bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-enzymic glycation of individual plasma proteins in normoglycemic and hyperglycemic patients.
Authors: Austin G E , Mullins R H , Morin L G
Published: January 2020

Diabetic patients in poor glycemic control show increased glycation of total plasma proteins, but little is yet known about the relative extents to which the various individual proteins are glycated. Thus, we studied the non-enzymic glycation of several major plasma proteins and plasma protein fractions in normal and diabetic patients. In vivo glycation for most plasma proteins was very low in non-diabetic patients, only gamma globulin showing more than 5% glycation. In diabetic plasmas, glycation was much greater, immunoglobulins again showing the greatest proportion, followed in descending order by albumin, complement C3, fibrinogen, transferrin, haptoglobin, and alpha-1-antitrypsin. When plasma proteins were glycated in vitro, this order was IgG greater than complement C3 greater than albumin greater than transferrin greater than haptoglobin greater than alpha-1-antitrypsin. In general, proteins with the longest biological half-lives, such as IgG and albumin, showed the greatest in vivo glycation. On the other hand, proteins with high intrinsic glycability, such as complement C3, showed moderate glycation, despite a short half-life. Except for albumin, more basic proteins showed greater glycation than acidic proteins, but there was poor correlation between mole percent lysine and glycation. Evidently the relative extents of glycation of different plasma proteins are a complex function of integrated glucose concentrations over time and of the half-life and chemical characteristics of each protein.
Determination of cyclosporine concentrations with monoclonal antibodies.
Authors: Schran H F , Rosano T G , Hassell A E , Pell M A
Published: January 2020

We measured cyclosporine in whole blood from normal volunteers administered single oral doses of the drug and from two renal-transplant patients on immunosuppressive maintenance therapy, by liquid chromatography (I) and by radioimmunoassay with use of nonspecific polyclonal (II), specific monoclonal (III), and nonspecific monoclonal (IV) antibodies. Concentrations determined by III were equivalent to I, irrespective of cyclosporine dose, concentration, time after dose, or time after transplant. Concentrations determined by II and IV were consistently higher than those by I, owing to cross reactivity with metabolites. Ratios of values by II and IV to those by I increased from less than 1.5 to about 3-4 between 0.5 and 12 h after a single cyclosporine dose, owing to differences in rates of appearance and disappearance of cyclosporine and cross-reacting metabolites, though for the constant 12-h dose intervals in the two renal-transplant patients at steady state these ratios (most within the range 3-4) were relatively stable. Ratios of concentrations measured by IV to those by II (mean of 1.2 for single-dose data, most within the range of 1.2 to 1.5 at steady state) were unaffected by time after dose or time after transplant, suggesting that, despite certain cross-reactivity differences between the two nonspecific antibodies, results are proportional throughout therapy. We therefore propose that III and IV offer alternatives, respectively, to the currently used I and II for cyclosporine monitoring.
A monoclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein.
Authors: Fraeyman N H , Van de Velde E J , De Smet F H
Published: January 2020

A "sandwich"-type enzyme-linked immunosorbent assay for determining concentrations of human alpha 1-acid glycoprotein (AGP) is described. Microtiter plates coated with a polyclonal rabbit antibody to human AGP were subsequently incubated with the antigen, with a specific murine monoclonal antibody, and with goat anti-mouse immunoglobulins conjugated to alkaline phosphatase. To evaluate the method for assay of AGP in human sera, we compared it with single radial immunodiffusion and "rocket" electroimmunoassay. The respective correlations were r = 0.988 (n = 45) and r = 0.973 (n = 47). Repeated assays of a human serum sample with an average AGP concentration of 859 mg/L yielded within-day and between-day CVs of 1.4% (n = 5) and 6.3% (n = 10), respectively. Because of its low detection limit (4.4 micrograms/L), this assay is also suitable for determination of AGP concentrations in other biological fluids, such as dialysates of patients being treated by continuous ambulatory peritoneal dialysis.
Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct.
Authors: Wong S H , Knight J A , Hopfer S M , Zaharia O , Leach C N , Sunderman F W
Published: January 2020

This assay of plasma lipoperoxides involves hydrolysis in dilute H3PO4 at 100 degrees C; complexation of malondialdehyde (MDA), a hydrolysis product, with thiobarbituric acid (TBA); methanol precipitation of plasma proteins; fractionation of the protein-free extract on a C18 column; and spectrophotometric quantification of the MDA-TBA adduct at 532 nm. The detection limit was 0.15 mumol of MDA per liter of plasma. Run-to-run precision (CV) averaged 8 to 13%. Analytical recovery of MDA after addition of tetraethoxypropane standards to 21 specimens of human or rat plasma averaged 98% (SD 7%). Lipoperoxide concentrations (as MDA) averaged 0.60 (SD 0.13) mumol/L in plasma specimens from 41 healthy persons and 1.4 (SD 0.3) mumol/L in plasma specimens from 12 control rats. Mean lipoperoxide concentrations were 1.5 to 2.3 times as great in plasma sampled from rats one to three days after subcutaneous administration of NiCl2 at dosages (250 to 750 mumol per kilogram body wt) previously shown to induce lipid peroxidation in lung, liver, and kidney.
Determination of inorganic fluoride in blood with a fluoride ion-selective electrode.
Authors: Kissa E
Published: January 2020

Inorganic fluoride in whole blood, serum, or plasma has been determined with a fluoride ion-selective electrode by adding the 0.5- to 2.0-mL sample to 20 mL of water containing a buffer and fluoride, 25 micrograms/L. The fluoride concentration in the sample is calculated from the resulting cell potential difference recorded after equilibrating for 10 min. This novel method has the advantages of simplicity, accuracy, and high precision, standard deviations for 5 to 7 replicate determinations of fluoride ranging from +/- 1.7 to +/- 2.4 micrograms/L. For whole blood from donors living in an area with fluorinated drinking water, the F- concentration was 20 to 60 micrograms/L.
Dilution of plasma with Tris buffer increases measured catecholamines in plasma.
Authors: Cuche J L , Selz F , Ruget G , Gentil M , Gaudin C
Published: January 2020

We investigated the effects of dilution of plasma samples on the measured concentrations of catecholamines. Diluting samples of human plasma 10-, 50-, and 100-fold with Tris buffer (100 mol/L, pH 8.6) improved analytical recovery of internal standards, suggesting that it decreases the commonly observed inhibition of methylation in radioenzymatic assays of catecholamines in plasma. However, the dilution is not associated with a proportional decrease in counted radioactivity. This extra amount of radioactivity, which is unlikely to be nonspecific in origin, accounts for a significant increase in the calculated catecholamine concentration. Tentatively, we suggest that Tris buffer releases both catecholamines and conjugated catecholamines bound to some unidentified low-molecular-mass component of plasma.
Evaluation of lipoproteins and apolipoproteins in serum of a Tangier patient by micro-scale two-dimensional electrophoresis.
Authors: Manabe T , Visvikis S , Dumon M F , Clerc M , Siest G
Published: January 2020

We examined lipoproteins and apolipoproteins in serum of a Tangier-disease patient. We used three different techniques of micro-scale two-dimensional electrophoresis: (a) no denaturants; (b) with sodium dodecyl sulfate (SDS) used only in the slab gel electrophoresis; (c) and with urea and a detergent used in isoelectric focusing and with SDS in slab gel electrophoresis. By technique a, an extremely low concentration of high-density lipoproteins (HDL) in the Tangier serum was seen, and lipoproteins that cannot form HDL complexes were detected as multiple spots in the acidic (pl 4 approximately 5) and relatively low apparent molecular mass (20,000 approximately 80,000) region. By technique b, Tangier low-molecular-mass lipoproteins were dissociated into their constituent apolipoproteins, and we observed a higher proportion of apoC-III, together with lower proportions of apoA-I and apoA-II, than in the normal HDL fraction. Technique c showed the total content of apolipoproteins in the whole Tangier serum, as several workers have reported. The presence of low-molecular-mass lipoproteins and a high concentration of apoC-III in this lipoprotein fraction characterized the Tangier serum.
Urinary organic acids: isolation and quantification for routine metabolic screening.
Authors: Greter J , Jacobson C E
Published: January 2020

A method for isolating organic acids from acidified urine on an equivolume mixture of Porapak Q and Porapak T is described, and results are compared with extraction with ethyl acetate and ion exchange on DEAE-Sephadex. Average recoveries of 14C-labeled oxalic acid, lactic acid, succinic acid, alpha-ketoglutaric acid, citric acid, and cinnamic acid were equal to or better than those obtained with the solvent-extraction method. The ion-exchange method gave higher recoveries for oxalic acid, lactic acid, and citric acid. The quantification of separated acids from reconstructed mass spectrometric ion traces is compared with quantification from the simultaneously recorded flame ionization detector response signals. A good correlation was obtained. With the present routine metabolic screening method we have detected several patients with inborn errors of metabolism.
Temperature-dependent binding of cyclosporine to an erythrocyte protein.
Authors: Agarwal R P , Threatte G A , McPherson R A
Published: January 2020

In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [3H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA.
An accelerated enzyme immunoassay for human choriogonadotropin in urine, involving reflow of specimen through capillary tubes.
Authors: Chandler H M , Fuller S A , Nakagawa C H , Nagainis P A , Hurrell J G
Published: January 2020

A very rapid and sensitive assay for human choriogonadotropin (hCG) has been developed involving two beta-subunit-specific monoclonal antibodies. In the assay the test specimen is passed backward and forward (reflow) through a monoclonal-antibody-coated capillary tube for 1 min, then incubated for 1 min with a second monoclonal antibody conjugated to urease (EC 3.5.1.5). After addition of a urease substrate solution, 10 int. units of hCG per liter can be detected visually within 5 min, which compares very favorably with other currently available hCG assay procedures. Advantages of the reflow/capillary tube assay system and optimization of the test procedure are discussed.
A simple silver-staining technique for detecting Bence Jones proteins in unconcentrated urine.
Authors: Sheat J M , Lorier M A
Published: January 2020

Detection of Bence Jones proteins in urine usually involves a concentration step, followed by electrophoresis and, if necessary, immunofixation. The time-consuming and expensive concentration step can be eliminated by use of the silver-stain technique described here. This procedure, routinely used for staining unconcentrated urine, is inexpensive, sensitive, and easily performed in a clinical laboratory. Bence Jones proteins can be detected in concentrations as low as 5 mg/L.
A double-monoclonal immunoenzymometric assay for alpha 1-fetoprotein modified for increased analytical precision.
Authors: Shahangian S , Fritsche H A , Hughes J I
Published: January 2020

We modified a one-step, two-site, double monoclonal immunoenzymometric assay (Abbott Laboratories) for serum alpha1-fetoprotein (AFP) to increase its sensitivity and improve test precision at the low end. We increased sample size, incubation interval, and reaction time and temperature, and decreased the final reaction volume. Interassay CVs for the modified method ranged from 6.2 to 8.0% for mean concentrations of AFP in serum of 5.2 to 34.2 micrograms/L--substantially better than those for the unmodified monoclonal method--and agreed well with those of the comparison method (modified Abbott polyclonal immunoenzymometric assay). AFP values by the modified monoclonal procedure (y) correlated well with results by the polyclonal method (x): y = 0.983x + 1.84 micrograms/L (r = 0.927, n = 59). The detection limit of the modified monoclonal test was 0.2 microgram/L, as compared with 1.0 and 1.4 micrograms/L, respectively, for the modified polyclonal and the unmodified monoclonal procedures. We recommend using the modified monoclonal method for monitoring cancer patients with low tumor burden.
Low A-esterase activity in serum of patients with fish-eye disease.
Authors: Mackness M I , Walker C H , Carlson L A
Published: January 2020

The activity of HDL-associated paraoxonase in the lipoprotein fraction of serum from two patients with fish-eye disease (FED) was only 11% of the mean value for control subjects. Similarly, concentrations of apolipoproteins A-I and A-II in the serum of FED subjects were only 10% of those in normal subjects. Thus the ratio of enzyme activity to A-I and A-II is virtually the same in these two groups of subjects. This suggests that paraoxonase activity of the lipoprotein fraction is associated with one or both of these apolipoproteins.
Caffeine-splitting of bilirubin/albumin complex: its relevance to the spectrophotometry of bilirubin in serum.
Authors: Franzini C , Cattozzo G
Published: January 2020

By means of gel-filtration of bilirubin/albumin mixtures, it is shown that unconjugated bilirubin remains completely linked to albumin (both human and bovine) in tetraborate buffer (pH 9.3), protein-free bilirubin appearing only when the bilirubin/albumin molar ratio exceeds two. On the other hand, bilirubin is completely set free from its protein link in the caffeine reagent. Additional chromatographic and spectrophotometric evidence is reported indicating the formation of a low-affinity complex between bilirubin and caffeine. These data explain why the spectrophotometric properties of bilirubin/albumin mixtures are matrix-dependent if measured in the tetraborate buffer but are no longer so when measured in the caffeine reagent. The relevance of these findings to the spectrophotometric "direct" assay of bilirubin in serum is discussed with reference to the occurrence of "delta-bilirubin" in pathological sera: this tightly protein-bound bilirubin fraction does not split in the presence of caffeine.
Evaluation and improvement of homovanillic acid methodology, and a critique of liquid chromatography with electrochemical detection.
Authors: Anton A H , Berk A I
Published: January 2020

Resolution of several problems encountered with some organic solvent extraction-fluorometric procedures commonly used to measure homovanillic acid (HVA) in urine resulted in a more specific and sensitive methodology, applicable to diverse biological material. Purification on an anion-exchange resin followed by extraction with organic solvent was the most reliable and specific for HVA. Eluting the resin with a near-boiling solution of NaCl concentrated the HVA in a smaller volume with no increase in the blank reading. In none of the other methods was enough oxidizing agent used to completely oxidize the excessive amounts of HVA in urines of patients with neuroblastoma. 5-Hydroxyindoleacetic acid interferes with the HVA fluorophor. Several of the advantages and problems with "high-pressure" liquid chromatography with electrochemical detection are identified, particularly the unpredictable nature of the working electrode.
Intra-individual variation of some analytes in serum of patients with chronic renal failure.
Authors: Hölzel W G
Published: January 2020

Biological intra-individual variation in the concentration of 15 biochemical analytes in serum was estimated for 17 patients with chronic renal failure (CRF) and compared with results for apparently healthy individuals. The ratio of the average intra-individual variation in CRF patients to that in normal subjects was 1.5 to 2.0 for sodium, chloride, calcium, and creatinine; 1.2 to 1.5 for hemoglobin, total protein, albumin, globulin, uric acid, cholesterol, and alpha-amylase. The intra-individual CVs for urea, high-density-lipoprotein cholesterol, triglycerides, and alkaline phosphatase did not differ significantly between groups. The intra-individual variation of calcium and the concentration of creatinine in serum correlate significantly (r = 0.661, p less than 0.01). Individual values showed a gaussian distribution for all analytes. There was no time dependence of the intra-individual variation during a three-week interval, except for calcium and cholesterol. The estimated biological component of intra-individual variation and the analytical variation can be used to derive decision-making criteria in monitoring CRF.
Enhanced-latex-agglutination assay for C-reactive protein in serum, with use of a centrifugal analyzer.
Authors: Winkles J , Lunec J , Deverill I
Published: January 2020

This is an improved assay of C-reactive protein in serum, for use with the Baker "Encore" centrifugal analyzer. Features of this assay include: 250-specimen throughput per hour, within-batch CV 2.2%, between-batch CV 2.7%, no antigen-excess problems up to 1000 mg/L, negligible interference from rheumatoid factor, and good correlation (r = 0.99) with radial immunodiffusion. The method is inexpensive and automated, involving no predilution steps. It can be adapted for use in a wide range of systems and can be used for single urgent estimations.
Analysis for carboxyhemoglobin by gas chromatography and multicomponent spectrophotometry compared.
Authors: Vreman H J , Stevenson D K , Zwart A
Published: January 2020

Measurements of carboxyhemoglobin (HbCO) by gas chromatography (GC) were compared with those obtained by multicomponent spectrophotometric analysis (MCA). Correlation was good for HbCO ranges (proportion of total hemoglobin) of 1.5 to less than or equal to 5% (GC = 0.94 MCA + 0.37%, n = 25, r = 0.98); 5.0 to less than or equal to 10.0% (GC = 0.89 MCA + 0.63%, n = 19, r = 0.93); greater than 10% (GC = 1.01 MCA - 0.02%, n = 22, r = 0.99); and greater than 0% (GC = 1.01 MCA - 0.10%, n = 95, r = 1.00). The correlation is lower for the clinically normal range of 0 to less than or equal to 1.5% (GC = 0.65 MCA + 0.24%, n = 29, r = 0.87). The gas-chromatographic method is linear for HbCO proportions of 30% to as little as 0.15%. Re-analysis of blood samples after two-month storage at 4 degrees C showed that samples remained stable under these conditions.
Two monoclonal-based assays for carcinoembryonic antigen compared.
Authors: Felder R A , MacMillan R H , Bruns D E
Published: January 2020

Monoclonal antibody-based methods can provide clinically different impressions for some patients than does an earlier method involving a conventional antiserum. Therefore, performance of two commercial double-monoclonal-antibody methods for carcinoembryonic antigen (CEA) were studied further, those of Abbott Diagnostics and Roche Diagnostic Systems. The two methods showed similar precision. Total CVs over 20 days at three concentrations were 16, 6, and 6% in the Roche assay, and 15, 8, and 7% in the Abbott assay (n = 20). Within-run CVs for quality-control levels 1, 2, and 3, respectively, in the Roche assay were 14, 8, and 9%, and in the Abbott 15, 9, and 10% (n = 20). Mean CEA concentrations were consistently lower in the Roche assay than in the Abbott assay. Results of the two assays correlated significantly in patients with CEA-secreting carcinomas (r = 0.88), and in serial samples from cancer patients who had low, medium, or high CEA concentrations in their sera (r = 0.92 for all serial data). About 10% of values were false-positive (greater than 5.0 micrograms/L) by each of the two methods in 89 patients with various non-malignant conditions. The two methods appear to be more specific for tumor-associated CEA than are polyclonal antibody-based assays previously available from the two manufacturers.
Laboratory performance and director qualifications.
Authors: Kenney M L
Published: January 2020

Since 1971, federal laboratory regulations have required that directors of approved laboratories possess earned doctorates. Private accrediting agencies and some states also require doctoral directorship of accredited laboratories. No empirical studies have demonstrated that a director's earned doctorate is necessary to assure laboratory quality. Laboratories in physicians' offices (POLs) are exempt from federal regulation but receive federal reimbursement on the basis of the physicians' medical degree. No empirical studies have demonstrated that unregulated laboratories perform comparably with regulated laboratories. This investigation found no statistically discernible differences in quality when 1983 proficiency test data were used to compare statistically the performance of doctoral- and non-doctoral-directed Medicare-certified independent laboratories in California. When regulated non-doctoral-directed full-service laboratories were statistically compared with unregulated limited service POLs, regulated non-doctoral-directed laboratories consistently demonstrated superior per formance to POLs. Evidently a director's earned doctorate is neither a necessary nor a sufficient condition to assure laboratory performance. Government regulation appears to provide substantial quality assurance in the clinical laboratory field.
Ability of the 1(2)s rule to detect substandard performance in interlaboratory proficiency testing.
Authors: Ehrmeyer S S , Laessig R H
Published: January 2020

The most common evaluation criterion used by regulatory and voluntary interlaboratory proficiency testing (PT) programs to judge the quality of chemistry results is based on the group mean +/- 2.0 standard deviations (SD), the 1(2)s rule. The mean and SD are those of the selected PT population. Through computer simulations we have quantified, for the first time, the ability of the 1(2)s criterion to detect "acceptable" as well as substandard ("unacceptable") intralaboratory performance by PT. We found that the 1(2)s rule correctly identifies "acceptable" performance--i.e., low intralaboratory coefficient of variation (CV) values and small bias values--greater than 90% of the time. However, this criterion fails to detect laboratories with "unacceptable" bias and (or) CV at least 23% of the time. The high failure rate of the 1(2)s rule raises serious questions about its appropriateness for use by PT programs.
Performance of quantitative urine analysis in Australasia critically assessed.
Authors: Shepard M D , Penberthy L A
Published: January 2020

The standard of quantitative urine analysis in Australasia has been assessed over a three-year period (1984-1986) through a national interlaboratory quality-assurance program, conducted jointly by the Royal College of Pathologists of Australasia and the Australian Association of Clinical Biochemists. We investigated the precision and accuracy of individual methods and measurement systems routinely used in 14 urine assays. Assays of sodium, potassium, creatinine, glucose, and chloride were performed satisfactorily. Further improvement is required in assays of urinary osmolality, phosphate, and 5-hydroxyindoleacetic acid. Determinations of oxalate, urea, calcium, and 4-hydroxy-3-methoxymandelic acid are improving, but assays of protein and urate in urine present major problems for Australasian laboratories. In this program the sulfosalicylic acid turbidimetric method for urinary protein has consistently displayed poor precision plus a significant positive bias and should be abandoned. In contrast, the trichloroacetic acid-Ponceau S manual method has continued to perform well.
Can a single measurement of carbamazepine suffice for therapeutic monitoring?
Authors: Arranz Peña M I , Saenz Lope E
Published: January 2020

Using fluorescence polarization immunoassay, we measured the peak and trough concentrations of total and free carbamazepine in serum and saliva from 33 epileptic children. We found that, except for patients who show side effects, measurement of trough values in a single sample, collected after an overnight fast and before the morning dose, sufficiently estimates clinical response. Moreover, total carbamazepine is as informative as free carbamazepine.
Effect of heparin and citrate on measured concentrations of various analytes in plasma.
Authors: Smith J C , Lewis S , Holbrook J , Seidel K , Rose A
Published: January 2020

Frequently it is assumed that concentrations of biochemical analytes are similar for serum and plasma, without regard to the anticoagulant. Recently it has been hypothesized that use of certain anticoagulants results in an osmotic redistribution of fluid between blood cells and plasma, causing some dilution of the plasma. We sought to determine the effect of two commonly used anticoagulants, heparin and citrate, on the measured concentrations of 13 clinical-chemical analytes, including selected trace elements, vitamins, lipids, and proteins. The data demonstrate that hyperosmolar citrate causes a significant dilution of all indices measured. In contrast, heparin had no osmotic effect; concentrations of most of the analytes measured in the heparinized plasma were statistically no different from those measured in the corresponding serum. Therefore, anticoagulants must be chosen carefully, especially if concentrations in plasma and serum are to be compared.
Detection of interference by cyclobenzaprine in liquid-chromatographic assays of tricyclic antidepressants.
Authors: Puopolo P R , Flood J G
Published: January 2020

We evaluated a technique for detecting cyclobenzaprine interference with liquid-chromatographic assays for tricyclic antidepressants. The technique involves dual-wavelength absorbance monitoring of the column effluent at 214 and 254 nm. Ratios of analyte peak heights at each wavelength are used to check for the presence of co-eluting interferences. With this technique, one can detect interference with an amitriptyline assay caused by 10 micrograms of cyclobenzaprine per liter.
Improved interassay correlation of digoxin results in patients with and without renal failure by elimination of digoxin-like immunoreactive factors.
Authors: Skogen W F , Rea M R , Valdes R
Published: January 2020

Use of immunoassays that do not detect endogenous digoxin-like immunoreactive factors (DLIF) in serum significantly improves the between-assay correlation of digoxin results for patients. We investigated five different immunoassay methods (Abbott, Clinical Assays, Corning, Du Pont, and Syva), measuring digoxin by all five assays in sera from 38 patients in renal failure and in 40 patients with normal renal function, all taking digoxin. The mean standard error of the estimate (Sy X x) of digoxin results (compared for all five assays) were significantly lower for patients with normal renal function than for patients in renal failure (0.148 vs 0.293 microgram/L, P less than 0.001). Assays previously shown (Clin Chem 1987;33:401) to be the least sensitive to DLIF (Syva and Corning) gave the lowest mean scatter about the regression (Sy X x = 0.192 microgram/L, renal failure; 0.114 microgram/L, normal renal function) for all 10 assay correlations. Evidently, discrepancies between digoxin values as measured by different immunoassay kits for patients with renal disease can be attributed to DLIF. Moreover, because inaccurate digoxin results attributed to DLIF may not be limited exclusively to groups of patients with known increased concentrations of DLIF, the possibility of "latent" DLIF interference may be a problem in many other human subjects.
Phenylalanine and tyrosine in serum and eluates from dried blood spots as determined by reversed-phase liquid chromatography.
Authors: Rudy J L , Rutledge J C , Lewis S L
Published: January 2020

We have developed a reversed-phase liquid-chromatographic procedure for simultaneously determining phenylalanine and tyrosine in serum and eluates of dried blood spots. Batch derivatization with phenylisothiocyanate and a 10-min linear gradient chromatographic assay with ultraviolet absorbance detection provide rapid sample throughput. Interrun precision (CV) is less than 12%; analytical recovery (from blood spot samples) exceeds 85%. Results for patients' samples correlate well with those from an amino acid analyzer and we encountered no apparent interferences. The speed and specificity of this assay facilitate the rapid diagnosis and monitoring of patients with phenylketonuria.
Kinetic immunonephelometric determination of protein concentrations in follicular fluid.
Authors: Gulamali-Majid F , Ackerman S , Veeck L , Acosta A , Pleban P
Published: January 2020

Nonsteroidal biochemical markers of oocyte maturity could presumably aid in oocyte selection and in the timing of insemination for in vitro fertilization. We assessed the usefulness of six potential markers found in follicular fluid. We used kinetic immunonephelometry to measure concentrations of alpha 1-antitrypsin, alpha 2-macroglobulin, antithrombin III, ceruloplasmin, fibrinogen, and plasminogen in 53 fluids and the corresponding plasma from 20 women undergoing in vitro fertilization. Specimens were obtained from both mature and immature follicles from each woman. The respective protein concentrations in follicular fluid could be assayed with a between-assay CV of 1.5% to 3.2%. Analytical-recovery studies indicated that only fibrinogen and antithrombin III concentrations were altered (by 120% and 75%, respectively) during aspiration of the follicle into saline. All protein concentrations were significantly increased in mature follicles, as were the follicular fluid:plasma concentration ratios for alpha 1-antitrypsin, antithrombin III, and ceruloplasmin.
Laboratory and field evaluation of three desktop instruments for assay of cholesterol and triglyceride.
Authors: von Schenck H , Treichl L , Tilling B , Olsson A G
Published: January 2020

We evaluated three desktop instruments suitable for decentralizing the assays of blood cholesterol and triglycerides to satellite and physician's office testing. The instruments, Ektachem DT 60 (E), Reflotron (R), and Seralyzer (S), were used according to their manufacturers' instructions to assay serum or capillary blood from outpatients at two physicians offices. Accuracy was assessed in the centralized laboratory by using an automated centrifugal analyzer (A). The bias of A was monitored with an international quality-control material. We found all instruments easy to handle. Regression equations for cholesterol determinations were: E = 0.92A + 0.7 (n = 331, r = 0.94), R (capillary blood) = 0.96A + 0.3 (n = 256, r = 0.95), and S = 0.93A + 0.6 (n = 260, r = 0.92). For triglycerides we obtained E = 1.02A (n = 331, r = 0.97), R (cap. blood) = 0.88A (n = 213, r = 0.97), R = 0.94A + 0.1 (n = 90, r = 0.99), and S = 0.96A (n = 266, r = 0.98). Duplicate and within-day precision was less than 8%. Between-day precision (during a month) was less than 10%. We stress the need of both laboratory and field evaluation and emphasize the benefit of quality control.
Surface modification with protein A for uniform binding of monoclonal antibodies.
Authors: Schramm W , Yang T , Midgley A R
Published: January 2020

We describe optimal conditions for immobilization of two monoclonal antibodies to progesterone for solid-phase assays. Polystyrene surfaces are refined with Protein A to achieve uniform, reproducible, stable, and sterically accessible immobilization of immunoglobulins (IgG). To this end, we optimized the amount of immobilized Protein A, the pH of the medium for immobilization, the concentration of antibody, and the polystyrene surface. We also investigated three carriers for solid-phase assays: 12 X 75 mm polystyrene test tubes, Macrowells (Skatron, Inc.; suitable for processing with multiple pipettors), and microwell strips (Immulon II, Dynatech Inc.). Immunoglobulin does not appreciably dissociate from any of these solid matrices, even if the assay procedure takes several hours. Therefore, we postulate that more than one molecule of immobilized Protein A binds to IgG, or that there is an additional interaction between the antibody and the polymer surface.
Simultaneous measurement of eight corticosteroids by liquid chromatography, and application of the procedure to diagnosis of congenital adrenal hyperplasia.
Authors: Wei J Q , Zhou X T , Wei J I
Published: January 2020

We describe a liquid-chromatographic procedure for simultaneously determining eight steroids in serum. We used a Zorbax ODS column and a mobile phase of methanol/isopropanol/water (44/10/46, by vol), which well resolves the steroids cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, androstenedione, 17-hydroxyprogesterone, and progesterone, but not 11-deoxycorticosterone and androstenedione. Analytical recoveries of the steroids ranged from 89.27% to 99.58%. CVs were less than 10%. Prednisone and dexamethasone do not interfere. Using this method, we studied the serum steroid profiles of six patients with congenital adrenal hyperplasia (CAH) due to deficiency of 21-hydroxylase or 17-hydroxylase. Not only could we make a clearcut diagnosis and distinguish the subtle types of CAH, but also we could investigate clinical and biochemical variants of CAH. For example, we confirmed 17-hydroxylase deficiency in a patient who was normotensive with primary amenorrhea and streak gonads.
Three immunoassay methods evaluated for quantifying prealbumin (transthyretin) in serum.
Authors: McCarthy R C , Fetterhoff T J , Luckey D W
Published: January 2020

We developed an immunoturbidimetric assay for prealbumin on the Cobas Bio centrifugal analyzer and compared results from this assay with those from a rate nephelometric assay (Beckman Instruments Inc.) and a radial immunodiffusion kit (Behring Diagnostics). All three assays were evaluated for precision, linearity, and correlation to each other for analysis of sera from pediatric patients. All assays gave similar results for patients' samples. Values were higher by the radial immunodiffusion assay than by the other two methods, which gave similar results for the same specimens. We conclude that the immunoturbidimetric and rate nephelometric assay for prealbumin are acceptable alternatives for quantifying prealbumin in serum and also have a faster turnaround time than radial immunodiffusion.
A lactate dehydrogenase-immunoglobulin G1 complex, not blocked by anti-idiotype antibody, in a patient with IgG1-lambda type M-proteinemia.
Authors: Fujita K , Sakurabayashi I , Kusanagi M , Kawai T
Published: January 2020

The serum of a patient with IgG1-lambda type M-proteinemia showed an abnormal isoenzyme pattern for lactate dehydrogenase (LDH, EC 1.1.1.27). By affinity chromatography, we showed that four isoenzymes (LDH2, LDH3, LDH4, and LDH5) were bound to the M-protein. This complex formation was not blocked by anti-idiotype antibody, even though the binding capacity of IgG was exclusively located in the Fab region of the molecule. Moreover, heavy and light chains of the patient's IgG, obtained by reduction, separately had affinities for each of the LDH isoenzymes. LDH-IgG complex was easily dissociated by affinity chromatography on 5'-AMP-Sepharose 4B or by added NADH. We propose the following hypothesis for the LDH-IgG complex formation: LDH can recognize the gamma-Fab region of IgG at the NAD+ binding site of the molecule, but the affinity of the LDH molecule for immunoglobulin is much weaker than that for NADH or 5'-AMP.
Simple, rapid enzymatic determination of glycerophosphocholine in human seminal plasma.
Authors: Chap H J , Moatti J P , Mieusset R , Nieto M , Laneelle G , Bennet P J , Mansat A , Pontonnier F , Douste-Blazy L
Published: January 2020

We have devised a new enzymatic determination of sn-glycero-3-phosphocholine (GPC) in human seminal plasma. This is based on GPC hydrolysis by a phosphodiesterase (PDE), free choline being then determined by the choline oxidase method. The whole procedure involves a first incubation in the presence of choline oxidase and catalase, to eliminate the excess of choline present in seminal plasma (10-fold, compared with GPC). Absorbance and concentration are linearly related up to at least 100 nmol per assay, analytical recovery ranges between 89% and 105%, and intra- and interassay CVs are 3.2% and 5.6%, respectively, at the highest substrate concentration. Using this procedure, we found seminal plasma from 21 fertile men to contain 5.22 (SD 3.33) mumol per ejaculate--within the same range as previously reported values obtained chromatographically. After vasectomy, GPC in seminal plasma decreased to 28% of its original value, as determined in 10 volunteers. Thus this new method displays appropriate characteristics of specificity, reliability, and convenience, allowing its use in routine evaluation of male fertility.
"High-resolution" mini-two-dimensional gel electrophoresis automatically run and stained in less than 6 h with small, ready-to-use slab gels.
Authors: Hochstrasser D , Augsburger V , Pun T , Weber D , Pellegrini C , Muller A F
Published: January 2020

Although two-dimensional (2-D) gel electrophoresis is one of the most powerful techniques for analyzing protein mixtures, its application in routine clinical laboratories is currently limited, because it is time-consuming, complex, and relatively expensive. Here we describe a method for automatically running and staining "high-resolution" mini 2-D electrophoresis gels in less than 6 h, by using "ready-to-use" slab gels and a PhastSystem electrophoresis apparatus. We present 2-D gel electrophoretograms of 25 nL of plasma, as well as their automatic computer analysis. For comparison, a conventional 2-D gel electrophoresis profile of 200 nL of a plasma sample is shown. The technique is easy to perform, highly sensitive, rapid, and potentially useful in semi-routine clinical chemistry laboratories.
Chemically blocked analog assays for free thyronines. II. Use of equilibrium dialysis to optimize the displacement by chemical blockers of T4 analog and T3 analog from albumin while avoiding displacement of T4 and T3 from thyroxin-binding globulin.
Authors: Witherspoon L R , el Shami A S , Shuler S E , Neely H , Sonnemaker R , Gilbert S S , Alyea K
Published: January 2020

Chemical blockers used to displace thyronine analog from albumin in analog kits for assay of free thyroxin (FT4) or free triiodothyronine (FT3) may also displace thyroxin (T4) or triiodothyronine (T3) from thyroxin-binding globulin (TBG), resulting in an apparent TBG dependence of results of free hormone estimates. We used equilibrium dialysis and antibody binding to assess the displacement of thyronine analogs and thyronines from albumin and TBG by use of chemical blockers. We chose a combination of two chemical blockers, which eliminated thyronine analog-albumin binding but minimized thyronine displacement from TBG for use in FT4 and FT3 assays. These blocked-analog free-hormone assays yielded accurate clinical results in euthyroid patients, hypo- and hyperthyroid patients, and in pregnant women. FT4 results were not entirely normalized in all nonthyroidally ill patients, indicating that decreased analog-albumin binding is not the only factor resulting in low FT4 results. In current Diagnostic Products Corp. (DPC) FT4 and FT3 blocked-analog kits, the blocker concentrations are the same as we used in these assays.
Delta Bilirubin: Absorption Spectra, Molar Absorptivity, and Reactivity in the Diazo Reaction
Authors: Doumas Basil T , Wu Tal-Wing , Jendrzejczak Bernadine
Published: January 2020

Vol. 33:

p 770: The next-to-last sentence in column one should read: "When the diazo concentration is increased from 1.53 to 2.55 mmol/L, there is no difference in color yield from Bδ and DTB, but there is α further increase in color with Bc."

p 1110: The correct price for the book Medical Uses of Statistics is $35.

p 1180: The legend for the horizontal axis of Figure 6 incorrectly indicates the unit of phospholipid concentration to be µg/L. The correct unit is mg/L.

p 1255: In the first tabulation in the Brief by Nanji et al., the figures after "Hemoglobin" should read 46, 38, and 55 (SDs are correct).

p 1351: In the fifth-from-last line of text in the right-hand column, "1.3" should read "3.3."

p 1962: The correct price for additional copies of the 1987 Beckman Conference Supplement Issue is $17.50.

Contents (Oct. issue, no. 10): The name of the author of the Opinion article (pp 1701-6), A. W. Jones, was inadvertently omitted from the Contents page; the author listed, G. Simpson, wrote a response to Dr. Jones' comments.
Effects of storage and handling conditions on concentrations of individual carotenoids, retinol, and tocopherol in plasma.
Authors: Craft N E , Brown E D , Smith J C
Published: January 2020

We investigated the effects of storage and handling on measured values for carotenoids, retinol, and tocopherol in plasma. We found no significant differences in the concentrations of these analytes measured in plasma samples that were frozen immediately after separation as compared with replicate samples maintained at room temperature in the dark for 24 h. Analytes were stable in solvents for at least 18 h at 23 degrees C after extraction. Purging samples with nitrogen gas before freezing had no detectable beneficial effects. All analytes were stable in plasma stored at -70 degrees C for at least 28 months or at -20 degrees C for five months. By 15 months the concentrations of carotenoids were significantly less (P less than 0.05) in plasma stored at -20 degrees C than in plasma stored at -70 degrees C, while retinol and tocopherol concentrations were not significantly different. Concomitant with the decrease in carotenoids was the appearance of unidentified peaks in the ultraviolet. Adding ascorbic acid or butylated hydroxytoluene antioxidants to the precipitating solvent did not alter the losses of carotenoids or alter the appearance of unidentified peaks. Under appropriate conditions, plasma carotenoids, retinol, and tocopherol are stable for more than two years.
Purification of anti-apolipoprotein CIII antibodies by exploiting their affinity for apolipoprotein CIII linked to polyacrylamide gel after isoelectric focusing.
Authors: Alsayed N , Drüeke T B , Lacour B
Published: January 2020

A new immunoaffinity technique for purifying anti-apolipoprotein CIII antibodies has been developed by isolating bands of apo CIII2 from isoelectric focusing (IEF) gel. Total apo C was obtained from delipidated very-low-density lipoprotein by chromatography on Sephacryl S 200. Apo CIII2 was separated from the apo CII band by IEF on polyacrylamide gel with use of pH 4-6 ampholytes. After sonication of the band in distilled water and various different washes, we directly mixed with antiserum a suspension of apo CIII2 bound to IEF-polyacrylamide gel. After their elution, we tested the specificity of the antibodies by an enzyme immunoassay technique, using plates coated with apolipoprotein, 100 ng per well. No cross-contamination of eluted anti-apo CIII antibodies by anti-apo CII or anti-apo E antibodies was observed. This affinity technique is easy to use, rapid to perform, and no sophisticated apparatus is needed. The gels can be used repeatedly and yield reproducible results with a very good analytical recovery (94%). We anticipate that this technique will prove useful for purification of other antibodies, particularly antibodies to apolipoproteins such as anti-apo CII and anti-apo E.
Time-resolved immunofluorometric assay of sex-hormone binding globulin.
Authors: Niemi S , Mäentausta O , Bolton N J , Hammond G L
Published: January 2020

A time-resolved immunofluorometric assay (trlFMA) for human sex-hormone binding globulin (SHBG) is described in which antibody-coated tubes or microliter strip-wells and a europium (Eu) chelate-labeled monoclonal antibody are used. The trlFMA sensitivity is similar to that of other SHBG immunoassays, and other analytical variables compare favorably with an SHBG immunoradiometric assay (IRMA) kit and a steroid binding capacity assay: the interassay coefficient of variation (CV) is less than 8% and the intra-assay CV is less than 6% for concentrations between 6 and 200 nmol/L. The reference intervals (means +/- SD) for SHBG concentrations (nmol/L) in serum from 10 men, 10 women, and 10 pregnant women were 23 +/- 12, 65 +/- 39, and 439 +/- 122, respectively. In 14 hirsute women the mean +/- SD serum SHBG concentration (37 +/- 21 nmol/L) was significantly lower (P less than 0.01) than the mean for an age-matched, nonhirsute female comparison group. The trlFMA is technically simple, requires no centrifugation or separation reagent, and takes a counting time of only 1 s. In addition, the Eu-label is nontoxic, presents no waste-disposal problems, and has a long shelf life.
Specific determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine by liquid chromatography with post-column reaction.
Authors: Abe K , Konaka R
Published: January 2020

We describe a "high-performance" liquid-chromatographic method for determining 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) in human urine. MHPG is separated on a reversed-phase column with isocratic elution, oxidized with sodium metaperiodate, and its absorbance measured at 365 nm. This method shows higher specificity, less interference for MHPG than methods involving electrochemical or fluorescence detection. Post-column derivatization of MHPG with periodate yields vanillin. The detection limit (twice the signal-to-noise ratio) in urine samples was 0.08 mg/L. Mean analytical recovery was 72%. Within-assay and day-to-day CVs were 2.9% and 6.5%, respectively. Reference intervals for MHPG in 24-h urine from apparently healthy subjects were 0.85-3.24 mg/day for men and 0.63-2.20 mg/day for women. In terms of creatinine excretion, the respective reference intervals were 0.55-1.99 and 0.70-1.96 mg per gram of creatinine.