bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of four "kit" immunoassay methods for determination of alpha-fetoprotein in serum during pregnancy.
Authors: Wong P Y , Doran T A , Ho F F , Mee A V
Published: January 2020

We evaluated four immunoassay methods for determination of alpha-fetoprotein in serum. All are commercially available in kit form in North American and we find them acceptable with respect to sensitivity, stability, precision, linearity, and analytical recovery. For the Dainabott method, the median values from 16 through 20 weeks of gestation are 34.4, 38.1, 42.1, 51.4, and 66.3 micrograms/L for each week, respectively. Corresponding values for the Amersham method are 27.9, 31.4, 34.9, 38.2, and 54.0 micrograms/L, respectively. For the Behring-RIA method they are 33.4, 37.4, 39.4, 49.9, and 66.3 micrograms/L, respectively. Results for four cases of confirmed neural tube defects are presented.
Quantitative gel-electrophoretic determination of serum amylase isoenzyme distributions.
Authors: Gillard B K
Published: January 2020

I report a direct, sensitive, quantitative method for determining serum amylase isoenzyme activity with commercially available reagents. Day-to-day reproducibility (CV) was 3--4% for the isoenzymes in normal serum; within-run precision was 8, 3, and 2% for low, normal and high isoenzyme activities. Amylase isoenzymes, separated into the pancreatic and salivary types by electrophoresis in polyacrylamide gel, are then quantified by directly incubating the gels in soluble-starch solution, staining with iodine, and densitometry. The proportion of pancreatic isoenzyme (47 normal sera) was 43 +/- 8% (mean +/- SD). Isoenzyme activities as low as 2% of normal can be measured accurately in 10 micro L of serum. The reproducibility, precision, and sensitivity indicate that the method is applicable to differential diagnosis of hyperamylasemia or hypoamylasemia, and is suited for monitoring the subtle changes in serum amylase isoenzyme distribution that may accompany disease progression or therapy.
Electrophoretic separation of high-density lipoprotein choelsterol evaluated and compared with the modified lipid research clinic procedure.
Authors: Stein E A , McNeely S , Steiner P
Published: January 2020

We evaluated a new agarose-gel-electrophoretic procedure (Corning) (I) for separating and quantitating of high-density lipoprotein cholesterol (HDLC), comparing it with the modified Lipid Research Clinics (LRC) procedure (heparin 183 kilounits/L, MnCl2 92 mmol/L) (II). Method I was insensitive to an HDLC concentration of 50 mg/L, but gave a linear dose-response curve between 130 and 1200 mg/L. Method II is sensitive to 50 mg/L and linear from 50--1200 mg/L. The within-plate CV for the Corning method varied from 26.2% for an HDLC of 168 mg/L to 6.8% for 580 mg/L. Within-day between-plate CV for the Corning method ranged from 22.1% at 155 mg/L to 8.0% at 651 mg/L, compared to 3.0 and 0.8% for the modified LRC procedure. Between-day CV for method I was 20, 12.6, 4.3, and 3.5% for HDLC concentrations of 175, 435, 542, and 678 mg/L, respectively; for method II it was 14, 5, 3.5, and 2.6%, respectively. Analysis of HDLC in 100 patients by both procedures showed mean HDLC values to be significantly lower (mean + SD, 27.8 +/- 1.7 mg/L; p less than 0.001) by method I. In 46 patients with HDLC less than 450 mg/L, this difference was accentuated (mean + SD = 40.5 +/- 2.6 mg/L) and clinically significant. Electrophoretic methods offer a promising further alternative method for HDLC separation and quantitation, but the negative bias, present limited sensitivity, and lack of precision at less than 450 mg/L indicate that they are not yet optimal for routine clinical use for patients with values less than 450 mg/L.
Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis.
Authors: Conlon D R , Blankstein L A , Pasakarnis P A , Steinberg C M , D'Amelio J E
Published: January 2020

We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.
Evaluation of a nephelometric assay for haptoglobin and its clinical usefulness.
Authors: Van Lente F , Marchand A , Galen R S
Published: January 2020

Serum haptoglobin has been advocated as an indicator of intravascular hemolysis. We have evaluated a nephelometric determination of serum haptoglobin. The assay is sensitive and exhibits within-run precision in the range of 2.5-7.4% coefficient of variation (CV) and between-run precision of 7.0% (CV). In addition, when haptoglobin values determined with the nephelometric assay were compared with hemoglobin-binding capacity determined by electrophoresis, the correlation coefficient was 0.968. The assay is essentially independent of phenotype and free of significant interference by hemolysis. The clinical correlation of haptoglobin values obtained for 100 selected patients with the nephelometric technique correlated well, if less than 250 mg/L, with the presence of hemolytic disease.
Radioimmunoassay for immunologlobulin G in serum and urine.
Authors: Woo J , Floyd M , Longley M A , Cannon D C
Published: January 2020

We describe a radioimmunoassay for immunoglobulin G (IgG) in serum and urine. Aliquots of diluted samples and 125I-labeled IgG were incubated in antibody-coated tubes at 37 degrees C for 24 h, the supernates were decanted, and the radioactivity in tubes containing the bound fraction was counted. The dose-response curve in the range of 0.4--500 mg/L of urine or 640--40 000 mg/L of serum was linear on logit-log transformation and iterative weighted regression. Assay sensitivity was 10 ng of IgG. Validation studies included testing for precision, accuracy, antibody specificity, and parallelism of the dose-response curves for standard and unknown. In a study of 14 apparently normal individuals, serum IgG = 4.0-10.9 gL, urine IgG = 1.1-4.8 mg/24 h, and IgG clearance = 0.2 X 10(-4) to 4.8 X 10(-4) mL/min. In 20 patients with renal allografts, serum IgG = 15.8-66 g/L, urine IgG - 9.6-626 mg/24 h, and IgG clearance = 9 X 10(-4) to 1.99 X 10(-1) mL/min. IgG values correlated well with severity of renal allograft rejection.
Kinetic determination of serum sorbitol dehydrogenase activity with a centrifugal analyzer.
Authors: Dooley J F , Turnquist L J , Racich L
Published: January 2020

We describe a mechanized method for centrifugal analyzer determination of sorbitol dehydrogenase in serum, based on conversion of D-fructose to sorbitol with simultaneous oxidation of NADH, in triethanolamine buffer at pH 7.4 and 30 degrees C. The standard curve for this assay is linear to 200 U of activity per liter of serum. The mean within-run precision (CV) of the assay is 0.8%. Results correlate well with those by a spectrophotometric method. In sera from 20 apparently healthy adult humans, sorbitol dehydrogenase activity averaged 1.7 (SD +/- 0.8; range, 1-3) U/L. The mean activity (U/L) for a group of 30 rats was 4.4 (SD, +/- 0.2; range, 3-6); for 20 dogs, 5.8 (SD, +/- 0.7; range 3-9); and for 30 mice, 26.8 (SD +/- 2.1; range, 22-34). To determine the utility of measuring this enzyme in the serum of rats for assessment of hepatotoxicity in drug-safety studies, we compared sorbitol dehydrogenase activity with that of alkaline phosphatase, aspartate aminotransferase, and alanine aminotranferase in the sera of rats treated with thioacetamide or in which the common bile duct has been ligated.
Radioimmunoassay of progesterone in saliva: application to the assessment of ovarian function.
Authors: Walker R F , Read G F , Riad-Fahmy D
Published: January 2020

We report a specific radioimmunoassay that has the required sensitivity (7 pg per assay tube) for determining progesterone concentrations in 400 microL of mixed saliva collected from normal women. The assay is precise: intra and inter-assay variation (CV) never exceeded 11.0 and 8.0%, respectively. The assay was used to determine progesterone in saliva samples collected daily for not less than 28 days by normal women and by patients having abnormal ovarian function. Four normal women provided matched saliva and plasma samples for accurate dating of the menstrual cycle by plasma progesterone, estradiol, lutropin, and follitropin. Nine further subjects collected saliva samples only, and from these data a provisional "normal range" was established. Progesterone concentrations in saliva during the follicular phase of the cycle were low (less than 100 pmol/L) but rose beginning on day 12 to reach peak values of 230-550 pmol/L on day 21. Thereafter, progesterone concentrations in saliva declined to values generally less than 170 pmol/L at the commencement of menses. Saliva samples from three patients attending an infertility clinic were also studied to assess ovarian function.
Assay of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid by liquid chromatography with electrochemical detection.
Authors: Morrisey J L , Shihabi Z K
Published: January 2020

We describe a method for the assay of urinary 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid), based on its separation on a microparticulate reversed-phase column and direct electrochemical detection. Patients with neuroblastoma, pheochromocytoma, and Parkinson's disease have increased amounts of this compound in their urine.
Blood thyroxine concentration is lower in low-birth-weight infants.
Authors: Dussault J H , Morissette J , Laberge C
Published: January 2020

From out data bank, we analyzed results for thyroxine concentration in blood samples spotted on filter paper from different age groups (one to nine days postpartum) of our low-birth-weight and normal population. It was significantly lower in the low-birth-weight infants in almost all the age groups, and in both groups it significantly declined after postnatal day 5. The influence of weight is more important in the low-birth-weight population, but does not completely explain the smaller values. The results indicate that blood samples used in screening for congenital hypothyroidism in the neonate should be taken on postnatal days 3 to 5, and if a recall specimen is collected on days 5 to 9, one should expect the thyroxine concentration in the second specimen to be lower than in the first.
Efficacy of a simplified primary screening procedure for detection of hyperlipoproteinemias in a pediatric population.
Authors: Srinivasan S R , Foster T A , Berenson G S
Published: January 2020

We examined the efficacy of a simple primary screening procedure for detecting beta- and pre-beta-lipoprotein abnormalities in 3183 children, ages 5-14, residing in Bogalusa, Louisiana. This procedure is based on the ability of beta- and pre-beta-lipoproteins to form insoluble complexes with heparin in the presence of Ca2+; the turbidity produced by the reaction was considered as an index of the concentration of these two classes of lipoproteins. Our results indicate a close relationship (r = 0.88) between the beta- + pre-beta-lipoprotein index (turbidity) and the concentrations of these lipoproteins. Comparison of serum lipid and beta- + pre-beta-lipoprotein values of 5% of the children whose results fell outside the normal limits (upper and lower 5%) indicated that serum total cholesterol was not reflecting the beta- + pre-beta-lipoprotein concentration of a given child. The variability of alpha-lipoprotein concentration in these children accounted for this discrepancy. Measuring the serum beta- + pre-beta-lipoprotein index may be more useful for large-scale screening and for detecting subtle abnormalities than are determinations of either cholesterol or triglycerides.
Manual and continuous-flow colorimetry of triacylglycerols by a fully enzymic method.
Authors: Megraw R E , Dunn D E , Biggs H G
Published: January 2020

We describe a fully enzymic method for manual and continuous-flow colorimetric assay of triacylglycerols (triglycerides) in serum. Triglycerides are enzymically hydrolyzed in 10 min by lipase and microbial esterase. The resulting free glycerol is measured enzymically by glycerol kinase and glycerol-3-phosphate dehydrogenase. The NADH so formed is oxidized by coupling with a tetrazolium salt/diaphorase system. The test follows Beer's law to 8 g/L, and the final color is stable for at least 1 h for serum, 15 min for aqueous triolein standards. The manual assay requires only 25 microliter of serum and few manipulations. A specific triolein standard was developed for calibrating the manual method. For the continuous-flow method, calibration is made with four concentrations of glycerol standard. The procedure is sensitive, has good precision and accuracy, and gives results that compare well with chemical and enzymic commercial kit methods.
Assays for chloramphenicol compared: radioenzymatic, gas chromatographic with electron capture, and gas chromatographic-mass spectrometric.
Authors: Pickering L K , Hoecker J L , Kramer W G , Liehr J G , Caprioli R M
Published: January 2020

We compared these three techniques for measuring chloramphenicol in serum or urine. Although each has its particular advantages, any of them is shown to be satisfactory and may appropriately be used by clinical laboratories, according to the facilities available.
Correlation between X-prolyl dipeptidyl-aminopeptidase and serum amine oxidase in serum of patients with post-burn keloids.
Authors: Nagatsu T , Iwase K , Kasahara Y , Kubono K , Sakakibara S , Aoyama H , Izawa Y
Published: January 2020

Activities of X-prolyl dipeptidyl-aminopeptidase (EC 3.4.14.1) and amine oxidase (EC 1.4.3.6) in serum were assayed in two groups of patients, children two to nine years old and adults 23 to 60 years old, with hypertrophic scars after severe burn. The peptidase activity tended to be low initially for several months after the burn, but then returned to normal after six months. These changes were marked in the child group, less so in the adult group. Similar but less-pronounced changes were also observed in serum amino oxidase activity. The two serum enzyme activities showed a significant positive correlation (r = 0.668, p less than 0.001, n = 27) in the patients.
Determination of total CO2 in plasma by automated flow-injection analysis.
Authors: Baadenhuijsen H , Seuren-Jacobs H E
Published: January 2020

We describe a procedure for measuring total CO2 in plasma, based on the principles of the flow-injection analysis technique, which makes use of unsegmented fast-flowing reagent streams, as developed by Růziĉka and co-workers. The further methodological design resembles the silicone-rubber membrane technique of Kenny and Cheng. CO2 in the sample is released by reaction with H2SO4. Appropriate amounts of CO2 permeate through the membrane that separates the acid reagent streams and a buffered cresol-red indicator system. The experimental set-up and functioning of this system are described.
Creatine kinase in serum: 6. Inhibition by endogenous polyvalent cations, and effect of chelators on the activity and stability of some assay components.
Authors: Szasz G , Waldenström J , Gruber W
Published: January 2020

We studied the effects of some chelators on creatine kinase activity. Creatine kinase is competitively inhibited by endogenous polyvalent cations (e.g., calcium, Ki = 4.5 mmol/L); this can be reversed by adding chelators to the reagent, resulting in a mean increase in activity of 1.14-fold at 25 degrees C and 1.18-fold at 30 and 37 degrees C. Adding chelators, 5 and 10 mmol/L, to serum stored at 37, 30, 25, 4, and -20 degrees C increased isoenzyme stability in some cases, but under certain conditions decreased it, especially at higher chelator concentrations, and more so for EGTA than for EDTA. Blood sampling into tubes prepared with chelators and storage of plasma has no advantage over serum stored in the presence of chelators. The most striking effect of chelators is their protective effect on thiols in the creatine kinase reagent. In the presence of EDTA, 2 mmol/L, the reagent is stable for at least a day at 25 degrees C or a week at 4 degrees C. The poor stability of glucose-6-phosphate dehydrogenase, which is nearly independent of chelators, is the limiting factor for reagent containing EDTA. Bis-Tris, a buffer recently recommended for assay of creatine kinase activity, is a weak chelator. Imidazole acetate buffer combined with EDTA yields activities identical to those found with Bis-Tris at assay temperatures of either 25 or 30 degrees C.
Determination of catecholamines and their 3-O-methylated metabolites in urine by mass fragmentography with use of deuterated internal standards.
Authors: Muskiet F A , Thomasson C G , Gerding A M , Fremouw-Ottevangers D C , Nagel G T , Wolthers B G
Published: January 2020

We report the determination of catecholamines (dopamine, norepinephrine, and epinephrine) and their 3-O-methylated metabolites (3-methoxytyramine, normetanephrine, and metanephrine) in urine by gas chromatography/mass spectrometry, with use of stable isotopically labeled internal standards. Normal excretion values in terms of creatinine, expressed as a function of age, are given, together with results obtained for patients with neuroblastoma, ganglioneuroblastoma, pheochromocytoma, parkinsonism treated with L-DOPA plus peripheral decarboxylase inhibitor, and a patient with cardiogenic shock treated with dopamine. The results obtained for normal adults are discussed in relation to other catecholamine metabolites and mean overall catecholamine turnover.
Improved lysozyme assay in biological fluids.
Authors: Grossowicz N , Ariel M , Weber T
Published: January 2020

We describe a simple, rapid, sensitive, and highly reproducible assay for lysozyme, with use of concentrated cell suspensions of Micrococcus lysodeikticus in Tris-buffered glycerol/water (40/60 by vol), pH 7.5. Stored at -20 degrees C, the cells' susceptibility to lysozyme remains unaltered over long periods. Almost identical concentration curves were obtained with different aliquots of the same preparation during eight months. Lysozyme activity was reflected in the decrease in absorbance of the reaction mixture after incubation for 15 min at 37 degrees C. Concentrations of egg-white lysozyme as low as 0.02 mg/L can be accurately assayed.
Increased concentrations of free fatty acids and glycerol and altered creatine kinase MM isoenzyme patterns in certain diabetic patients.
Authors: Schwertner H A , McLaren R C , Arnold E L
Published: January 2020

We report unusually high concentrations of free fatty acids and glycerol in sera of patients with adult-onset diabetes, and the accompanying alterations in creatine kinase isoenzyme MM patterns associated with such patients. The serum samples with increased free fatty acids also showed increased electrophoretic movement of the MM isoenzyme on cellulose acetate membranes. Fatty acid concentrations found in such samples averaged 9.88 +/- 5.65 (SD) meq/L and the average glycerol concentration was 153 +/- 115 (SD) mg/L. The serum glycerol concentrations correlated with those of the free fatty acids (r = 0.886, slope = 0.309, intercept = 4.76).
A solid-phase enzyme-linked immunosorbent assay for the quantitation of human plasma alpha 1-acid glycoprotein.
Authors: Wang H P , Chu C Y
Published: January 2020

We describe a solid-phase enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in human plasma. Plasma samples are incubated with alkaline phosphatase-linked, purified alpha 1-acid glycoprotein in alpha 1-acid glycoprotein-specific antibody-coated polystyrene tubes. The alkaline phosphatase that becomes attached to the tube via an immunological reaction between the alpha 1-acid glycoprotein and the specific antibody is measured spectrophotometrically. This assay is accurate reproducible, simple, and economical. As little as 4 microgram of alpha 1-acid glycoprotein per liter can be detected. The normal range for alpha 1-acid glycoprotein in the plasma of healthy adults, as measured by this method, is 0.48-1.27 g/L; the range is significantly different, 0.29-0.73 g/L, for women who are taking oral contraceptive pills.
Effect of reagent pH on determination of high-density lipoprotein cholesterol by precipitation with sodium phosphotungstate-magnesium.
Authors: Grove T H
Published: January 2020

When determining high-density lipoprotein cholesterol by use of sodium phosphotungstate-magnesium precipitation method, I found that the pH of the sodium phosphotungstate reagent was a critical factor in the method. Unless the pH of the reagent was less than 7.6, the precipitation of low-density lipoprotein and very-low-density lipoprotein was incomplete. When the specimen pH was between 7.35 and 8.65, the pH of the serum of plasma did not influence the completeness of precipitation. Optimum concentrations of precipitation reagents, determined after the pH of the sodium phosphotungstate reagent was standardized to pH 6. 15, were 40 g/L for sodium phosphotungstate and 2 mol/L for MgCl2. The distribution of high-density lipoprotein cholesterol in a healthy adult population was skewed to the left for women (n = 34; mean = 660 mg/L) and bi-modal for men (n = 44; mean = 460 mg/mL). The central 95% reference interval was 280 to 880 mg/L for women and 250 to 750 mg/L for men.
Simplified determination of urinary delta-aminolevulinic acid in a wide range of concentrations.
Authors: Fiorentina H D , Grogna M , Dewiest F
Published: January 2020

We describe a simple method for measuring delta-aminole-vulinic acid in urine without the need for the seldom-used but time-consuming internal-standard addition step. It combines the analytical procedure described by Tomokuni and Ogata [Clin. Chem. 18, 1534 (1972)] and a correction based on urine density to obtain, more rapidly and less expensively, results as accurate as those given by the Davis and Andelman [Arch. Environ. Health 15, 53 (1967)] chromatographic method.
Assay of 1,25-dihydroxy vitamin D3 by isotope dilution--mass fragmentography.
Authors: Björkhem I , Holmberg I , Kristiansen T , Pedersen J I
Published: January 2020

We describe a specific assay of 1,25-dihydroxy vitamin D3 in human serum, based on isotope dilution-mass fragmentography. We added [26-2H3]-1,25-dihydroxy vitamin D3 to a fixed amount of serum. The steroids were extracted with chloroform/methanol and purified by liquid chromatography. The purified materal was converted into the trimethylsilyl ether and analyzed. Unlabeled 1,25-dihydroxy vitamin D3 was quantitated from the ratio between the tracings at m/e 452 and 455. The two ions used correspond to loss of two trimethylsilyloxo functions from the molecular ion. Essentially the same results, with some problems of interference, were obtained when we used the more intense ions at m/e 131 and 134 (corresponding to cleavage between C-24 and C-25). The detection limit was about 5 ng/L of serum; the coefficient of variation was about 6%. The accuracy of the method was assessed by recovery experiments. 1,25-Dihydroxy vitamin D3 in sera from 15 healthy subjects was found to average 55 +/- 10 ng/L(+/- SD). We believe this represents the first determination of 1,25-dihydroxy vitamin D3 in serum by use of a method not based on radioimmunoassay or receptor assay. Slightly lower values have been reported by those latter techniques.
Separation of protoporphyrins and related compounds by reversed-phase liquid chromatography.
Authors: Culbreth P , Walter G , Carter R , Burtis C
Published: January 2020

We describe the separation of protoporphyrin and related porphyrins by reversed-phase "high-performance" liquid chromatography, with fluorometric detection. We used the method to demonstrate that acid hydrolysis of the dimethyl ester of protoporphyrin IX is complete in 2 to 3 h and is followed by the acid-catalyzed conversion of protoporphyrin IX to a chemical species that chromatographic evidence indicates to be hematoporphyrin IX. In addition, the method was used to evaluate the purity of a commercial preparation of protoporphyrin IX and was also demonstrated to have the sensitivity and specificity needed for measuring the protoporphyrin IX content of whole-blood.
Phosphatidylglycerol in 261 samples of amniotic fluid from normal and diabetic pregnancies, as measured by one-dimensional thin-layer chromatography.
Authors: Tsai M Y , Marshall J G
Published: January 2020

We describe a one-dimensional thin-layer chromatographic method for separating lecithin, sphingomyelin, phosphatidylglycerol, and other phospholipids. The occurrence of phosphatidylglycerol in relation to the lecithin/sphingomyelin ratio is reported for 261 amniotic fluid samples. This compound does not consistently appear until the ratio exceeds about 3.1, and occurs less often in samples from diabetic patients than in those from normal patients. The respiratory distress syndrome did not occur when phosphatidylglycerol was present in amniotic fluid although the reverse was not necessarily true. Thus the presence of phosphatidylglycerol offers additional assurance of pulmonary maturity.
Substrate-labeled fluorescent immunoassay for phenytoin in human serum.
Authors: Wong R C , Burd J F , Carrico R J , Buckler R T , Thoma J , Boguslaski R C
Published: January 2020

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.
Electrolytes of breast-cyst fluid.
Authors: Gatzy J T , Zaytoun M P , Gaskins K , Pearlman W H
Published: January 2020

We constructed an electrolyte balance sheet for 11 individual specimens and two pooled specimens of breast-cyst fluid. Na+ and K+ ranged in concentrations from those representative of extracellular fluids (147 and 9 mmol/L, respectively) to those of intracellular fluid or a high K+ secretion (23 and 168 mmol/L, respectively). Cl- concentrations ranged from 9 to 98 mmol/L (mean, 31), bicarbonate concentrations from 0 to 34 mmol/L (mean, 16). Ca2+ concentrations did not exceed 2.1 mmol/L. Total protein ranged from 14.7 to 35.8 gL. Fluid osmolality ranged from 249 to 300 mOsm/kg of water (mean, 280). The difference between measured cations and anions indicates an "anion gap" of 140 mmol/L, which must be carried by 46 mOsm/kg of water. We suggest that tricarboxylic acids of the Krebs cycle may account for the missing trivalent anions.
Positive interference of catecholamine metabolites with quantitation of urinary uric acid by the direct acid ferric-reduction method.
Authors: Liu T Z , Khayam-Bashi H
Published: January 2020

We report the effect of major catecholamine metabolites such as homovanillic acid and vanilmandelic acid, normally present in urine, on quantitation of urinary uric acid by the direct acid ferric-reduction procedure [(Am. J. Clin. Pathol. 60, 691 (1973)]. When aqueous uric acid standards containing a mixture of these two metabolites, in concentrations comparable to those in a normal human 24-h urine, were added to Fe3+-phenanthroline or Fe3+-2,4,6-tripyridyl-s-triazine reaction mixture, the resulting absorbances at 505 or 593 nm considerably exceeded those of standards without such additions. Moreover, quantitation of urinary uric acid by the direct acid ferric-reduction procedure gave values that significantly exceeded those determined by the uricase method. Interference by the two metabolites was eliminated by extracting urine specimens with ethyl acetate before quantitation. Spectral scans revealed that homovanillic acid or vanilmandelic acid alone could also effectively reduce Fe3+-chelator complex and that the effect of these compounds on the analytical reaction was additive.
Polychromatic analysis: new applications of an old technique.
Authors: Hahn B , Vlastelica D L , Snyder L R , Furda J , Rao K J
Published: January 2020

As a means of correcting for many potential interferences, polychromatic analysis offers an effective alternative to either sample pretreatment or separate blank determinations. Because of the differing spectral characteristics of each interfering species, and the nature of these interferences (static vs. kinetic), the application of polychromatic analysis must be optimized for each analytical procedure to minimize all residual errors. Overall assay precision can be maximized by proper use of flagging procedure to minimize all residual errors. Overall assay precision can be maximized by proper use of flagging procedures (also based on polychromatic analysis). By using a second wavelength measurement, the analyst can also verify the accuracy of an analytical measurement in selected situations. These applications of polychromatic analysis are a departure from conventional analytical procedures in allowing for correction of various interferences in the actual reaction mixture, without making assumptions about the recovery of analyte during pretreatment or the equivalent absorptivity of interferent in both blank and assay solutions.
Enzymic assay of total cholesterol involving chemical or enzymic hydrolysis--a comparison of methods.
Authors: Deacon A C , Dawson P J
Published: January 2020

Manual procedures for estimating serum total cholesterol by use of cholesterol oxidase (EC 1.1.3.6) and the phenol--aminophenazone--peroxidase chromogenic system are described, in which cholesteryl esters are hydrolyzed either by use of pancreatic cholesterol ester hydrolase (EC 3.1.1.13) or saponification by ethanolic potassium hydroxide. Both methods are linear up to a cholesterol concentration of 12 mmol/L and are reproducible (between-run CV, about 1.1%). The chemical hydrolysis method yields results that are about 10% lower than those obtained by enzymic hydrolysis, because of incomplete removal of interfering thiols generated during the saponification of serum. The chemical hydrolysis procedure is much less susceptible to interferences, particularly by bilirubin, but the enzymic hydrolysis system is simpler to perform and therefore has a greater potential for mechanization.
Use of specific inhibitors to disciminate alkaline phosphatase isoenzymes originating from human liver, placenta and intestine: absence of meconial alkaline phosphatase in maternal serum.
Authors: Doellgast G J , Meis P J
Published: January 2020

We used the inhibitors bromotetramisole, L-phenylalanine amide, and L-phenylalanine in combination to measure intestinal phosphatase in maternal serum and amniotic fluid. By using high concentrations of these inhibitors, it was possible to measure the three isoenzymes separately. We found no evidence of the presence of meconial alkaline phosphatase in the serum of the mother (six cases) after meconial passage in utero.
Relations between reported alcohol consumption and certain biological variables in an "unselected" population.
Authors: Bagrel A , d'Houtaud A , Gueguen R , Siest G
Published: January 2020

We looked for variables that could serve as indexes of alcohol consumption. We tested iron, gamma-glutamyltransferase, mean erythrocyte volume, mean erythrocyte hemoglobin concentration, urea, uric acid, and alanine aminotransferase, among others, in an unselected population of men, and by principal components analysis we singled out the correlation between the two enzymes and the two hematologic values. On the other hand, calculation of the coefficients of correlation between the total amount of alcohol consumed and the 10 variables studied, together with multiple regression analysis, showed that the three variables that correlated most significantly with alcohol consumption were gamma-glutamyltransferase, mean erythrocyte volume, and the use of tobacco. Critical evaluation of the results leads us to conclude that still more discriminative biological indices msut be sought and that the use of psychosocial data is also desirable.
Analysis for diazepam and nordiazepam by electron-capture gas chromatography and by liquid chromatography.
Authors: Wallace J E , Schwertner H A , Shimek E L
Published: January 2020

We describe the use of electron-capture gas chromatography or reversed-phase "high-performance" liquid chromatography for concurrent analysis for diazepam and nordiazepam in serum. In the gas-chromatographic analysis our use of a new chemically deactivated stationary liquid phase, SP 2250-DB (Supelco, Inc.), resulted in improved chromatographic sensitivity and peak symmetry for the two benzodiazepines as compared to that obtained with either OV-17or OV-1 phases. Steady-state concentrations of diazepam and nordiazepam in serum as determined by gas-liquid chromatography correlated closely with those found by liquid-liquid chromatography.
Evaluation of commercial heparin preparations for use in the heparin-Mn2+ method for measuring cholesterol in high-sensity lipoprotein.
Authors: Mayfield C , Warnick G R , Albers J J
Published: January 2020

Commercial heparin preparations (18 lots) from seven manufacturers were compared in the heparin-Mn2+ procedure for high-density-lipoprotein cholesterol quantitation. With normotriglyceridemic samples, 16 heparin lots, isolated from porcine intestinal mucosa, gave mean values for supernatant cholesterol that did not differ statistically; all were within 7 mg/L. Two heparin preparations from bovine lung gave results that were slightly (16 mg/L, average) but significantly (p less than 0.005) lower. With hypertriglyceridemic samples, we observed greater variation in supernatant cholesterol among the heparin preparations, which was ascribable to variable sedimentation by centrifugation of very-low-density and low-density lipoproteins precipitated by heparin-Mn2+ treatment. If the precipitated lipoproteins were completely removed by an ultrafiltration procedure, we saw no significant difference among the heparin preparations for results with hypertriglyceridemic samples.
New micro-turbidimetric method for determination of protein in cerebrospinal fluid and urine.
Authors: Iwata J , Nishikaze O
Published: January 2020

We report a new micro-scale (0.1-mL sample) turbidimetric method for determination of protein by use of benzethonium chloride in alkali. The method is highly specific for protein, has a higher sensitivity than the classic method of Lowry et al., and shows satisfactory reproducibility and recovery. The turbidity produced in our method is the same for albumin and gamma-globulin and is more stable than in Meulemans' method (in which sulfosalicylic acid is used) or in the method of Bossak et al. (in which trichloracetic acid is used). In contrast to Pesce and Strande's method, there is no manipulative loss of protein.
Radioimmunoassay of total urinary estrogens.
Authors: Bordens R W , Engel R H
Published: January 2020

We describe a radioimmunoassay for total estrogens in urine. Estrogens are extracted by adsorption on XAD-2 resin, eluted with methanol, and acid-hydrolyzed. Estrogens are then determined in a diluted aliquot of the hydrolysate. Within-run coefficients of variation for estriol values between 6.0 and 100 micrograms/L were less than 5%; between-run CV was 19.6% at an estriol concentration of 16.8 microgram/L. A correlation coefficient of 0.96 was found on comparison of the radioimmunoassay (y) with a standard fluorometric assay (x); the regression equation is y = 2.18 x - 6.70 micrograms/L. Determination on pregnancy and non-pregnancy urine can be done within 4 h.
Determination of aminoglycoside antibiotics in biological fluids: a review.
Authors: Maitra S K , Yoshikawa T T , Guze L B , Schotz M C
Published: January 2020

We describe current procedures for measuring aminoglycoside antibiotics. Although many of these techniques are suitable for clinical adaptation, selection of a method for use in a clinical laboratory will depend on the budget and availability of equipment and expertise.
Improved determination of phenobarbital, primidone, and phenytoin by use of a preparative instrument for extraction, followed by gas chromatography.
Authors: St Onge L M , Dolar E , Anglim M A , Least C J
Published: January 2020

A microprocessor-controlled automatic extractor, (PREMTM I) is described with which lipophilic components may be extracted from physiological fluids by means of a selective, solid-phase extraction cartridge, and the extracts presented in dry form for subsequent analysis. This instrument has been successfully evaluated for the extraction of phenobarbital, primidone, and phenytoin from human serum before chromatographic analysis. We describe a specific method for determination of these anticonvulsants, involving the PREP I and analysis, after flash-heater methylation, in a gas chromatograph equipped with a nitrogen-sensitive detector. Absolute recoveries ranged from 90 to 102%. Within-day and day-to-day precision (CV) ranged from 4 to 7% for all three drugs in therapeutic concentrations. Comparison of this method with results obtained with a liquid--liquid micro-scale extraction and a similar gas-chromatographic method resulted in correlation coefficients of 0.988 (phenobarbital), 0.978 (primidone), and 0.982 (phenytoin). These drugs in therapeutic concentrations can be simultaneously extracted and measured with confidence.
Micro-scale enzymic determination of ethanol in plasma with a discrete analyzer, the ABA-100.
Authors: Whitehouse L W , Paul C J
Published: January 2020

An enzymic method for estimating ethanol has been adapted to the Abbott Bichromatic Analyzer-100. Fully automated, the method can accommodate up to 192 assays per hour. It requires only 2.5 muL of plasma and therefore is applicable to analysis for ethanol in blood sampled by finger-prick.
Routine glucose determination in serum by use of an immobilized glucose dehydrogenase nylon-tube reactor.
Authors: Sundaram P V , Blumenberg B , Hinsch W
Published: January 2020

We report a method for immobilizing glucose dehydrogenase on the inside surface of nylon tubes to produce an immobilized-enzyme nylon-tube reactor. The glucose dehydrogenase reactor is integrated into the flow system of a continuous-flow analyzer to facilitate routine analysis of serum glucose at 50 samples/h. We compared results with those by the reference hexokinase/glucose-6-phosphate dehydrogenase solution method. The coefficient of correlation was r = 0.996. A glucose dehydrogenase reactor made starting with 1 mg (250 U) of enzyme was stable during eight weeks of continuous use, that is, for nearly 3500 tests. This reduced the cost of the assay by at least 50-fold, compared with that for a commercial glucose dehydrogenase test pack method.
A simplified, colorimetric micromethod for xylose in serum or urine, with phloroglucinol.
Authors: Eberts T J , Sample R H , Glick M R , Ellis G H
Published: January 2020

We have developed a simplified xylose assay procedure that requires only 10 min and requires 50 microL of serum or 5 microL of urine. The reaction with phloroglucinol is more sensitive than the classic p-bromaniline color reaction, and requires only 4 min of heating for color development. A single reagent is mixed with the specimen directly, without prior protein precipitation. Analytical recovery of xylose added to serum was quantitative; precision studies resulted in a between-day coefficient of variation of 5.2%. Glucose, which has significant potential for interference in most other xylose procedures, reacts under the test conditions only to the extent of 70 mumol of apparent xylose per liter for a 5.5 mmol/L solution of glucose. The new procedure has been valuable in the assessment of malabsorption, especially in children and infants, where serum xylose is the preferred measurement.
Assay of plant sterols by use of cholesterol oxidase.
Authors: Kritchevsky D , Tepper S A
Published: January 2020

The cholesterol esterase/cholesterol oxidase enzyme kit can be used for analysis of plant sterols. The results are comparable to those obtained by conventional colorimetric analysis.
Improved method for simultaneous determination of cholesterol in high- and low-density lipoproteins.
Authors: Noma A , Okabe H , Netsu-Nakayama K , Ueno Y , Shinohara H
Published: January 2020

A previously described procedure for simultaneous determination of cholesterol in high- and low-density lipoproteins (HDL and LDL) [Clin. Chem. 24, 1504--1508 (1978)] has been improved by using centrifugation instead of ultrafiltration. Addition of NICl2 (2.0 mmol/L) to the reagent produced a good separation of HDL from the complex of low- and very-low-density lipoprotein-heparin-Ca2+ on centrifugation at 3000 rpm for 15 min. Replicate analyses for high-density lipoprotein cholesterol by the present method demonstrated the following intra-assay precision: mean = 389 mg/L, SD = 11 mg/L, CV = 2.8%. The present (y) and original (x) methods gave results that agreed reasonably well (n = 50, r = 0.960, y = 1.0x + 0.9). The enzymic method for HDL- or (HDL + LDL)-cholesterol after the separation of their fractions gave erroneous results, in particular in the cases of hyperbilirubinemic sera and in (HDL + LDL) fractions.
Low proportions of glycosylated hemoglobin associated with hemoglobin S and hemoglobin C.
Authors: Aleyassine H
Published: January 2020

Using a cation-exchange chromatographic method, we found normal or subnormal values for glycosylated hemoglobin in a few diabetic patients with persistent hyperglycemia. Subsequent investigations revealed that these unexpected results had originated from black patients with diabetes. In view of common occurrence of abnormal hemoglobins in the Negro population, we subjected blood preparations to electrophoresis on cellulose acetate and acrylamide gel. The results have shown the presence of hemoglobin S or hemoglobin C in each patient. When allowance was made for the percentage of the abnormal hemoglobin, the "corrected values" of glycosylated hemoglobin increased to the diabetic range. Furthermore, the corrected values agreed well with the "expected values" calculated from a regression line correlating fasting blood glucose concentrations and proportions of glycosylated hemoglobin in more than 300 diabetics with no evidence of hemoglobinopathy. We conclude that in diabetic patients presenting with hemoglobin S or hemoglobin C, there is a considerable decrease in the values for glycosylated hemoglobin as measured by cation-exchange chromatographic methods, and that this decrease is proportional to the percentage of the abnormal hemoglobin.
A systems approach to fluorescent immunoassay: general principles and representative applications.
Authors: Curry R E , Heitzman H , Riege D H , Sweet R V , Simonsen M G
Published: January 2020

We have developed an automated system for the immunoassay of subnanogram quantities of clinically interesting compounds by molecular fluorescence. The system includes all the necessary reagents and an automated fluorometer. The microprocessor-based instrument consists of a measurement and data-processing module and an automated sampling unit. With use of 10 pmol/L amounts of fluorescent dyes such as fluorescein, measurements with precision and accuracy of 1--3% are attained. In a competitive-binding fluorescence immunoassay, antigen labeled with a fluorescent dye competes with antigen in the sample or standard for a limited amount of antibody immobilized on a polyacrylamide bead 2--5 micrometers in diameter. After separating antibody-bound from free tracer, we measure the amount of fluorescence bound to the beads. In representative example assays, correlation of fluorescence immunoassay (y) with a reference radioimmunoassay (x) of thyroxine was y = 1.01x + 13 nmol/L, r = 0.98. Correlation of fluorescence immunoassay (y) with a reference radioimmunoassay (x) of triiodothyronine was y = 0.99x + 0.004 nmol/L, r = 0.96.
Improved method for detecting hemagglutination by centrifugal analysis.
Authors: Wenz B , Feng C S , Karmen A
Published: January 2020

Centrifugal analysis can be used to detect hepatitis B surface antigen, antibody to rubella virus, and fibrin-related antigen. The procedure is performed with the same reagents used in conventional hemagglutination studies. Positive and negative reactions are distinguished by the rates of erythrocyte clearance in the centrifugal field (delta A/delta time); positive cells move more rapidly than negative cells, and this difference varies directly with the concentration of detectable antigen or antibody. This phenomenon is thought to be a result of the greater adhesion of negative cells to the cuvette's surface. Sensitivity and specificity are greater in the centrifugal analysis technique than in the more conventional hemagglutination tests. False-positive reactions are eliminated and the quantitative data are accurate and reproducible.
Instrumentation and techniques for analysis of hydrogen peroxide and peroxide-producing reactions involving earthworm (Diplocardia longa) bioluminescence.
Authors: Wampler J E , Mulkerrin M G , Rich E S
Published: January 2020

Earthworm bioluminescence (species Diplocardia longa) is a sensitive tool for determination of peroxide titers in biological systems, with linearity of the standard curve for hydrogen peroxide extending from 10 nmol/L in the assay to greater than 1 mmol/L, and with sensitivity to 2 pmol of peroxide per assay. This assay's insensitivity to extraneous protein, turbidity, and solutes that are not redox active makes it particularly useful for automated or crude sample analysis and for analysis of oxidases and their substrates in coupled reactions. Examples of this latter application we discuss are analyses of glucose and putrescine oxidases and their substrates. In each case linear standard curves are obtained, covering several decades of concentration. The coupled reactions also allow in situ detection of oxidases in electrophoresis gels. We also describe a small, portable photometer system and an on-line computer-controlled instrument system that has facilitated development of these assays and will also allow automatic scanning of bioluminescence in gels. The data-collection and decision-making algorithms of this system are simple and easily adaptable for use in an inexpensive computer-based photometer system.