bluuebunny/crossref_metadata_2025 huggingface dataset:
Seven different digoxin immunoassay kits compared with respect to interference by a digoxin-like immunoreactive substance in serum from premature and full-term infants.
Authors: Pudek M R , Seccombe D W , Jacobson B E , Whitfield M F
Published: January 2020

Seven different digoxin immunoassay kits showed cross reactivity with an endogenous digoxin-like immunoreactive substance consistently present in serum of neonates, whether premature or full-term. The degree of interference, in decreasing order was: NML greater than New England Nuclear greater than Bio-Rad greater than Clinical Assays greater than Becton Dickinson greater than Serono greater than Syva (EMIT). More recently purchased NML kits showed less sensitivity to the substance, evidently reflecting lot-to-lot differences in antibody. A single baseline determination of the substance before digoxin is administered inadequately compensates for this interference, because the interferent concentrations can differ from day to day, with evidence that it may be most concentrated on the fourth to sixth postnatal day. Its concentration in the serum of neonates is unrelated to the concentration of dehydroepiandrostenedione sulfate, an indicator of fetal adrenal-cortical activity.
Determination of ultrafiltrable zinc in plasma by flameless atomic absorption spectrophotometry.
Authors: Whitehouse R C , Prasad A S , Cossack Z T
Published: January 2020

Ultrafiltrable zinc can be determined in small volumes of plasma by using Amicon ultrafiltration membrane cones and either flameless atomic absorption spectrophotometry or 65Zn. The ultrafiltration membrane cone is made sufficiently zinc-free by acid washing, followed by rinsing with de-ionized water. Within-run CVs were 12 and 10%, respectively, for the two methods. Matrix effects are compensated for by using standards in solutions with an inorganic matrix that simulates the sample matrix. Results for ultrafiltrable zinc by the two techniques agreed, and agreed with previously published data obtained by different ultrafiltration techniques. The mean (and SD) percentage of plasma zinc that was ultrafiltrable in 12 control subjects was 1.0 (0.5) by AAS and 0.7 (0.3) by 65Zn.
Use of immunoaffinity chromatography for purification of 125I-labeled human prolactin.
Authors: Stuart M C , Boscato L M , Underwood P A
Published: January 2020

We assessed a simple method for purifying 125I-labeled human prolactin, taking advantage of the abundant supplies of monoclonal antibodies available. 125I-Labeled human prolactin purified by immunoaffinity chromatography is compared with that purified by gel filtration on Sephadex G-100. We used monoclonal antibodies to prolactin to prepare the affinity chromatography columns. Prolactin was radiolabeled by the Chloramine T method, purified by each of the above procedures, and the binding and displacement characteristics were studied in radioimmunoassays in which either monoclonal antibodies or a rabbit anti-prolactin serum was the first antibody. A nonimmune fraction of 125I-labeled prolactin that co-eluted with the immunoreactive hormone from Sephadex G-100 was removed by affinity chromatography, which increased the antibody binding of 125I-labeled prolactin in the radioimmunoassay in the absence of unlabeled antigen (B/T0, in percent) twofold or more, increased the assay sensitivity, and increased the slope of antigen displacement measured by the 50% intercept. Several advantages make this the purification method of choice.
A direct radioimmunoassay for aldosterone in plasma.
Authors: Lun S , Espiner E A , Nicholls M G , Yandle T G
Published: January 2020

This rapid radioimmunoassay for aldosterone is performed directly on 100 microL of unprocessed plasma, with 125I-labeled aldosterone as the labeled antigen. Our use of steroid-free plasma in preparing the standard curve resulted in an overestimate of aldosterone; this problem was overcome by adding to such plasma a mixture of other steroids to provide a constant steroid/aldosterone ratio. Over a wide range of aldosterone concentrations, results agreed well between the present assay and a routine method involving solvent extraction and paper chromatography (r = 0.85), and sensitivity (20 ng/L) and inter- (10.4%) and intra- (3.9%) assay CVs were better with the present assay. Our assay is especially useful for multiple samples and (or) when only small-volume samples are available.
Determination of the antidepressants maprotiline and amoxapine, and their metabolites, in plasma by liquid chromatography.
Authors: Wong S H , Waugh S W
Published: January 2020

Maprotiline, amoxapine, and their metabolites were determined in plasma by three separate procedures, each of which involves a three-step extraction followed by reversed-phase separations. Amoxapine, maprotiline, and N-desmethylmaprotiline can be measured with use of a conventional C-18 column with 10% carbon load, but analyses for 7- and 8-hydroxyamoxapines require a small (5-microns) particle size, high carbon load, 20% C-18 column with 0.2 mol/L phosphate as mobile phase. Within-run and day-to-day CVs were 5% and 8%, respectively. Peak-height ratios were linearly correlated with concentrations between 0 and 400 micrograms/L. Detection limits ranged from 1 to 3 ng. Interference studies indicated that nortriptyline co-elutes with maprotiline. Assays of patients' plasma showed substantial amounts of these drugs and most of their metabolites, but only trace amounts of 7-hydroxyamoxapine.
Gas-chromatographic measurement of 3-hydroxybutyrate and lactate in plasma and 3-hydroxybutyrate in whole blood.
Authors: Cosmi G , Di Corcia A , Samperi R , Vinci G
Published: January 2020

We describe two simple, rapid, and sensitive gas-chromatographic methods. One is for determining 3-hydroxybutyrate and lactate in plasma, the other for determining 3-hydroxybutyrate in perchloric acid extracts of blood. Samples are purified by adsorption onto graphitized carbon black (Carbopak B). After desorption, the two hydroxyacids are gas-chromatographically measured. Chromatography is on Carbopack B/polyethylene glycol (Mr 20 000)/trimesic acid, 94.4/ 4.8/0.8 by weight. A series of determinations of 3-hydroxybutyrate in whole blood from 15 subjects gave a mean of 59 (SD 46) mumol/L, with 97.8% analytical recovery (range 94.4-101.8%). In 15 plasma samples, we determined a mean value for 3-hydroxybutyrate of 62 (SD 72) mumol/L, with an analytical recovery of 96.4% (range 93.5-97.6%) and a mean value for lactate of 0.99 (SD 0.55) mmol/L, with an analytical recovery of 92.0 (range 91.3-97.6%). Six replicate analyses of plasma containing typical concentrations of lactate and 3-hydroxybutyrate gave respective CVs of 3.2, 2.1, 1.6, and 1.4%, and 4.9, 3.7, 2.8, and 2.0% for low, medium, high, and very high concentrations. Total analysis time for either method is less than 1 h.
Creatine kinase B subunit as measured with a radioimmunoassay kit in detection of acute myocardial infarction.
Authors: Witherspoon L R , Shuler S E , Genre C F , Gilbert S S , Moore R J , Meihaus V , Hurry E K
Published: January 2020

Results with a commercial radioimmunoassay (RIA) reagent kit for quantification of the creatine kinase B subunit (CK-B) (Nuclear-Medical Laboratories, Irving, TX 75061) were compared with results obtained by electrophoresis for patients consecutively admitted to our coronary care unit for suspected acute myocardial infarction. Analytical sensitivity, precision, and specificity of the RIA were satisfactory. Its clinical efficacy was assessed in 97 patients suspected of having had an acute myocardial infarction. Of 30 patients who had had an acute myocardial infarction, increased CK-B was detected by RIA in 30 and by electrophoresis in 27. The temporal relationship between CK-B by RIA and CK-MB by electrophoresis was similar. Of 66 admissions where infarction was not established, CK-B was negligibly increased in samples from four patients by RIA, and from one by electrophoresis. Although not abnormally increased (greater than 5 U/L), CK-MB was detected by electrophoresis in samples from another five of these 66 patients. We conclude that estimation of CK-B by this RIA is an excellent alternative to estimation of CK-MB by electrophoresis in patients suspected of having had an acute myocardial infarction.
Usefulness of serial determinations of myoglobin and creatine kinase in serum compared for assessment of acute myocardial infarction.
Authors: Cairns J A , Missirlis E , Walker W H
Published: January 2020

Twenty-one patients with their first myocardial infarction underwent serial blood sampling every 2 h for determination of serum creatine kinase (CK) and myoglobin during the first 48-72 h after onset of pain. The first blood sample, obtained at a mean time of 4.4 h after infarct onset, invariably showed increased myoglobin (mean, 8.3-fold normal), whereas CK was often normal (mean, 1.6-fold normal). Peak myoglobin values occurred earlier than peak CK values (9.9 h vs 21.6 h, p less than 0.0005), but there was a significant correlation of peak values (myoglobin = 0.384CK - 0.264, r = 0.794, p less than 0.0005). The mean exponential disappearance rate (Kd) of CK was 0.00106 min-1 and of myoglobin was 0.00265 min-1 (p less than 0.0005). The disappearance of myoglobin was well described by a mono-exponential expression except in two patients. The total duration of the increase in myoglobin was significantly less than that of CK (34.7 h vs 74.4 h, p less than 0.0005).
Optimized atomic absorption spectrophotometry of zinc in cerebrospinal fluid.
Authors: Palm R , Sjöström R , Hallmans G
Published: January 2020

This method for direct determination of Zn in cerebrospinal fluid (CSF) involves flame atomic absorption spectrophotometry with a pulse nebulizer technique. Standard solutions of Zn in 150 mmol/L NaCl were used. We could account for 88% of added standard with the method in individual samples from 10 patients and in pooled CSF. The method is acceptably precise, CVs in pooled CSF ranging from 4 to 12%. The mean CSF-Zn concentration for nine healthy men was 0.18 (SD 0.04) mumol/L and for nine healthy women 0.15 (SD 0.03) mumol/L, a statistically insignificant difference. These values are lower than those in previous reports, which may have been the result of contamination problems, nonatomic absorption, or nonstandardized sampling. In the healthy volunteers, the CSF-Zn concentration was positively correlated with serum-Zn, CSF-protein, and CSF-albumin concentrations, as well as with the CSF/serum ratio for albumin.
Lipid concentrations in serum and EDTA-treated plasma from fasting and nonfasting normal persons, with particular regard to high-density lipoprotein cholesterol.
Authors: Folsom A R , Kuba K , Leupker R V , Jacobs D R , Frantz I D
Published: January 2020

Serum/plasma sample pairs, collected both during (12-h) fasting and nonfasting from 44 healthy subjects, were analyzed for total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. Values for total cholesterol and triglycerides averaged 2.1% and 5.2%, respectively, higher for serum than for plasma. Values for HDL cholesterol were not different. Triglyceride concentrations in nonfasting subjects were 40% higher than concentrations during fasting, but total cholesterol and HDL cholesterol were not significantly affected by fasting. These findings suggest that concentrations of HDL cholesterol may be directly compared in clinical and epidemiological settings without regard to whether serum or plasma was sampled or whether the subjects were fasting or nonfasting.
A peroxidase-coupled method for the colorimetric determination of serum triglycerides.
Authors: McGowan M W , Artiss J D , Strandbergh D R , Zak B
Published: January 2020

We describe an enzymatic method for rapid, precise measurement of serum triglycerides with use of sample:reagent ratios as large as 1:200. Hydrolysis of triglycerides is catalyzed by lipase to produce glycerol and free fatty acids. The glycerol generated is then phosphorylated by adenosine 5'-triphosphate in the presence of glycerol kinase. Oxidation of the resulting glycerol 3-phosphate to produce hydrogen peroxide is catalyzed by L-alpha-glycerophosphate oxidase. An intense red chromogen is produced by the peroxidase-catalyzed coupling of 4-aminoantipyrene and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate with hydrogen peroxide. This sensitive chromogen system not only permits use of unusually small sample volumes, it also facilitates a linear response to serum triglyceride concentrations up to at least 10 g/L while displaying good Ringbom (measure of accuracy) characteristics.
Identification of the serum binding proteins for iron, zinc, cadmium, nickel, and calcium.
Authors: Scott B J , Bradwell A R
Published: January 2020

The binding of five biologically important metals to serum proteins has been studied. After suitable radioactive isotopes were added to serum proteins separated and precipitated by two-dimensional immunoelectrophoresis, the sample plates were exposed to roentgenogram film. 59Fe bound to transferrin alone; 65Zn bound mostly to albumin, but also to another 12 proteins; 109Cd was mostly associated with alpha 2-macroglobulin, but was also present on albumin, immunoglobulins G and A, and prealbumin; 63Ni, added in high concentration, was associated with an alpha 2-mobility protein and albumin; and, finally, 45Ca was mostly bound to albumin, but seven other binding proteins were also identified, with transferrin predominant. The results are not quantitative, but the technique is simple and specific, and the information gained can direct further studies on isolated proteins.
Relationships among human amniotic fluid dipalmitoyl lecithin, postpartum respiratory compliance, and neonatal respiratory distress syndrome.
Authors: Lohninger A , Salzer H , Simbruner G , Husslein P , Martin G
Published: January 2020

The major molecular species of amniotic fluid phosphatidylcholine were determined as diacylglycerol trimethylsilyl ether derivatives by gas-liquid chromatography with use of glass capillary columns. We studied amniotic fluid specimens from 48 pregnancies. As expected, the major disaturated species of amniotic fluid phosphatidylcholine, dipalmitoylphosphatidylcholine, and palmitoylmyristoylphosphatidylcholine increased with gestational age, whereas phosphatidylcholine species with 34, 36, and 38 carbon atoms in the acyl radicals decreased. Although the amniotic fluid samples were obtained shortly before parturition (4.2 +/- 3.7 h, mean +/- SD), the correlation between dipalmitoylphosphatidylcholine concentration in amniotic fluid and gestational age was better than between dipalmitoylphosphatidylcholine concentration and the compliance of the respiratory system of the newborns. In addition, the percentage of amniotic fluid phosphatidylcholine species present as dipalmitoylphosphatidylcholine seemed to be a more reliable predictor of lung maturity than was the absolute dipalmitoylphosphatidylcholine concentration in amniotic fluid.
Does lactate dehydrogenase isoenzyme-5 contribute to the predictive power of total lactate dehydrogenase in myocardial infarction?
Authors: Chapelle J P , Albert A , Smeets J P , Maréchal J P , Heusghem C , Kulbertus H E
Published: January 2020

In 385 patients with acute myocardial infarction, lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes were determined electrophoretically 24, 48, and 72 h after admission. At those times, LD-1/LD-2 ratios exceeding 1 were recorded in 78.9, 88.8, and 92.2% of the cases, respectively. LD-1 ranged from 181 to 2674 U/L, or 21.9 to 66.1% of the total activity. On the first day of hospitalization, 27.3% of the patients demonstrated abnormal LD-5 (greater than 6% of total LD); this finding dropped to 20.5% and 17.4% in the two following days. Early increases in LD-5 were most frequently observed in patients associating inferior infarcts with posterior or lateral extension and having a previous history of myocardial infarction. On day 1, LD-5 was significantly increased in early deceased patients as compared to long-term survivors (9.7% vs 4.9% of total LD, p less than 0.01). LD-5 definitely contributes to the prognostic efficiency of total LD in acute myocardial infarction, but does not replace it as a risk predictor. This study confirms the superiority of total LD over the isoenzyme measurements to achieve short-term prognostication.
Utility of isoelectric focusing of cerebrospinal fluid and serum on agarose evaluated for neurological patients.
Authors: Link H , Kostulas V
Published: January 2020

Agarose isoelectric focusing was used to demonstrate oligoclonal bands in cerebrospinal fluid (CSF) and serum from 998 consecutive neurological patients. Compared with agarose electrophoresis, agarose isoelectric focusing was slightly more sensitive, showing more (and more easily discernible) oligoclonal bands. Agarose isoelectric focusing, which has good reproducibility, revealed oligoclonal bands in CSF in 95% of 43 patients with multiple sclerosis, 44% of 39 with aseptic meningoencephalitis, and 14% of 906 with other neurological diseases. Interestingly, oligoclonal bands were found in CSF from 12% of 162 patients with acute cerebral infarction and 23% of 53 with polyneuropathy, and also in 29% of 17 with dementia, while only 4% of 206 patients with headache, vertigo, or psychoneurosis had this CSF abnormality. We recommend this procedure for the routine examination of paired CSF and serum specimens for the presence of oligoclonal bands.
Salicylate determined with a microcentrifugal analyzer, and compared with Du Pont aca, trinder, and liquid-chromatographic methods.
Authors: Eckfeldt J H , Nelson K M
Published: January 2020

We describe a new automated method for measuring serum salicylate in the Multistat III microcentrifugal analyzer. Ferric nitrate reagent and serum blanking are used. We compare this new method, the automated Du Pont aca method, and the manual Trinder method with a "high-performance" liquid-chromatographic method. The unblanked Trinder method had the poorest correlation (r = 0.980, Sy X x = 19.1) with the chromatographic method. The serum-blanked aca and Multistat III methods showed better correlation (r = 0.995, Sy X x = 9.5 mg/L, and r = 0.991, Sy X x = 13.0 mg/L, respectively) with the chromatographic method. However, we conclude that all three colorimetric methods give clinically useful results and that the increased time, expense, and expertise required for chromatographic salicylate analysis are difficult to justify in a routine clinical laboratory.
Interpretation of cerebrospinal fluid protein assays in various neurologic diseases.
Authors: Christenson R H , Behlmer P , Howard J F , Winfield J B , Silverman L M
Published: January 2020

Albumin and immunoglobulin G (IgG) were determined in cerebrospinal fluid (CSF) and serum, and the CSF/serum albumin Index (CSF X 10(3)/serum albumin concentration ratio) and IgG Index [(CSF/serum IgG)/(CSF/serum albumin)] were calculated. Data for these indices and oligoclonal banding are described in 23 cases of multiple sclerosis (MS), 19 of systemic lupus erythematosus (SLE), eight of sarcoidosis, 48 cases of miscellaneous disease, and 25 control patients with nonspecific complaints. Of the MS, SLE, and sarcoidosis patient groups, 8.5%, 26%, and 12.5% showed an abnormally high CSF/serum albumin Index; 87%, 16%, and 0% an increased IgG Index; and 87.5%, 42% and 0% showed positive oligoclonal banding. IgG Index and oligoclonal banding results for MS patients differed significantly from the sarcoidosis (p less than .001) and SLE (p less than .05) groups. When the CSF/serum albumin Index is considered also, the control and sarcoidosis patient results differ significantly from the MS group (p less than .001 and p less than .01). A strong correlation between the IgG Index and oligoclonal banding is implicated.
Stability of estrogen- and progesterone-receptor concentrations in human uterus tissue.
Authors: Zachariah N Y , Chakmakjian Z H
Published: January 2020

Homogeneous control specimens for estrogen receptor (ER) and progesterone receptor (PR) assays were prepared from freshly collected human uterus. After removal of the connective tissue, the specimen was washed with isotonic saline, cut into small pieces, quickly frozen in liquid nitrogen, and stored at -70 degrees C until analyzed. Cytosol prepared from this specimen was lyophilized and stored at -70 degrees C. A single step of reconstitution, with glycerol (100 mL/L) in water, is sufficient to prepare a control. Two specimens prepared this way were found to be reasonably stable for 20 months (first specimen, mean +/- SD: ER = 22.1 +/- 2.9 fmol/mg, PR = 136.5 +/- 26.9 fmol/mg; second specimen: ER = 107.2 +/- 11.7 fmol/mg, PR = 922 +/- 71.6 fmol/mg). Another specimen, prepared similarly but not frozen in liquid nitrogen soon after collection, was less stable; its ER and PR concentrations deteriorated faster.
New "on-line" sample-pretreatment procedure for routine liquid-chromatographic assay of low-concentration compounds in body fluids, illustrated by triamcinolone assay.
Authors: Schöneshöfer M , Kage A , Weber B
Published: January 2020

In this fully automated technique for sample cleanup before chromatographic or other quantitation steps, analytes in body fluids are enriched and semi-purified on a first column. After their selective elution, analytes are "transformed" by admixing appropriate solvents in such a way that they are focused on the top of a second column. By backflush, they then are transferred to an analytical liquid-chromatographic column (or simply eluted for quantification by other techniques). This technique is illustrated by the liquid-chromatographic assay of triamcinolone from a 1-mL urine sample, with ultraviolet detection. Because analytical recovery is almost complete and precision high, no internal standardization is necessary. Interference is eliminated as well as or better than with manual techniques. Chief advantages of this technique are online operation, processing of samples of larger volume, low cost with respect to extraction devices, and nearly universal applicability for exogenous or endogenous compounds of clinical relevance. It potentially may be widely applied.
"Unbound analog" radioimmunoassays for free thyroxin measure the albumin-bound hormone fraction.
Authors: Stockigt J R , Stevens V , White E L , Barlow J W
Published: January 2020

We have assessed the influence of albumin-bound thyroxin (T4) on apparent free T4 values obtained by two "unbound analog" free T4 methods (AmerlexR Free T4 and Clinical Assays one-step Free T4). We evaluated sera showing three different albumin anomalies: total hereditary analbuminemia, partially corrected analbuminemia, and familial dysalbuminemic hyperthyroxinemia, where abnormal albumin-binding of analog tracer is associated with high apparent free T4 values by these methods. In hereditary analbuminemia, free T4 was almost undetectable by both assays; in contrast, free T4 by equilibrium dialysis was normal. After addition of T4-free human serum albumin, the apparent free T4 concentration in total hereditary analbuminemia became normal by the analog methods. Immunoprecipitation of [125I]T4 and the unidentified labeled kit analogs by antiserum to human albumin was negligible in untreated total hereditary analbuminemia and approximately twice normal in familial dysalbuminemic hyperthyroxinemia. Therefore, alterations in tracer binding to albumin correlate with the apparent free T4 concentrations obtained by the analog methods. The interactions of the unidentified analog tracers and T4 with albumin are such that these techniques principally reflect the albumin-bound T4 moiety.
Nonaqueous reversed-phase liquid chromatography and fluorimetry compared for determination of retinol in serum.
Authors: Nelis H J , De Roose J , Vandenbavière H , De Leenheer A P
Published: January 2020

A new assay for retinol in human serum, based on nonaqueous reversed-phase liquid chromatography, is presented. Sample preparation includes addition of the internal standard, retinyl propionate, deproteinization of 100 microL of serum with acetonitrile, and extraction with hexane. The standard curve is linear up to 2 mg/L. The assay is characterized by excellent sensitivity (detection limit, 15 micrograms/L) and good within-run and between-run precision (CVs of 2.6 and 2.7%, respectively), and results compare favorably with those by fluorimetry. We assayed 135 samples from hospitalized patients by both methods. Although the two sets of values correlated well (r = 0.955) the fluorimetric method occasionally suffers from interferences. In practice, fluorimetry proves valuable as a routine method, while liquid chromatography meets the criteria of a potential reference method.
Use of laser-excited fluorescence to measure mixed-function oxidase activity.
Authors: Egan B Z , Lee N E , Burtis C A , Kao J Y , Holland J M
Published: January 2020

The microsomal mixed-function oxidase (MFO) system is involved in the metabolism of various chemical compounds. Polycyclic aromatic hydrocarbons are metabolized by the cytochrome P-448 enzyme system, which contains MFOs. Induction of this MFO activity may be useful as an indicator of the toxicity of the inducer material. We have successfully used a portable centrifugal analyzer equipped with an argonion laser light source to quantify cytochrome P-448 activity induced in mouse-liver microsomes by exposing the animal's skin to different doses of liquids derived from a coal-liquefaction process. The MFO activity was determined kinetically by measuring the rate at which the highly fluorescent compound, resorufin, produced by the oxidation of 7-ethoxyresorufin substrate, was formed. The 514.5-nm laser excitation beam was directed with a fiber-optic bundle from the laser to the cuvets of a specially designed rotor; emitted fluorescence was monitored at 90 degrees to the incident beam through a 560-nm cut-on secondary filter. Use of the laser excitation source allowed very low MFO activities to be measured: picomole quantities of resorufin could be determined. We anticipate that the increased sensitivity of the method described here will allow MFO activities to be determined in body fluids and skin.
Molecular markers of hemostatic disorders: implications in the diagnosis and therapeutic management of thrombotic and bleeding disorders.
Authors: Fareed J , Bick R L , Squillaci G , Walenga J M , Bermes E W
Published: January 2020

With current technological advances, it is now possible to measure in less than 50 microL of plasma picomolar amounts of circulating products of platelet activation, products of protease activation related to coagulation and fibrinolytic pathways, and prostaglandin metabolites formed during a physiologic or pathologic process. Most of these markers, which circulate in blood in nanogram or picogram amounts per milliliter during or after pathologic activation, provide pertinent information on the status of a patient in terms of specificity and early detection, and will be of crucial value in the diagnosis of hemostatic defects and the management of newer antithrombotic drugs that cannot be monitored by currently available assays. Currently, 125I- and 3H-based simple radioimmunoassays are available for platelet factor 4, beta-thromboglobulin, fibrinopeptide A, B beta 15-42 related peptides, thromboxane B2, and the prostaglandins 6-keto-PGF1 alpha and PGE2. Nonisotopic methods such as enzyme-linked immunosorbent assays and fluoroimmunoassays are being developed. Serotonin and ADP, products of platelet activation, are measurable by liquid-chromatographic, immunoenzymatic, and spectrophotofluorometric methods. Analytical methods for fibrin split products (fragments D and E) and serine protease inhibitor complexes such as thrombin-antithrombin-III, factor Xa-antithrombin-III, and kallikrein-C1-esterase are also being developed. We have evaluated all of these methods and found them to be very sensitive to those components of endogenous activation of the hemostatic system listed above.
Two commercial methods evaluated for eliminating the labile fraction from the assay for glycated hemoglobin (glycohemoglobin).
Authors: Nathan D M , Dunn B S , Francis T B
Published: January 2020

Two recently introduced commercially available methods, a minicolumn and an electrophoresis assay, both effectively remove labile glycated hemoglobin generated in vitro and give results for patients' samples similar to those by liquid chromatography with samples pre-incubated in saline.
Salivary estriol concentrations during normal pregnancies, and a comparison with plasma estriol.
Authors: Evans J J , Wilkinson A R , Aickin D R
Published: January 2020

Saliva would have advantages over plasma or urine for monitoring estriol during pregnancy. Specimen collection, after stimulation of flow by citric acid, is non-invasive and simple. We measured concentrations of unconjugated estriol in saliva and compared them with those in plasma in normal pregnancies, and found a good correlation (r = 0.79). In addition, trends of concentrations in saliva and plasma were statistically compared and found to be highly correlated. The variation among individuals in the saliva/plasma concentration ratio suggested that some inter-individual factor(s) may affect this relationship. The normal reference interval for unconjugated estriol concentration in saliva from 20 weeks of gestation to term was established.
Liquid and lyophilized quality-control materials compared for use in continuous-flow analysis.
Authors: Elin R J , Gray B A
Published: January 2020

Using a Technicon SMAC continuous-flow instrument, we compared a liquid quality-control material ("Decision level 3," Beckman) with two different lyophilized quality-control materials. The variances for 19 analytes in the three materials were significantly greater for Decision than for the Ortho Abnormal and the Omega II lyophilized control sera (p less than 0.00001 for each comparison by the omnibus test). The imprecision for Decision was not due to incomplete mixing or carryover. The viscosity of Decision, however, was more than twice that of the two lyophilized control sera or of pooled human serum.
Simplified determination of serotonin in plasma by liquid chromatography with electrochemical detection.
Authors: Tagari P C , Boullin D J , Davies C L
Published: January 2020

This simplified determination of serotonin in human plasma involves "high-performance" liquid chromatography with electrochemical detection. Deproteinated samples of platelet-poor plasma are injected directly into the chromatograph without preliminary chromatographic purification. The limit of quantitative detection of serotonin in plasma is 5 nmol/L (equivalent to 250 fmol injected). Intra- and inter-assay CVs ranged from 5.6% to 7.7% and 3.5% to 14.9%, respectively, for concentrations of serotonin from 0.02 to 10.0 mumol/L. Results agreed well with those by spectrofluorometry, both for plasma standards (r = 0.985, range 100 nmol/L to 100 mumol/L) and for pathological samples obtained from four carcinoid syndrome patients (r = 0.964). The method is also applicable to the concurrent measurement of 5-hydroxytryptophan and 5-hydroxyindoleacetic acid.
Differences in radioimmunoassay results for thyroglobulin that affect management of patients with thyroid cancer.
Authors: Bayer M F , McDougall I R
Published: January 2020

We compared two commercial assays for measurement of serum thyroglobulin [Nuclear Medical Systems (NMS) and "CIS" (Damon Diagnostics)] with each other and with one developed at Stanford (J Clin Endocrinol Metab 49:557-564, 1979). The NMS assay is a competitive-binding RIA, the CIS and Stanford assays are two-site immunoradiometric assays. The kit standards varied in thyroglobulin concentration. The NMS standards differed in immunoreactivity from thyroglobulin in clinical specimens and from the other standards. Also, nonparallelism between standards and patients' sera in the NMS assay suggested a less-specific antiserum. Results by the CIS and Stanford assays correlated well (n = 120, r = 0.964), those by the NMS assay less strongly (n = 101, r = 0.855 vs CIS, r = 0.888 vs Stanford). Clinical evaluation in 50 patients treated for differentiated thyroid carcinoma (10 with metastases and 40 currently disease-free) indicated good agreement for positive results by the three assays. The CIS and the Stanford assay both gave high results (greater than or equal to 25 micrograms/L) in all 10 cases with metastases; the NMS RIA identified eight of these patients (thyroglobulin greater than or equal to 30 micrograms/L), but excluded two with anti-thyroglobulin autoantibodies. In subjects without disease, however, the percentage of undetectable thyroglobulin (negative result), as opposed to low measurable thyroglobulin (inconclusive result) varied considerably: 85% by CIS, 30% by NMS, and 75% by the Stanford assay.
Study of a mechanism responsible for anomalous results for free thyroxin by some analog assays for subjects with albumin deficiency.
Authors: Wilke T J
Published: January 2020

I examined the effect of albumin deficiency, as might occur in pregnancy and nonthyroidal illness, on four analog-based assays for free thyroxin (Amersham's Amerlex, Diagnostic Products Corporation's Coat-A-Count "one-step," Clinical Assays' Gammacoat "one-step," and Corning's Immophase "single-step"). In subjects with albumin deficiency per se, Amerlex and Coat-A-Count results for free thyroxin were not significantly different from normal, whereas free thyroxin values by Gammacoat and Immophase assays were lower than that of the control. Furthermore, in the albumin-deficient group, the mean amount of thyroxin associated with antisera in each assay was significantly higher than that of the control and correlated well with the degree of decrease in free thyroxin. I conclude that subnormal results for thyroxin by analog assays, in euthyroid patients with albumin deficiency, is a function of the specific assays and is not a characteristic of analog assays per se. In addition, the study suggests that albumin deficiency alters the distribution between thyroxin and its binders in all four analog assays, but only in the Gammacoat and Immophase methods is this alteration sufficiently large as to distort assay results.
Homogeneous enzyme immunoassay for cortisol with a centrifugal analyzer.
Authors: Izquierdo J M , Quirós A , Alvarez-Uría J , Sotorrío P
Published: January 2020

We determine serum cortisol by a homogeneous enzyme immunoassay in the Cobas Bio centrifugal analyzer. To unbind cortisol from its protein complex, serum is treated for 15 min with an acidic solution. The reaction then proceeds automatically in the analyzer at 37 degrees C. To 50 microL of sample mixture is added 125 microL of reagent (cortisol antibodies, glucose 6-phosphate, and NAD+). This mixture is incubated for 60 s, after which 25 microL of a cortisol derivative labeled with glucose 6-phosphate is added; the increase in absorbance is monitored at 340 nm. The standard curve was linear from 10 to 500 micrograms of cortisol per liter. Within-assay precision (CV) varied from 0.2 to 0.6%, between-assay precision from 6.2 to 10.6%. Analytical recovery ranged from 100 to 103%. Results for control samples deviated from target values by 1.4 to 7.8%. Results compared well with those by radioimmunoassay. The method is reliable and practicable and will usefully replace previous routine methods for serum cortisol.
Chromatograph with external column evaluated for determining breath hydrogen in clinical studies.
Authors: Rosado J L , Torres L , Solomons N W
Published: January 2020

A novel breath-hydrogen analyzer involving an external chromatographic column in the form of a cartridge packed with molecular-sieve material and dessicant is described. The instrument incorporates a sintered SnO2 solid-state sensor into the detector, which registers a change in electrical potential in response to the adsorption of reducing gases; ambient room air is drawn through an activated charcoal filter by a small, internal pump and used as the carrier gas. Once calibrated with a gas of known H2 concentration, a digital meter registers the concentration of H2 in an unknown sample of breath air. The response is linear with concentration up to 160 microL/L. The external column is both necessary and effective for screening out contaminating reducing gases. The correlation coefficient for 250 gas samples measured simultaneously with an external-cartridge instrument and with an internal column chromatography is greater than 0.99. Results by the new analyzer corresponded 100% with those by the conventional machine for detecting carbohydrate malabsorption; for diagnosing efficient carbohydrate absorption, results by the two instruments agreed for 96% of the tests.
The distribution of chymotrypsin within the feces and description of a new device for the preparation of stool samples.
Authors: Kaspar P , Neumann U
Published: January 2020

We measured the distribution of chymotrypsin within feces by comparing duplicate 100-mg stool aliquots from 48 stool specimens. For practical purposes, the distribution of the enzyme in stool appears sufficiently homogeneous to assure representative results even from 100-mg stool aliquots. Given this likelihood, we have developed a new disposable device that measures 100-mg stool aliquots hygienically, prepares this material for shipping by mail, homogenizes and suspends the stool sample, and facilitates centrifugation of the fecal suspension, all within the same instrument. Performance studies with this new device revealed that stools of different consistency can be measured out precisely and conveniently by volume. The difference in the specific weight of different stools appears to be so low that for practical purposes a 100-mg mass of stool can be weighed out by volume.
Effect of heat and sodium dodecyl sulfate on solubilization of proteins before two-dimensional polyacrylamide gel electrophoresis.
Authors: Hodges S C , Hirata A A
Published: January 2020

To solubilize biological samples, sodium dodecyl sulfate (SDS) frequently is added and the mixture heated at 70-100 degrees C. However, two-dimensional polyacrylamide gel electrophoresis of a single protein after SDS treatment has not been reported. When rabbit-muscle creatine kinase was so run, we saw considerable difference in the gel staining pattern for the heated and nonheated enzyme dissolved in the SDS solution. After heating for 10 min at 95 degrees C the number of silver-stained spots apparent increased, and staining of several spots intensified. After 60 min, most of the discrete spots disappeared. Evidently the peptide backbone had been hydrolyzed. When the enzyme was simply left at room temperature for four days, the effects were similar. Appearance of new spots and loss of spots apparently are caused by heating alone but are intensified by SDS. Experiments with human serum albumin yielded similar results.
A new succinylcholine-based assay of plasma cholinesterase.
Authors: Abernethy M H , George P M , Melton V E
Published: January 2020

We describe a new method for measuring the in vitro rate of hydrolysis of the muscle relaxant succinylcholine. This substrate is hydrolyzed by plasma cholinesterase (EC 3.1.1.8). The resulting choline is determined by measuring the hydrogen peroxide formed on its oxidation by choline oxidase (EC 1.1.3.17). This is done by use of phenol and aminoantipyrine coupled to peroxidase, and yields an intense chromophore, Amax 500 nm. The assay requires 0.1 mL of plasma, and is precise and specific. The CV was 2.7% within run, 7.3% between run. For the usual (U variant) enzyme the Km is 53 mumol/L. Enzyme activity is removed by anticholinesterase antiserum, and is inhibited by dibucaine with a Ki of 2 mumol/L. Ten samples can be assayed in duplicate in an hour. This method is suited to routine use in any laboratory that has a simple spectrophotometer. The mean activity in 11 individuals with the cholinesterase phenotype UU was 105 U/L, for seven UA heterozygotes 61 U/L, and for three AA homozygotes 4 U/L. To the extent allowed by extrapolation from in vitro to in vivo results, this method should increase diagnostic accuracy and may directly predict duration of succinylcholine-induced apnea.
Plasma prolidase activity: a possible index of collagen catabolism in chronic liver disease.
Authors: Myara I , Myara A , Mangeot M , Fabre M , Charpentier C , Lemonnier A
Published: January 2020

We describe here an easy method of determining prolidase (EC 3.4.13.9) in plasma after preincubation with Mn2+ for 24 h at 37 degrees C to maximize prolidase activity. The mean activity in 338 patients who were either in hospital or outpatients was 900 U/L +/- 520 (2 SD), unrelated to sex or age. In 25 of these 338 samples tested, prolidase activity was between 1500 and 2000 U/L. It exceeded 2000 U/L in eight, all of whom were patients with chronic liver disease. Plasma prolidase activity was normal in cytolytic syndromes such as liver or heart disease. Of the 27 patients with cirrhosis, only five exhibited prolidase activity greater than 2000 U/L. Plasma prolidase activity was uncorrelated with six biochemical indexes to liver function (the aminotransferases, alkaline phosphatase, glutamyltransferase, total bilirubin, and serum albumin) or with the degree of cirrhotic fibrosis. We believe that plasma prolidase activity may be high only in the early stage of fibrosis. This hypothesis would be consistent with the data on rat-liver collagenolytic activities during CCl4 administration. Monitoring of plasma prolidase activity might be useful in evaluating fibrotic processes in chronic liver disease in the human.
Determination of creatine kinase MB activity with the Du Pont aca: interferences from the sample matrix.
Authors: Stein W , Bohner J
Published: January 2020

During the last three years we and other have observed discrepancies between results for creatine kinase isoenzyme MB as measured with the mechanized ion-exchange chromatographic method in the Du Pont aca and those by other techniques. These observations prompted us to investigate the influence of the matrix on the Du Pont CK-MB assay. We conclude that, apart from possible interferences by CK-MM, CK-BB, and both types of macro CK, the aca will give apparent CK-MB activities that are directly related to protein concentration and inversely related to sodium chloride concentration. Practical consequences for the routine and emergency laboratories are: no diluted samples are allowed; application is restricted to samples from patients suspected of acute myocardial infarction which show upper-normal total CK activity; and multiple timed samples are run, in order to recognize the typical change in enzyme pattern with time.
Improved diagnosis of classical vs atypical phenylketonuria by liquid chromatography.
Authors: Matsubara Y , Heininger J A , Lin Y Y
Published: January 2020

Atypical phenylketonuria among infants with hyperphenylalaninemia must be promptly diagnosed and differentiated from classical phenylketonuria, because these patients require different treatment to prevent irreversible neurological damage. Measurement of pteridines in urine by liquid chromatography has been widely used for this purpose. Here we report a rapid, simplified liquid-chromatographic method for simultaneously measuring urinary biopterin, neopterin, and creatinine. Unlike previous methods, this method involves neither tedious cleanup procedures nor special equipment.
Enzymatic determination of D-mannose in serum.
Authors: Soyama K
Published: January 2020

A new and simple enzymatic assay for measuring D-mannose in serum is described. Endogenous glucose is eliminated from serum by use of glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6). D-Mannose concentration is calculated from the increase in NADH formation after mannosephosphate isomerase (EC 5.3.1.8) is added. This increase is a result of coupling the following series of enzymes: hexokinase (EC 2.7.1.1), glucosephosphate isomerase (EC 5.3.1.9), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, NAD+-dependent). The study included subjects who were healthy volunteers and patients with suspected or proven fungal infections.
Automated kinetic method for D-3-hydroxybutyrate in plasma or serum.
Authors: McMurray C H , Blanchflower W J , Rice D A
Published: January 2020

This assay for 3-hydroxybutyrate in plasma or serum, based on kinetic initial-rate techniques, has been optimized with respect to initial rate and linearity as follows: pH 8.5, hydroxybutyrate dehydrogenase 62.5 U/L, and NAD+ concentration 20 mmol/L. We have used the assay satisfactorily with both the Gilford 103 and Hitachi 705 discrete analyzers, obtaining results that compare well with those by a segmented-flow method. Within-assay precision (CV) varied from 7.8 to 0.6%, depending on both the analyzer used and on the concentration of 3-hydroxybutyrate. Analytical recovery was also dependent on 3-hydroxybutyrate concentration, varying from 99% at 2 mmol/L to 91% at 9.5 mmol/L. Lactate dehydrogenase/lactate interference in this direct assay is eliminated by incorporating oxalate in the assay reagents. Hydrazine, commonly used in equilibrium methods of analysis for 3-hydroxybutyrate, produced no significant advantage in this assay and was omitted. The mixed reagents for the assay are stable at 4 degrees C for at least a week. The advantages of analysis for this metabolite may now be realized, so that clinical and subclinical ketosis can be identified in humans and animals.
Use of high-resolution proton nuclear magnetic resonance spectroscopy for rapid multi-component analysis of urine.
Authors: Bales J R , Higham D P , Howe I , Nicholson J K , Sadler P J
Published: January 2020

Numerous low-Mr metabolites--including creatinine, citrate, hippurate, glucose, ketone bodies, and various amino acids--have been identified in 400- and 500-MHz proton nuclear magnetic resonance (1H NMR) spectra of intact human urine. The presence of many of these was related to the specific condition of the donors: humans in different physiological states (resting, fasting, or post-exercise) and pathological conditions (e.g., diabetes mellitus, cadmium-induced renal dysfunction). We have also monitored the metabolism of simple nonendogenous compounds (methanol and ethanol) and of acetaminophen. The pH-dependencies of the NMR chemical shifts of some urine components are reported. Our studies show that high-resolution 1H NMR spectroscopy provides a fast, simple method for "fingerprint" identification of urinary compounds. In some cases, analytes can be quantified by standard additions or by comparing integrated peak areas for the metabolites with those for creatinine. Determinations of creatinine by 1H NMR spectroscopy compared well with those by an independent chemical assay based on the Jaffé reaction.
A thyrotropin radioimmunoassay kit evaluated vs two reference assays.
Authors: Bigos S T , Pekary A E , MacLean J , Pierce C E , Reed A W , Hershman J M
Published: January 2020

We evaluated a double-antibody radioimmunoassay kit for thyrotropin that includes calibrators prepared in a matrix of human serum and involves overnight nonequilibrium. Results were compared with those from two reference assays for thyrotropin. The range of within-assay CVs for the kit for thyrotropin values between 0.9 and 2.4 milli-int. units/L was 2.2 to 5.3%, that for between-assay CVs was 8.3 to 30%. The estimated minimum detectable concentration of thyrotropin was 0.6 milli-int. unit/L. We saw no cross reactivity with human choriogonadotropin by any of 48 sera from pregnant women. The original lot of serum specified as thyrotropin-free contained small but measurable amounts of thyrotropin; a second lot did not. Clinical data generated with the kit and the reference assays correlated well and were consistent with the clinical status of various categories of patients.