bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of autoantibodies to creatine kinase-BB on assays for MB isoenzyme.
Authors: Stein W , Bohner J
Published: January 2020

We describe the influence of autoantibodies that bind creatine kinase BB (CK-BB) on the methods for MB isoenzyme. If these autoantibodies are present in patients' sera, they cause the formation of macro CK type 1 (immunoglobulin-linked CK-BB). In some of these cases they can bind not only endogenous CK-BB but also CK-MB without significantly affecting enzyme activity. Although these antibodies show distinctly less affinity for CK-MB than for CK-BB, they nevertheless bind CK-MB in these particular sera, because their concentration exceeds that of CK-BB isoenzyme. If a person with such autoantibodies has an acute myocardial infarction, the immunoinhibition method for CK-MB, which does not discriminate between CK-MB and CK-BB, will recognize the increase and peak of CK-MB with time, although persistent macro CK activity will be superimposed on the typical isoenzyme pattern. However, isoenzyme electrophoresis and recently introduced immunoenzymometric assays for CK-MB in these cases may be less sensitive for detecting myocardial infarctions, because the typical increase in CK-MB activity may be identified later in the progression of symptoms, or even be missed.
Use of biotinylated probes for detecting sickle cell anemia.
Authors: Garbutt G J , Wilson J T , Schuster G S , Leary J J , Ward D C
Published: January 2020

Earlier, we reported that the 5' end of the normal beta-globin gene (beta) resides on a 1.14-kilobase DNA fragment, whereas the 5' end of the sickle cell gene (beta s) resides on a 1.34-kilobase fragment. In that blot hybridization analysis, we used genomic DNA digested with restricted endonuclease Mst II, and radioactive probes with short half-life. We demonstrate here that, if a biotinylated probe is used instead in a slightly modified procedure, sickle cell anemia can be quickly and directly detected if there is as much as 5 micrograms of total genomic DNA in the sample. This procedure obviates the special precautions necessary when radioactive materials are used.
Studies on the "labile-bound" glucose compartment in erythrocytes: studies on Psammomys obesus (sand rat) and preliminary studies on human erythrocytes.
Authors: Halperin M , Adler J H
Published: January 2020

Enzymatic (glucose oxidase) measurement of glucose concentration in the fluid compartment of Psammomys erythrocytes (Gfe) and of its concentration in the fluid compartment of blood plasma (Gfp) gives the ratio (mean +/- SD): Gfe/Gfp = 1.50 +/- 0.43 (n = 12, 23 degrees C). However, when we added 3H-labeled glucose (G*) in vitro to the whole blood, the ratio after 2 min was G*fe/G*fp = 0.90 (SD 0.11) and after 5 min G*fe/G*fp = 0.97 (SD 0.12). These calculations were based on previous determination of the fractional volumes of the fluid and non-fluid compartments in Psammomys blood. The results suggest that there is more than one compartment of measurable glucose in Psammomys erythrocytes. Glucose undergoes a fast free transfer between the plasma and the erythrocyte fluids, and a much slower transmission to another measurable compartment in the erythrocyte, where it is loosely bound to other molecules. This loosely bound glucose does not participate in the fast kinetic transmission across the erythrocyte membrane, but it is measurable by the glucose-oxidase-based method. Preliminary studies on human erythrocytes lead to similar conclusions.
A 2,4-dichlorophenyl diazonium-based method for total bilirubin without interference from indican in uremic sera.
Authors: McPhaul L , Kershaw M , Tilque D , Eckfeldt J H
Published: January 2020

Comparison of total bilirubin quantification by a 2,4-dichlorophenyl diazonium method (2,4-DCPD) with a Jendrassik-Grof type of method showed excellent correlation for randomly selected sera. However, sera from uremic patients on chronic hemodialysis showed a marked positive bias for the 2,4-DCPD result as compared with the Jendrassik-Grof result. The mean difference was 5.3 mg/L, and resulted in about 20% of the hemodialysis patients having bilirubin values greater than 13 mg/L, the upper limit of our reference range. Indican in uremic sera reportedly reacts with certain diazo reagents, so we investigated indican's reactivity in the above methods. In vitro addition of indican caused no interference in the Jendrassik-Grof method, but produced a significant positive interference in the 2,4-DCPD method, 1 mmol of indican per liter appearing as about 36 mg of total bilirubin per liter. Long reaction times with the 2,4-DCPD reagent accentuate the problem. By shortening the reaction time with the 2,4-DCPD reagent to 1.7 min, we find that the indican interference can be eliminated, without affecting quantification of total bilirubin in either normal or uremic sera.
Clinical Effectiveness of the Du Pont acaMeasurement of Creatine Cinase MB in Serum from Patients in a Coronary-Care Unit
Authors: Leroux Michael L , Rabson John , Desjardins Paul R E
Published: January 2020

Vol. 30

p 1553, Insert the symbol "○" in the sublegend to Fig. 1, third line, to read "...method: ○, activity..."

Vol. 31

p 572, middle of right column: delete the three repeated lines of type.

p 575, in "Table 1 (continued)," the first column heading hould read "Sample size, ND = ND·"

p 616, left column, first text paragraph: the second sentence should read, "This nonlinear protein binding explains the dependence of renal clearance of disopyramide on total disopyramide concentration in serum and the lack of dependence of renal clearance on the concentration of free disopyramide (5). Furthermore, the pharmacological response to this drug is better correlated with the concentration of the free drug in serum than with its total concentration (4, 6); we thus condude that both the free fraction and the total concentration should be measured when one is monitoring arrhythmic patients who are being treated with his drug."

p 617, left column: delete the two lines of text following the first paragraph of Results, and delete the three lines following the second paragraph.

p 640: Three lines are missing in the section on Ferritin standards, as follows: "Ferritin from human liver, 9.7 µg per bottle, i.e., 9.1 mg/L (National Institute for Biological Standards and Controls, Hampstead-Holly Hill, London, reference 80/602)."

p 641, left column, third line of text at bottom: "3988" should read "398.8" and "4140" should read "414.0."

p 647, middle column, preceding the 10th line from bottom, insert: "Using this method, we found the ... "

p 655, right column: the concluding paragraph (preceding the references) should read as follows: "Our results indicate that assay of serum SHBG is of value in the analysis of risk factors for mortality in post-menopausal women. Whether low SHBG concentration in these women primarily reflects androgen excess, estrogen deficiency, or other endocrine or metabolic changes remains a subject for further study, as does the relationship to adipose tissue distribution."
Radioimmunoassay of active pancreatic enzymes in sera from patients with acute pancreatitis. I. Active carboxypeptidase B.
Authors: Delk A S , Durie P R , Fletcher T S , Largman C
Published: January 2020

Previous studies have suggested that measurement of active enzymes in relation to proenzymes in serum of patients with pancreatitis may reflect the degree of zymogen activation in the gland. Here we describe the first single-tube assay for an active form of a pancreatic enzyme that is ordinarily synthesized as a proenzyme. Human procarboxypeptidase B, which we purified to near homogeneity, is approximately 13 000 Da larger than the active enzyme (EC 3.4.17.2). Antibodies specific for active carboxypeptidase B were obtained by affinity chromatography of anti-carboxypeptidase B antisera on a gel containing procarboxypeptidase B, then used to develop a single-tube radioimmunoassay for measuring active carboxypeptidase B in serum. Using this assay, we were able to detect, for the first time, active carboxypeptidase B in sera from patients with acute pancreatitis. Preliminary data show a correlation between the serum concentrations of active carboxypeptidase B and those of active trypsin complexed with serum inhibitors, but no correlation with serum amylase values.
Amino acid concentrations in plasma and skeletal muscle of patients with acute hemorrhagic necrotizing pancreatitis.
Authors: Roth E , Zöch G , Schulz F , Karner J , Mühlbacher F , Hamilton G , Mauritz W , Sporn P , Funovics J
Published: January 2020

We measured amino acid concentrations in plasma and skeletal muscle of three groups of patients with acute hemorrhagic pancreatitis: (a) patients without secondary organ lesions, (b) patients also suffering from kidney damage, and (c) patients in whom the pancreatitis was accompanied by sepsis and multiple organ failure. In all three groups, especially the third group, the amino acid concentrations in both plasma and muscle were below normal. Glutamine was only 14% of normal in muscle tissue of the third group. Onset of renal insufficiency was indicated by increasing values for 3-methylhistidine and cystathionine; multiple organ failure, by increased concentrations of methionine and phenylalanine in plasma. The low amino acid concentrations of patients with acute pancreatitis can be explained as a combined effect of semistarvation and hypercatabolism. Changes in the plasma concentrations of amino acids did not reflect necessarily the concentrations in muscle tissue.
Hydroxyurea interferes negatively with triglyceride measurement by a glycerol oxidase method.
Authors: McPherson R A , Brown K D , Agarwal R P , Threatte G A , Jacobson R J
Published: January 2020

Measured triglyceride concentrations were extremely low (less than 100 mg/L) in the serum of some patients who were receiving hydroxyurea for myeloproliferative diseases. The assay being used to quantify triglycerides was a "cascaded" enzymatic method involving (a) lipase, to generate glycerol from triglycerides; (b) glycerol oxidase, to convert glycerol to glyceraldehyde, with generation of hydrogen peroxide; and (c) peroxidase, which acts on the hydrogen peroxide with subsequent coupled generation of a red-violet quinone (reagent system used in the Technicon RA-1000). Hydroxyurea added to serum samples appeared to inhibit the action of glycerol oxidase, with a stoichiometric relation to the concentration of substrate (a decrease of roughly 2.4 mmol/L in measured triglyceride per 1 mmol of hydroxyurea per liter). A different enzymatic assay for triglycerides, which involves glycerol kinase (Beckman Instruments) did not show this effect of hydroxyurea.
A radioimmunoassay for retinol-binding protein in serum and urine.
Authors: Beetham R , Dawnay A , Landon J , Cattell W R
Published: January 2020

In this radioimmunoassay for human retinol-binding protein (RBP), sample (serum, diluted as much as 3600-fold; or urine, diluted or undiluted) or calibrant (purified RBP in phosphate buffer containing bovine serum albumin) is incubated with 125I-labeled RBP and rabbit anti-human RBP antiserum for 24 h before separation with second antibody/polyethylene glycol. The assay's sensitivity is 5 micrograms/L, its useful working range 10 to 200 micrograms/L. The between-batch CV is 12.5% at 12 micrograms/L, 7% at 120 micrograms/L. Results correlate well (r = 0.99) with those by radial immunodiffusion. The reference interval for RBP in serum of men is 39-75 mg/L (mean 57), significantly (p less than 0.01) broader than for women (29-66 mg/L; mean 48). The 2.5-97.5 centile interval for RBP in urine is from less than 0.5 to 12.2 micrograms per millimole of creatinine (median 5.8). RBP in urine is unstable below pH 5.0 at 37 degrees C.
Colorimetric determination of potassium in whole blood, serum, and plasma.
Authors: Wong S T , Spoo J , Kerst K C , Spring T G
Published: January 2020

This spectrophotometric method for the direct determination of potassium in serum or plasma is based on the selective complexing of potassium by a specific macrocyclic polyether, with the subsequent formation of an ion-pair with a colored anion. The colored anion is extracted into an organic solvent, clarified by centrifugation, and then measured at 415 nm. The absorbance of the chromogen varies linearly with [K+] to at least 15 mmol/L. Results of this colorimetric method (y) correlate well with the results obtained by a flame-photometric method (y = 1.04x - 0.22, r = 0.97, n = 81), with CVs ranging from 2 to 4%. We observed no interferences from lipemia, added bilirubin, or various electrolytes. We also evaluated the use of this reagent in a new automated blood analyzer developed by Abbott, a two-dimensional centrifugal system (Clin Chem 31:1457-1463, 1985). Potassium determined with this system (y) correlated well with results by flame photometry: y = 1.02x + 0.02 (r = 0.94, n = 168). With this system one can use whole-blood specimens in measuring potassium.
Liquid-phase reactions started by rehydrating lyophilized reagents in a centrifugal analyzer.
Authors: Truchaud A , Glikmanas G , Gourmelin Y , Hersant J , Trepo D , Foray G , Collombel C
Published: January 2020

In the disposable rotor of the SAM microcentrifugal analyzer, various lyophilized reagents are predistributed in 24 33-microL cuvets, for determination of multiple analytes in one specimen (e.g., for patient profiles). We evaluated a prototype of this system, which can be used at 25, 30, or 37 degrees C; absorbance readings at 340, 405, and 500 nm varied linearly up to 2.0 A. Starting the reactions by rehydrating the reagents with diluted serum is adequate because absorbance readings do not begin until 180 s after initiating the rehydration. Analytical performances of kinetic determinations at 30 degrees C showed good accuracy and correlation with other methods for creatine kinase, amylase, aspartate aminotransferase, and gamma-glutamyltransferase. Kinetic determinations for urea, and equilibrium determinations with blank corrections for glucose, cholesterol, and triglycerides gave excellent results for glucose and correct results for the other analytes. This compact analyzer combines the analytical performances of a centrifugal analyzer with the practicability of instruments having predistributed reagents.
Radiolabeled drugs as probes of central nervous system neurons.
Authors: Wagner H N
Published: January 2020

Methylspiperone, carfentanil, and suriclone, labeled with 11C, can be used in assessment of dopamine, mu-type opiate receptors, and benzodiazapine receptors in the brains of living human beings. These relatively specific neuronal probes permit measurement of the number and state of occupancy of receptors in the brain in health and disease, and may be useful to assess endogenous neurotransmitter secretion by decreasing competitive binding of the labeled ligand. To date, the most striking finding has been a decrease in the indices of dopamine and serotonin receptor densities as a function of age in normal men and women. A possible relationship between this decrease and vitamin B6 deficiency is currently under study.
Intra-erythrocytic sodium in uremic patients, as determined by "high-resolution" 23Na nuclear magnetic resonance.
Authors: Monti J P , Gallice P , Crevat A , el Mehdi M , Durand C , Murisasco A
Published: January 2020

The use of 23Na nuclear magnetic resonance with aqueous shift reagent has made it possible to determine intracellular sodium concentrations in living erythrocytes. We applied this technique to samples from 16 healthy subjects and 41 uremic patients. The results seem to show distinct populations among the latter. Classically, two different relaxation times are obtained for intracellular sodium in biological media, according to relaxation NMR theory. Some patients, however, exhibit abnormal results that cannot be accounted for by this theory.
On the albumin dependence of measurements of free thyroxin. I. Technical performance of seven methods.
Authors: Csako G , Zweig M H , Benson C , Ruddel M
Published: January 2020

We evaluated three one-step (analog) and two two-step radioimmunoassay for free thyroxin (FT4), and a FT4 index calculated from the total T4 (TT4) and thyroxin-binding globulin (TBG) ratio for technical performance, for correlation with the reference FT4 method (equilibrium dialysis), and for dependence on TBG and albumin concentrations. The one-step methods (Amerlex, Coat-A-Count, and GammaCoat) showed greater precision than the two-step procedures (GammaCoat and Spiria). Results by the latter two techniques, however, correlated better with those by equilibrium dialysis than did those by the analog methods or by TT4/TBG. Only the GammaCoat two-step method had a slight but statistically significant (inverse) correlation with TBG concentration. All three analog methods and TT4/TBG showed a marked dependence on albumin concentration, whereas the Spiria technique showed only a slight dependence. Only equilibrium dialysis was independent of both the TBG and albumin concentration. Thus, despite their good precision, the analog (one-step) FT4 methods and the TT4/TBG approach cannot be expected to produce valid results when the concentration of albumin in serum is abnormally low or high.
Lipids, lipoproteins, and apoproteins in serum during infection.
Authors: Alvarez C , Ramos A
Published: January 2020

We studied the alterations in the concentrations of total cholesterol, high-density lipoprotein cholesterol, triglycerides, and apoproteins A and B in serum of 54 patients hospitalized for various reasons, who developed sepsis during their stay. Forty of these patients required intensive care, 14 did not. Another group of patients with the same underlying pathological conditions was used as a control. We found the following: Sepsis causes the concentrations of total cholesterol, high-density lipoprotein cholesterol, and apoproteins A and B in serum to decrease, whereas triglycerides increase. However, these changes are not related to the infectious agent, the underlying illness, or the clinical situation of the patients. The return of serum lipids to more normal concentrations parallels the recovery from sepsis. The positive correlation between the drastically decreased concentrations of high-density lipoprotein cholesterol and the severe hypoalbuminemia in these patients suggests a common pathway for these two abnormalities.
Concentrations of sex-hormone binding globulin and corticosteroid binding globulin in serum in relation to cardiovascular risk factors and to 12-year incidence of cardiovascular disease and overall mortality in postmenopausal women.
Authors: Lapidus L , Lindstedt G , Lundberg P A , Bengtsson C , Gredmark T
Published: January 2020

We determined sex-hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) by radioimmunoassay of serum samples from a group of 253 women, who were 54 or 60 years old when first studied in 1968-69. The SHBG concentration was highly significantly and inversely related to body mass, body mass index, waist-to-hip circumference ratio, and serum triglyceride concentration; CBG concentration was inversely related to body mass and body mass index. The concentration of neither protein was related to whether or not the subject smoked. Decrease in the concentration of SHBG, but not of CBG, was a significant risk factor for 12-year overall mortality. The plot of the 12-year incidence of myocardial infarction vs SHBG concentration was U-shaped. We recommend that SHBG be included when serum androgens or estrogens are being evaluated as risk factors for cardiovascular disease and death.
Competitive solid-phase enzyme immunoassay for measuring digoxin in serum.
Authors: Hinds J A , Pincombe C F , Morris H , Duffy P
Published: January 2020

In this clinically useful enzyme immunoassay of digoxin in serum, we mix sample, beta-galactosidase-labeled digoxin, and anti-digoxin Fab fragments for 30 min at room temperature, then use Sepharose-bound second antibody for phase separation, and measure the unbound enzyme activity directly in the supernate of the equilibrium reaction mixture. The immunoassay buffer--phosphate-buffered isotonic saline with added rabbit globulin (4 g/L), hydrolyzed gelatin (2 g/L), Brij 96 detergent (5 g/L), glycerol (0.25 mol/L), and N-acetyl-8-anilinonaphthalene-1-sulfonic acid (2 mmol/L)--minimizes serum matrix effects for convenient measuring of unbound enzyme--digoxin conjugate. The immunoassay developed with Fab fragments has better displacement characteristics than that with intact antibody. Performance of the assay compares favorably with that of other manual digoxin immunoassays; in comparison studies with EMIT involving 110 clinical specimens, the coefficient of correlation was 0.97.
Monitoring the stability of wavelength calibration of spectrophotometers.
Authors: Korzun W J , Miller W G
Published: January 2020

The difference in absorbance (delta A) between equimolar acid and alkaline solutions of methyl red, at a wavelength near the isosbestic point of the indicator, is reproducible. Furthermore, this delta A is sensitive to changes in the wavelength calibration of the instrument used to make the measurement. The delta A of methyl red can be used to monitor wavelength accuracy in both manual and automated spectrophotometric instruments. Although this measurement does not establish wavelength calibration, it is useful for monitoring the wavelength accuracy of previously calibrated, automated spectrophotometers that do not easily lend themselves to calibration checks by conventional techniques.
Photodiode array detection for liquid chromatographic profiling of carboxylic acids in physiological fluids: 3-hydroxy-3-methylglutaric aciduria.
Authors: Buchanan D N , Thoene J G
Published: January 2020

3-Hydroxy-3-methylglutaric aciduria was detected in a newborn. The progress of the dietary therapy for the disorder was monitored by dual-column "high-performance" liquid chromatography with a computer-controlled photodiode array spectrophotometric detector. This procedure is a quick way to detect and monitor the progress of 3-hydroxy-3-methylglutaric aciduria.
Unusual findings related to atypical creatine kinases in two hospitalized patients.
Authors: Loshon C A , Rittenhouse S E , Bowers G N , McComb R B
Published: January 2020

We describe two cases, hospitalized patients, in whom the activity of creatine kinase (EC 2.7.3.2) isoenzyme-MB was above normal. Both are particularly noteworthy in that creatine kinase-BB and a macro creatine kinase form thought to be type II of mitochondrial origin were also present. The macro creatine kinase component in both cases co-migrated electrophoretically with creatine kinase-MM but was easily identified after the latter was removed by precipitation with M-subunit-specific antibodies. In the first case, the patient had a readily diagnosable acute myocardial infarction while under observation in the cardiac intensive care unit: electrocardiographic changes and the rapid increase and decrease in total creatine kinase were as would be expected. In marked contrast, in the second case, we saw no abrupt changes in either of these characteristics. The latter patient's primary disease was a rectal carcinoma with massive metastases to the liver; however, the presence of abnormally high creatine kinase-MB activity raised the question of possible myocardial infarction.
Two homologous radioimmunoassays for parathyrin compared and applied to disorders of calcium metabolism.
Authors: Schmidt-Gayk H , Schmitt-Fiebig M , Hitzler W , Armbruster F P , Mayer E
Published: January 2020

These 48-h homologous equilibrium radioimmunoassays for human parathyrin (hPTH) are based on the use of two antisera, MS 6 and MS 7, raised in guinea pigs against human parathyroid adenoma extract. For the assay with MS 6, Tyr43-hPTH(44-68) and hPTH(44-68) were radioiodinated for use as the assay tracer. Labeled peptides were separated from free iodine by passage through Sep-Pak C18 cartridges. This RIA appears to be mid-region specific: hPTH(28-48) and hPTH(64-84) were not recognized, whereas hPTH(53-84) was 100% cross reactive with hPTH(44-68). Thus, the PTH recognition site of antiserum MS 6 must be between amino-acid residues 53 and 63. With antiserum MS 7, which recognized the PTH molecule between amino-acid residues 69 and 84, we used hPTH(53-84) for preparing the standard curve and 125I-labeled Tyr52-hPTH(53-84) as the assay tracer. Because this RIA recognized PTH fragments containing residues 69-84 of hPTH, we termed it a C-terminal assay. Both assays were useful for diagnosis of primary and secondary hyperparathyroidism.
Salivary estriol as an index to fetal wellbeing.
Authors: Kirkish L S , Compton A A , McCann D S
Published: January 2020

We adapted two commercially available kits for measuring serum estriol to use in assaying salivary estriol in the third trimester of pregnancy. Salivary estriol increases during gestation, as does unconjugated estriol in serum. With a meticulous collection protocol, fetal wellbeing can be monitored as successfully with saliva samples as with serum. A fetal demise was charted similarly by the salivary and the serum estriol assay. Inter- and intra-assay reproducibility (CV) of the saliva assay was 6.2%. Day-to-day CV, based on a fasting sample before breakfast but obtained 1 to 3 h after rising, was 14.8% (SD 5.8%). Values for salivary estriol obtained immediately after ingestion of food or drink dropped by 19.0% (SD 17.9%) and 32.1% (SD 21.9%), respectively, as compared with values determined immediately before intake of food or water.
Immunofluorometry of thyrotropin, from whole-blood spots on filter paper, to screen for congenital hypothyroidism.
Authors: Arends J , Nørgaard-Pedersen B
Published: January 2020

We have evaluated a time-resolved immunofluorometric assay (IFMA) for determining thyrotropin. This "sandwich"-type system involves two monoclonal antibodies directed against different epitopes. A linear relationship between signal and thyrotropin concentration was observed up to 6000 milli-int. units/L. This procedure takes one day, vs six days with our present RIA technique, and requires only a tenth as much sample. Furthermore, intra- and interassay CVs are lower than with RIA. Assay of 19 paper-disc blood specimens from newborns identified as having congenital hypothyroidism, both by RIA and by clinical evidence, also gave positive results with IFMA. In prospective assay of 3944 specimens by both methods we identified one case of congenital hypothyroidism, which was detected by both techniques. Technical false-positive reactions, identified as such by repeated analyses, were fewer with the IFMA method than with RIA.
Radioimmunoassay of metanephrine and normetanephrine for diagnosis of pheochromocytoma.
Authors: Iinuma K , Ikeda I , Ogihara T , Hashizume K , Kurata K , Kumahara Y
Published: January 2020

Sensitive and specific radioimmunoassays of metanephrine and normetanephrine were developed by use of 125I-labeled synephrine and specific metanephrine antibody, and 125I-labeled octopamine and specific normetanephrine antibody. Specific antibody for both metanephrine and normetanephrine was raised in rabbits by immunization with bovine serum albumin conjugated with the corresponding hapten, prepared by the method of Grota and Brown (Endocrinology 1976;98:615). The detection limits of the metanephrine and the normetanephrine radioimmunoassays were 2 and 6 pg/tube, respectively. Mean plasma metanephrine and normetanephrine values for 24 normal subjects were 62 (SD 14) and 100 (SD 40) ng/L, respectively. Mean urinary metanephrine and normetanephrine values for 22 normal subjects were 154 (SD 74) and 217 (SD 109) micrograms/day. For 14 pheochromocytoma patients, plasma metanephrine and normetanephrine values ranged from 29 to 683 and from 28 to 7850 ng/L, and urinary metanephrine and normetanephrine values were 606 to 6630 and 296 to 4800 micrograms/day, respectively. The present methods are simple and suitable for routine tests or for mass screening for pheochromocytoma.
Production and certification of secondary enzyme reference materials (ERMs). Part 1: Preparation of the sera and some of their properties.
Authors: Koedam J C , Steentjes G M , Buitenhuis S , Schmidt E , Klauke R
Published: January 2020

We produced three batches of a human-serum-based enzyme reference material (ERM) enriched with human aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), creatine kinase (EC 2.7.3.2), and lactate dehydrogenase (EC 1.1.1.27). The added enzymes were not exhaustively purified; thus the final ERMs contained some enzymes as contaminants, of which only glutamate dehydrogenase activity might interfere. The stability during storage and after reconstitution was good. The commutability of the four enzymes in the three ERM batches was also good, except when German or Scandinavian methods for aminotransferases were involved. The temperature-conversion factors for the ERMs were equivalent to those for patients' sera. Reactivation after reconstitution was complete within 5 min and was independent of the temperature of the reconstitution fluid. We believe that these secondary ERMs will aid in the transfer of accuracy between well-defined reference methods and daily working methods so that clinical enzymology results will become more comparable from laboratory to laboratory.
Production and certification of secondary enzyme reference materials (ERMs). Part 2: The need for meaningful protocols that assure photometric accuracy and a well-defined process for value assignment.
Authors: Koedam J C , van Dreumel H J , v d Ham W , Steentjes G M , Terlingen J B
Published: January 2020

A collaborative study to assign values to two enzyme reference materials (ERMs) was performed by 18 laboratories whose spectrophotometers were checked by us, just before the study. We measured the wavelength accuracy and repeatability, the accuracy and linearity of the absorbance curves, the cuvette pathlength, equilibration time, equilibrium temperature, and a few other variables. Five spectrophotometers exhibited a marked wavelength-dependent nonlinearity. Most instruments were rather slow in bringing the sample to the correct temperature and the final temperature was often too high. In the collaborative study, each participant performed the same manual, well-described methods on four occasions in triplicate, using reagents prepared locally. The relation between the photometric checks and the analytical results is discussed, as well as the treatment of outliers and the effects on the variances. Suggestions are made about various facets of collaborative studies. The values assigned to the two ERMs carry a 95% uncertainty interval of +/- 1-4% of the mean.
New liquid-chromatographic method for measuring polyethylene glycol in urine.
Authors: Delahunty T , Hollander D
Published: January 2020

The various Mr fractions of polyethylene glycol (PEG) in human urine are quantified by "high-pressure" liquid chromatography. A simple preparation step involving lyophilization and chloroform extraction of the sample is required. Aliquots (10 microL) are chromatographed isocratically in equivolume mixtures of methanol and water on a column of styrene divinylbenzene and the refractive index of the effluent is measured. The results vary linearly with the concentrations of standards up to at least 10 g/L, and the six major fractions are clearly identifiable in injected samples containing 2 g or more of total PEG per liter. As little as 4 g/L can be precisely quantified, but for assessing the individual fractions, we recommend a minimum sample concentration of 7 g/L. Analytical recovery of PEG added to urine controls was 90%. Urines collected during 6 h from 11 human subjects after each had ingested 5.6 g of PEG showed no interference from endogenous compounds. We find this method to be a simple, labor-saving means of quantifying urinary PEG in the clinical laboratory.
Interlaboratory standardization of glycated hemoglobin determinations.
Authors: Little R R , England J D , Wiedmeyer H M , McKenzie E M , Mitra R , Erhart P M , Durham J B , Goldstein D E
Published: January 2020

As the clinical utility of glycated hemoglobin (gHb) measurement increases, so does the need for standardization of values between different methods and different laboratories. Using three different methods, we examined the feasibility of interlaboratory standardization of gHb measurement. A liquid-chromatographic (HPLC) system from our research laboratory was designated the reference method. For gHb standards we used erythrocyte hemolysates prepared from blood samples from nondiabetic and diabetic subjects. Values assigned to each standard were based on the mean of multiple gHb determinations by the HPLC method. A clinical laboratory routinely prepared hemolysates and assayed gHb by commercially available ion-exchange ("mini column") and affinity chromatographic methods. For each assay a standard curve was constructed and gHb values were derived from these curves. Samples analyzed in the clinical laboratory were also analyzed in the research laboratory and the curve-derived values were compared with the HPLC-measured values, to determine the accuracy of our interlaboratory standardization procedure. Correlations were excellent (r = 0.99). The lack of significant differences between calculated and HPLC-measured values indicates that interlaboratory standardization is feasible.
Practical "stat" method for radioimmunoassay.
Authors: Cembal S J
Published: January 2020

A prompt and reasonably precise radioimmunoassay result is often urgently required, but it is difficult to satisfy such requests without running a complete standard curve, even when only a single sample is to be assayed. To obtain a greater constancy in the response of the RIA system, I propose a method for use of RIA in a "stat" (i.e., urgent) method: maximum binding and nonspecific binding for RIA data processing are "calculated" from response indexes obtained in a previous assay run with the same reagents and from the binding values of two or three standards processed together with a single sample. These calculated values are then used to calculate the response index for the single sample, which is directly interpolated in the calibration curve of the previous non-stat assay. I checked the validity of this stat method for eight analytes, comparing a total of 552 analytical results obtained by the conventional and the stat method. This method is shown to give adequately precise results for use in clinical emergencies.
Progesterone in saliva does not parallel unbound progesterone in plasma.
Authors: Evans J J
Published: January 2020

To assess the potential application of salivary progesterone, in light of the voluminous published information on blood progesterone, I compared plasma and salivary progesterone in paired samples and found that they exhibit similar patterns, both having high concentrations in the second half of the cycle. However, the ratio between values for plasma and saliva changed during the cycle. I observed no concomitant changes in binding of progesterone to plasma proteins, which might have been expected if the concentrations in saliva reflect those of non-protein-bound steroid in plasma. There was some binding of progesterone by salivary proteins, but the proportion of progesterone so bound apparently remained constant during the cycle. Data on progesterone in saliva must therefore be interpreted with awareness of the properties of saliva, and not simply as "free" plasma progesterone.
Fluorescence polarization immunoassay for theophylline modified for use with dried blood spots on filter paper.
Authors: Li P K , Lee J T , Conboy K A , Ellis E F
Published: January 2020

We used dried blood spots successfully as alternative specimens for quantifying concentrations of theophylline in the circulation by a modified fluorescence polarization immunoassay (FPIA) with the Abbott TDx instrument. The method described is simple, rapid, and acceptably precise. More importantly, it can provide results comparable with those of the conventional serum assay. Results for 64 pairs of dried blood spots (y) and serum (x) specimens analyzed by the respective FPIA methods yielded the following regression parameters: y = 0.94x + 0.71, r = 0.988, and Sxy = 0.92. A major advantage of FPIA is that it requires only basic laboratory skills. When coupled with the use of dried blood spots, this system can be effective in remote theophylline monitoring, particularly suited for domiciliary care.
Binding characteristics of thyroxin binding globulin in serum of normal, pregnant, and severely ill euthyroid patients.
Authors: Maberly G , Waite K , Ma G , Soni N , Eastman C
Published: January 2020

Serum from normal, pregnant, and severely ill patients was stripped of endogenous thyroid hormones and diluted 1100-fold in barbital buffer. We then used it to study the binding characteristics of thyroxin binding globulin (TBG), noting significant differences in binding capacities among the groups. The mean (+/- SD) triiodothyronine/thyroxin ratio for binding capacity was 18 +/- 4 for normal subjects. The ratio was significantly increased in pregnant patients, 21 +/- 4 (p less than 0.05), and significantly lower in severely ill patients, 12 +/- 4 (p less than 0.05). When serum was diluted before assay, to give a uniform TBG concentration among groups, these apparent differences in binding characteristics were eliminated. It therefore is unlikely that different molecular species of TBG account for the variations in binding characteristics in these clinical states. Apparently, the distribution of thyroxin and triiodothyronine among the binding sites on TBG changes with variations in TBG concentration. This may explain the discrepancies observed in the concentrations of free thyroid hormones as estimated by various methodologies.
A kinetic colorimetric procedure for quantifying magnesium in serum.
Authors: Wimmer M C , Artiss J D , Zak B
Published: January 2020

We have developed a kinetic colorimetric procedure for determination of magnesium in serum. The magnesium-dependent enzyme glycerol kinase is used to phosphorylate glycerol to glycerol 3-phosphate, the latter being oxidized to dihydroxyacetone phosphate and hydrogen peroxide by glycerophosphate oxidase. The generated hydrogen peroxide is then reduced by peroxidase with the simultaneous oxidative coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate, producing a red reaction product with an absorption maximum at 510 nm. The rate of color production is proportional to the concentration of the Mg X ATP complex, which is, in turn, proportional to the magnesium concentration in serum. This method is rapid and precise, avoids the use of expensive instrumentation, is easily automated, and results compare well with those by the Du Pont aca and manual Magon sulfonate methods.
Isolation and characterization of an abnormal alpha slow-moving high-density lipoprotein subfraction in serum from children with long-standing cholestasis.
Authors: Peynet J , Fénéant-Thibault M , Legrand A , Marot D , Rousselet F , Lemonnier A
Published: January 2020

An abnormal high-density lipoprotein (HDL) subfraction, detected during periods of mild jaundice in the serum of seven children with chronic cholestasis from birth, was isolated and characterized. This fraction, identified by its slow alpha electrophoretic migration, is present in addition to normal HDL and differs from the abnormal HDL previously described in cholestatic syndromes. It is devoid of apolipoprotein B but is precipitated by phosphotungstate-MgCl2. These properties allowed its isolation by double selective precipitation. This subfraction is undetectable with this procedure in the serum of healthy subjects, is rich in cholesterol, and contains a large amount of apolipoprotein E, which may explain its precipitation by phosphotungstate-MgCl2. These apo E-containing HDL may play a major role in the lipid metabolism of patients with long-standing cholestasis during periods of mild jaundice.
Determination of vanillylmandelic acid with ion-pair chromatography and fluorescence detection.
Authors: Lindgren K , Rodopoulos N
Published: January 2020

We describe a chromatographic procedure for sensitive (2.0 mumol/L), specific quantification of vanillylmandelic acid (VMA) in urine with iso-VMA as an internal standard. After rapid extraction from urine, the VMA is determined by isocratic reversed-phase ion-pair chromatography on a bonded C8 column and detection of the native fluorescence on excitation at 285 nm. The fluorescence signal is quite dependent on the pH of the mobile phase. Results by the method vary linearly with VMA concentration up to 320 mumol/L and correlate well (r = 0.9880) with those obtained by conventional ultraviolet spectrophotometry. The mean 24-h excretion of VMA from 29 healthy volunteers was 21.4 (SD 5.4) mumol.
Use of purified lyophilized human lactate dehydrogenase isoenzymes in a study of the measurement of lactate dehydrogenase activity.
Authors: Smith D A , Moses G C , Henderson A R
Published: January 2020

We examined the stability of human lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes 1, 2, and 3--purified to specific activities of about 200 kU/g--when lyophilized in a buffered stabilized matrix of bovine albumin. Each isoenzyme was prepared at two activity concentrations and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. LD-1 activity decayed with zero-order kinetics, LD-2 and LD-3 with first-order kinetics. The extrapolated half-lives of these preparations at -20 degrees C varied between 80 and 530 years. Stability of reconstituted samples stored at 4 degrees C was excellent for LD-1 but poor for LD-2 and LD-3. We suggest that preparations of human LD-1 be further investigated as a possible reference material.
Myoglobinemia after burn injury: relationship to creatine kinase activity in serum.
Authors: Guechot J , Lioret N , Cynober L , Letort C , Saizy R , Giboudeau J
Published: January 2020

Myoglobin is released into the blood after burn injury. We measured it and other analytes in blood collected from 22 burn patients two to seven times during their recovery. There was a significant correlation between myoglobinemia and creatine kinase (CK) activity in serum (r = 0.764; p less than 0.001). In a group of 14 thermal-burn subjects a correlation was found between burn depth (clinically expressed as Unit Burn Surface) and both myoglobinemia (r = 0.825; p less than 0.01) and CK activity (r = 0.686; p less than 0.01). In eight thermal-burn patients who were recovering satisfactorily, myoglobin and CK activity measured on days 2, 4, 7, 10, and 13 after injury were significantly increased (p less than 0.05) on days 2, 4, and 7. Evidently myoglobinemia and CK activity are good biological markers of burn depth, and reflect muscle damage equally well.
Collection of blood on filter paper for measurement of glycated hemoglobin by affinity chromatography.
Authors: Little R R , McKenzie E M , Wiedmeyer H M , England J D , Goldstein D E
Published: January 2020

We present data on a filter-paper collection and assay method for measurement of glycated hemoglobin (gHb). Onto filter paper dipped into a solution of glucose oxidase (EC 1.1.3.4) and allowed to dry, approximately 20 microL of capillary blood was spotted. For analysis, we eluted the dried blood spot from the paper by soaking in water for 1 h, then measured the gHb in the eluate by affinity chromatography. Because the gHb significantly increased from day 1 to day 14 of storage, it was necessary to standardize the day of elution from the paper. We found a high correlation between gHb measured from samples stored for 14 days on treated paper and gHb measured by affinity chromatography from frozen whole blood or hemolysates (r = 0.96). This method is convenient, requiring small amounts of blood and little sample handling and assay time, and may be particularly useful in certain situations such as large-scale screening for diabetes.
Simple liquid-chromatographic determination of urinary coproporphyrin in workers exposed to lead.
Authors: Tomokuni K , Hirai Y
Published: January 2020

We developed a simple method for the determination of urinary coproporphyrin (CP) in lead workers, using a "high-performance" liquid chromatograph (HPLC) equipped with a fluorescence detector. The detection limit of urinary CP in this method was 5 micrograms per liter of urine. The working linear range of urinary CP concentration was 5 to 1500 micrograms/L. In 41 lead-exposed workers, the urinary CP values obtained by the present HPLC method were well correlated with those obtained by a conventional spectrophotometric method (r = 0.94). The present method is useful for screening workers exposed to lead.
Liquid-chromatographic determination of 4-hydroxyproline in urine.
Authors: Hughes H , Hagen L , Sutton R A
Published: January 2020

In this method for 4-hydroxyproline in urine, hydroxyproline is derivatized with 4-chloro-7-nitrobenzofurazan, with subsequent estimation by reversed-phase "high-performance" liquid chromatography. The ranges for excretion of free and total hydroxyproline while the subjects were ingesting unrestricted diets were 2-29 and 122-374 mumol/24 h (n = 21), respectively, with no significant sex-related difference. A comparison with results by colorimetry indicated no significant differences: mean (n = 18) concentrations (mumol/L) of hydroxyproline in urine were 180 (SD 149) by the present method, 163 (SD 166) by colorimetry. For protein hydrolysate the respective values were 5.9 (SD 2.7) and 6.7 (SD 2.9).
Serum thyrotropin as measured with a one-step monoclonal-antibody radioimmunometric assay compared with two commercial radioimmunoassay kits.
Authors: Pekary A E , Hershman J M
Published: January 2020

We compared results obtained with a commercial immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used (Tandem -R TSH (ONE STEP) ImmunoRadioMetricAssay; Hybritech Inc.) with those obtained with two commercial radioimmunoassay methods: GAMMA-DAB [125I]HS-hTSH RIA (Clinical Assays) and Thyro-SHure TSH Diagnostic Kit (Nuclear Medical Laboratories). The correlation of all results obtained in the Hybritech assay with those of the two commercial RIAs exceeded 95%. Mean values for 100 euthyroid samples measured in the Hybritech assay were lower than for the other two methods. The separation between hyperthyroid and euthyroid patients was much clearer with the Hybritech assay than with the RIA methods. The Hybritech assay was far more sensitive than the Clinical Assays or the Nuclear Medical Laboratories assay.
Perchloric acid interference in enzymatic-fluorimetric-continuous-flow assay methods for measuring glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate in blood.
Authors: Stappenbeck R , Hodson A W , Skillen A W
Published: January 2020

Perchloric acid is commonly used to denature and precipitate proteins in samples before various metabolites are measured in tissue, blood, and other body fluids. However, perchloric acid can interfere in the analytical process, possibly by inhibiting the enzymes used. We have determined the effects of perchloric acid on measurements of glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate in blood by enzymatic-fluorimetric-continuous-flow assays. There was a net increase or decrease in the apparent concentration of some of these metabolites when the perchloric acid concentration in the samples differed from that of the reference standards-some of these differences were due to the concentration of perchlorate ion and some to the pH of the acid extracts. The results show the need either to add a fixed amount of blood to perchloric acid or to neutralize and remove the perchlorate.