be completed in 30 min, and greater than 1 mL of plasma is required for the procedure. The within-run CV in the chromatographic determination of pooled plasma was greater than 3%, and the analytical overall recovery of the two compounds was 95%. The concentrations in plasma of medical students at rest were 0.32 (SE 0.06) nmol/L for epinephrine and 0.98 (SE 0.08) nmol/L for norepinephrine.
Improved method for quantitative determination in serum of alkaline phosphatase of skeletal origin.
Authors: Farley J R , Chesnut C H , Baylink D J
Published: January 2020

In this quantitative method for detection of skeletal alkaline phosphatase (EC 3.1.3.1) activity in human serum, intestinal and placental alkaline phosphatase activities are recognized by their susceptibility to inhibition by L-phenylalanine, and skeletal and hepatic alkaline phosphatases are distinguished by their different sensitivities to inactivation by heat. Alkaline phosphatase isoenzymes prepared from organ sources may behave differently from the corresponding isoenzymes in serum. Our procedure allows us to include organ-derived internal standards of skeletal, intestinal, and biliary alkaline phosphatase to minimize between-assay variation. In preliminary applications, we have found that (a) total serum alkaline phosphatase activity is extremely variable in post-menopausal osteoporotic subjects and is not a reliable index of skeletal alkaline phosphatase activity; (b) seven osteoporotic patients responding to therapy with sodium fluoride with increased bone formation showed increased skeletal alkaline phosphatase activity in their serum as compared with age-matched controls (p less than 0.005); and (c) 10 post-menopausal osteoporotic patients responding to therapy with stanozolol with increased total body calcium showed an increase in circulating skeletal alkaline phosphatase activity (p less than 0.001).
Detection of aspartylglycosaminuria by gas--liquid chromatography.
Authors: Maury C P
Published: January 2020

I describe a rapid gas-chromatographic method for specific detection of the lysosomal storage disease aspartylglycosaminuria, based on the identification of the major storage compound, 2-acetamido-1-N-(4'-L-aspartyl)-2-deoxy-beta-D-glucopyranosylamine (GlcNAc-Asn) in the urine of affected individuals. A 50-microL sample of urine is methylated without prior purification; the methylation products are then analyzed by gas--liquid chromatography. Under these conditions a diagnostic GlcNAc-Asn peak can be seen in the urine of patients with aspartylglycosaminuria, but not in the urine of control subjects or patients with related storage diseases.
Urinary free catecholamines determined by liquid chromatography--fluorometry.
Authors: Anderson G M , Young J G , Jatlow P I , Cohen D J
Published: January 2020

Free norepinephrine, epinephrine, and dopamine are determined in urine after purification on a small ion-exchange column and concentration on alumina. The catecholamines are separated by reversed-phase ion-pair chromatography and are quantitated by measuring their native fluorescence (lambda ex 285 nm, lambda em 305 nm). The internal standard is dihydroxybenzylamine. Within-day CVs for the compounds ranged from 1.6 to 5.6%; between-day CVs were 6.6 to 9.8%.
Effects of temperature on the steady-state kinetics and measurement of aspartate aminotransferases.
Authors: Rej R , Vanderlinde R E
Published: January 2020

We examined the effects of temperature on the activity and steady-state kinetics of aspartate aminotransferase (EC 2.6.1.1), using purified human soluble (s-AspAT) and mitochondrial (m-AspAT) isoenzymes, human serum, and porcine s-AspAT. All enzymes obeyed similar linear Arrhenius relationships over the range 20-40 degrees C. Apparent energies of activation (52.3 kJ.mol-1) and ratios of activity between 30 and 37 degrees C (0.626) were identical for the human s- and m-AspAT. This ratio was 0.623 (SEM 0.004) for human sera; deviation from the predicted ratio by individual sera was within analytical error. Similar activity/temperature relationships were observed for porcine s-AspAT. The use of factors to convert AspAT activities at 30 and 37 degrees C influenced neither precision of measurement of frequency distributions of results. The apparent Michaelis constants for the human isoenzymes increased with temperature. The least-influenced Km was for 2-oxoglutarate and s-AspAT: K2-oxoglutarate was 0.24 mmol.L-1 at 25 degrees C and 0.29 mmol.L-1 at 37 degrees C; apparent enthalpy change for substrate binding (delta HS) was 12.1 kJ.mol-1. The largest variation was for 2-oxoglutarate and m-AspAT: K2-oxoglutarate was 0.46 mmol.L-1 at 25 degrees C and 1.02 mmol.L-1 at 37 degrees C; delta HS was 50.8 kJ.mol-1. Incubation of the human isoenzymes with substrate mixture (without 2-oxoglutarate) at 23 and 37 degrees C did not affect activity during 60 min if tris(hydroxymethyl)aminomethane buffer was used. When the isoenzymes were diluted to 10 nmol-L-1 (about 200 U.L-1) in buffer alone and incubated at 50 degrees C, m-AspAT activity was decreased by 20% after 120 min; the cytoplasmic enzyme was unaffected.
Isolation and purification of aspartate aminotransferase isoenzymes from human liver by chromatography and isoelectric focusing.
Authors: Leung F Y , Henderson A R
Published: January 2020

To purify cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from human liver. We used heat treatment, ammonium sulfate precipitation, anion- and cation-exchange chromatography, affinity chromatography, and isoelectric focusing. Final preparations of the isoenzymes were homogeneous, with specific activities of 198 and 208 kU/g for the cytoplasmic and the mitochondrial enzymes, respectively. The mitochondrial isoenzyme focused as a single band with a pl value of 9.60, whereas the cytoplasmic isoenzyme had subforms with pl values of 5.22, 5.42, and 5.62 at 4 degrees C. In Tris . HCl buffer, both isoenzymes have an activity maximum at pH 7.8. In [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bistris) buffer, however, the mitochondrial isoenzyme also showed an optimum pH of 6.7.
Simple laboratory determination of excess oligosacchariduria.
Authors: Sewell A C
Published: January 2020

I describe a simple set of procedures for the screening of patients' urine to detect oligosaccharide-storage diseases. Urines from patients with mucolipidosis I, mannosidosis, fucosidosis, aspartylglycosaminuria, and type VI glycogen-storage disease can be distinguished by thin-layer chromatography. Patients with beta-galactosidase deficiency can be detected by use of a combination of ion-exchange and thin-layer chromatography. Excess sialyloligosaccharide excretion is detected by using gel filtration and a quantitative assay for neuraminic acid. The advantages of the system are detection of virtually all known disorders in which oligosaccharides are over-excreted, production of characteristic patterns, and small sample requirement.
Quantification of damage to striated muscle after normothermic or hypothermic ischemia.
Authors: Presta M , Ragnotti G
Published: January 2020

Muscular ischemia of different duration was induced by use of a tourniquet applied to the rear left leg of rabbits under normothermic or hypothermic conditions, and variations in serum creatine kinase, lactate dehydrogenase, K+, Ca2+, and lactate were determined at various intervals after blood flow was restored. A monocompartmental model analysis applied to the activities of the two enzymes in the serum demonstrated that: (a) the total enzyme activity leaked into the blood is proportional to the duration of normothermic but not of hypothermic ischemia; (b) this enzyme leakage persists until at least the third day after blood flow is restored; (c) a substantial part of the ischemic damage seems to occur as a consequence of re-perfusion, not of the ischemia itself; and (d) hypothermia greatly minimizes the ischemic damage. The data we obtained for K+ and for lactate confirm the protective effect of hypothermia.
Nephelometry of human IgG subclass concentration in serum.
Authors: Beck O E , Kaiser P E
Published: January 2020

An immunoassay method for determination of human IgG subclass concentration by rate nephelometry was developed by using the Beckman Immunochemistry Analyzer, subclass-specific antisera, and a human serum standard. Twelve sera derived from normal as well as gammapathological patients were analyzed. The total IgG concentration, as determined by using a readily available kit, correlated with the sum of the concentration of the individual subclasses. Most of the gammapathological cases were shown to be the IgG1 subclass, which was confirmed by radial immunodiffusion.
Elimination of inhibitors of alkaline phosphatase from 2-amino-2-methyl-1-propanol.
Authors: Derks H J , Borrias-van Tongeren V , Terlingen J B , Koedam J C
Published: January 2020

When 2-amino-2-methyl-1-propanol buffer solutions are extracted with chloroform, impurities inhibiting alkaline phosphatase are selectively removed and this source of variation is thereby greatly decreased. We also studied the effect of adding zinc sulfate to this buffer. A combination of chloroform extraction and zinc sulfate, added to give concentrations from 5 to 10 mumol/L, results in buffer of high and consistent quality. The alkaline phosphatase activities of 12 reference sera of different origin were determined with use of the purified buffers. Those sera, which closely resembled human serum in terms of alkaline phosphatase isoenzyme composition, behaved similarly to human serum. Use of such control sera is strongly recommended.
Absence of beta-globulin band in the serum protein electropherogram of a patient with liver disease.
Authors: Vladutiu A O , Kim J S
Published: January 2020

Agarose-gel electrophoresis of serum of a 72-year-old woman with liver cirrhosis showed virtually no beta-globulins two weeks before the patient's death. There was marked decrease in the concentrations of transferrin, beta-lipoproteins, hemopexin, complement component C3, beta-glycoprotein I, and cholesterol in serum. Absence of a beta-globulin band appears to signify an ominous prognosis.
New approach to cystic fibrosis diagnosis by use of an improved sweat-induction/collection system and osmometry.
Authors: Webster H L , Barlow W K
Published: January 2020

A comprehensive equipment system is described that induces, collects, and analyzes sweat as a diagnostic procedure for cystic fibrosis. The safety and convenience of the iontophoretic induction phase have been improved by specially designed circuitry and electrodes. The problem of potentially serious error as a result of condensation within sweat-collection cups has been eliminated by use of a thermostatically controlled heated cup. The osmolality of the collected sweat is measured with a vapor-pressure osmometer. Data are presented to support the validity of osmolality as an alternative to electrolyte assay or electrical conductivity measurement and normal and abnormal ranges are provided. The complete system was tested in a pediatric hospital and data are presented to illustrate the relative advantages of the system with respect to safety, convenience, diagnostic reliability, and decreased potential for human error.
Improved determination of D-glucaric acid in urine.
Authors: Jung K , Scholz D , Schreiber G
Published: January 2020

Using a simplex optimization procedure, we have elaborated a sensitive and reliable micromethod for determining D-glucaric acid, based on inhibition of glucuronidase activity by 1,4-D-glucarolactone, produced from D-glucaric acid by heating. The maximum attainable difference between glucuronidase activity in the reaction mixture with and without D-glucaric acid was used as an optimization criterion. From the optimization scheme, 1 mmol/L 4-nitrophenyl-beta-D-glucuronide and pH 5.0 were found to be optimal conditions for determination of D-glucaric acid with this test. In contrast to other similar methods, in which a logarithmic calibration curve is used, we calculate D-glucaric acid with a linear calibration curve by using the Dixon (Biochem. J. 55: 170, 1953) plot. The coefficient of variation for the method (within-run and between-day precision) was from 3.1 to 5.8%. Men excrete more D-glucaric acid than do women per 24 hours, but we saw no sex-related differences in excretion of D-glucaric acid when it was expressed in terms of urinary creatinine.
A modified radioimmunoassay for 1,25-dihydroxycholecalciferol.
Authors: Gray T K , McAdoo T , Pool D , Lester G E , Williams M E , Jones G
Published: January 2020

A radioimmunoassay for 1,25-dihydroxycholecalciferol which did not cross react with 1,25-dihydroxyergocalciferol is described. IgG fractions were prepared from the serum of rabbits that had been immunized with 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine albumin. Radioligand binding by the IgG fractions was time-, temperature-, and pH-dependent. The IgG fractions had a high affinity for 1,25-dihydroxycholecalciferol but cross reacted with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Vitamin D2 metabolites did not cross react in the assay when amounts up to 9 ng per tube were tested. The determination of 1,25-dihydroxycholecalciferol in human serum required an organic extraction and chromatographic isolation of the metabolite. Radioligand binding was influenced by the presence and concentration of the proteins in the phosphate buffer. The mean concentration of 1,25-dihydroxycholecalciferol in serum from normal adults was 56 (SEM 5.7) ng/L. 1,25-Dihydroxycholecalciferol was not detectable in serum from a nephrectomized subject and the concentration in serum was lower than normal in hypoparathyroid patients. Ingestion of 1,25-dihydroxycholecalciferol by nephrectomized or hypoparathyroid patients restored the concentration of 1,25-dihydroxycholecalciferol in serum to the normal range. The stability of the IgG fraction, the relatively short incubation interval, and the ability to measure 1,25-dihydroxycholecalciferol without interference from 1,25-dihydroxyergocalciferol are unique aspects of this radioimmunoassay.