bluuebunny/crossref_metadata_2025 huggingface dataset:
Human amylase isoenzymes separated on concanavalin A--Sepharose.
Authors: Takeuchi T
Published: January 2020

Human salivary amylase and pancreatic amylase were purified and characterized. These amylases gave two bands and one band, respectively, each staining for both protein and sugar, after electrophoresis on sodium dodecyl sulfate--polyacrylamide gel. The relative molecular mass (Mr) or pancreatic amylase was calculated to be 60 000; for the two components (A and B) of salivary amylase the Mr were 61 000 and 64 000. The two salivary amylases were separated by chromatography on concanavalin A--Sepharose; only component B bound to concanavalin A. The carbohydrate content of pancreatic amylase was 1.61 +/- 1.02% (SD), and of salivary amylases A and B 2. 18 +/- 0.71% and 8.77 +/- 2.28%, respectively. The salivary and pancreatic amylases had completely identical antigenicities against antibody to either. On isoelectric focusing, pancreatic amylase showed one peak at pH 7.0, salivary amylase A showed a major peak at pH 6.4 WITH A TRACE OF MATERIAL At pH 5.9, and salivary amylase B a major peak at pH 5.9 and one minor peak at pH 6.4. Serum amylase was separated into two major peaks with isoelectric points (pl) of 6.4 and 7.0, respectively, and one minor peak, with a pl of 5.9. Only a small part of the serum amylase with a pl of 5.9 combined with concanavalin A; the two other serum amylases did not.
Mechanized determination of the apparent unbound unconjugated bilirubin concentration in serum.
Authors: Wennberg R P , Rasmussen L F , Ahlfors C E , Valaes T
Published: January 2020

The peroxidase method for determining the apparent unbound bilirubin concentration in serum has been automated by use of a programmable, computer-directed spectrophotometer. This mechanized assay determines the total bilirubin concentration and apparent unbound bilirubin concentration in serum samples and titrates the serum with bilirubin to estimate the effect of increasing total bilirubin concentrations on the apparent unbound bilirubin concentration. The entire analysis requires 0.1 mL of serum and 4 min operation time, as compared with about 30 min for the manual method. The coefficients of variation for determination of the apparent unbound bilirubin concentration in bilirubin-enriched commercial control serum were 2.8% within-day and 5.6% between-day. Bilirubin--albumin binding in serum samples from infants with severe hyperbilirubinemia was analyzed by the manual peroxidase method, the automated peroxidase method, and Sephadex gel filtration. Good correlation was found among all three methods.
Enzyme-labeled immunosorbent assay for serum ferritin: method evaluation and comparison with two radioassays.
Authors: Fortier R L , McGrath W P , Twomey S L
Published: January 2020

We report our evaluation of a commercially available procedure and reagents for determination of ferritin in serum by enzyme-labeled immunosorbent assay (ELISA). Results by our procedure and the "Fer Iron" (Ramco Labs) procedure shows a degree of association (r) of 0.95 and a regression equation of y = 1.03x - 33. Similarly, our procedure compared to the "Gamma Dab" (Clinical Assays) ferritin procedure shows a degree of association of 0.98 and a regression equation of y = 0.93x - 11. Between-day standard deviations were 6 and 22 micrograms/L (n = 24 and 20) for ferritin concentrations of 20 and 300 micrograms/L, respectively. Ferritin values showed no correlation with total iron concentration, but show a broad inverse relationship with iron-binding capacities. The favorable correlation with existing procedures and the speed of the analysis commend the use of ELISA for measurement of ferritin in serum.
Analytical luminescence: its potential in the clinical laboratory.
Authors: Whitehead T P , Kricka L J , Carter T J , Thorpe G H
Published: January 2020

The various types of chemiluminescent and bioluminescent reactions are described. Applications of luminescence in the analysis of substancs of clinical interest are surveyed. The advantages, disadvantages, and prospects for luminescent assays are discussed.
Bioluminescence assay of creatine kinase and its isoenzymes in serum and cerebrospinal fluid.
Authors: Tarkkanen P , Komor S , Cornely C , Hacke W , Greiling H
Published: January 2020

We examined the sensitivity of bioluminescence for the determination of very low concentrations of creatine kinase brain-type subunit (CK-BB) in serum and in cerebrospinal fluid. To optimize the sensitivity of CK-isoenzyme assays and eliminate possible sources of error, we separated the isoenzyme fractions by using inhibiting anti-MM and precipitating anti-MM and anti-BB antibodies. The results with the bioluminescence assay correlated with spectrophotometric values such that r = 0.97 for the total CK activity and r = 0.98 for the CK-B activity. The reproducibility of the present method was comparable with the spectrophotometric method and was even better at low enzyme activities. The within-series precision for assay of total CK activity at 2 U/L corresponded to a CV of 9%; at 13 U/L the CV was 5.8%. All the assays were carried out at 25 degrees C. Even at this low temperature, CK activities as low as 0.2 U/L could be determined. In eight patients without any evidence of cerebral cell damage, total CK activity in cerebrospinal fluid was x = 1.05 +/- 0.6 U/L, and CK-BB activity was x = 0.7 +/- 0.4 U/L. In sera of these patients CK-BB activity was x = 0.6 +/- 0.5 U/L. Differences in CK and CK-BB activities in four patients with transient or progressive brain-cell damage are discussed.
Viable and total cell masses in dental plaque as measured by bioluminescence methods.
Authors: Robrish S A , Kemp C W , Chopp D E , Bowen W H
Published: January 2020

Bioluminescence methods have been applied to the measurement of the viable and total cell masses of small samples of dental plaque. The total adenine nucleotide content of dental plaque samples and of a pure culture of bacteria was determined and the adenylate energy charge calculated from this. When a pure culture of bacteria was killed with heat, the adenylate energy charge decreased exponentially with duration of treatment and corresponded with a decrease in the count of viable organisms.
Simultaneous liquid-chromatographic quantitation of salicylic acid, salicyluric acid, and gentisic acid in urine.
Authors: Cham B E , Bochner F , Imhoff D M , Johns D , Rowland M
Published: January 2020

We have developed a specific and sensitive method for the determination of salicylic acid, salicyluric acid, and gentisic acid in urine. Any proteins present are precipitated with methyl cyanide. After centrifugation, an aliquot of the supernate is directly injected into an octadecyl silane reversed-phase chromatographic column, then eluted with a mixture of water, butanol, acetic acid, and sodium sulfate, and quantitated at 313 nm by ultraviolet detection according to peak-height ratios (with internal standard, o-methoxybenzoic acid) or peak heights (no internal standard). The method allows estimates within 25 min. Sensitivity was 0.2 mg/L for gentisic acid, and 0.5 mg/L for both salicyluric and salicylic acid (20-micro L injection volume); response was linear with concentration to at least 2.000 g/L for salicylic acid and metabolites. Analytical recovery of salicylic acid and metabolites from urine is complete. Intra-assay precision (coefficient of variation) is 5.52% at 7.5 mg/L for salicylic acid, 5.01% at 9.33 mg/L for salicyluric acid, and 3.07% at 7.96 mg/L for gentisic acid. Interassay precision is 7.32% at 7.51 mg/L for salicylic acid, 5.52% at 8.58 mg/L for salicyluric acid, and 3.97% at 8.32 mg/L for gentisic acid. We saw no significant interference in urine from patients being treated with various drugs other than aspirin.
Liquid-chromatographic determination of 5-fluorocytosine.
Authors: Miners J O , Foenander T , Birkett D J
Published: January 2020

We report a "high-performance" liquid-chromatographic method for measuring 5-fluorocytosine in plasma and cerebrospinal fluid. After deproteinization with trichloroacetic acid, the supernates are chromatographed on a reversed-phase (C18) column. Response to concentration is linear in the range of 5 to 200 mg/L, with ultraviolet detection at 276 nm. The assay requires only 0.1 mL of plasma, is reproducible, and may be performed in less than 12 min. 5-Fluorocytosine concentrations determined by this procedure correlated well with those obtained by spectrofluorometry, although the present method is more specific with no observable interference from co-administered amphotericin B and most other commonly encountered drugs, including salicylat:. This method is applicable to the routine therapeutic monitoring of pediatric and adult patients as well as to pharmacokinetic studies.
Drug-induced dyslipoproteinemia: a report of two cases.
Authors: Naito H K , McHenry M C , Lewis L A
Published: January 2020

We describe two cases of atypical dyslipoproteinemia due to drug-induction. This secondary form of lipoprotein abnormality is unique because the newly available drug, miconazole, apparently directly delipidated the alpha-lipoproteins in the bloodstream. On closer study we found that the delipidation was caused by the vehicle rather than the fungicide--more specifically, only by the polyethoxylated castor oil in the vehicle. It affects serum lipoproteins both in vitro and in vivo, and the effect is species-specific. In vitro studies indicate that it preferentially delipidates high-density lipoprotein rather than low-density lipoprotein. Because its effects on the serum lipoproteins of rats resemble those on man, and because aortic lesions were produced in rats injected daily (90 mL/L) with this substance, caution is indicated in long-term use of drugs containing this chemical component in the vehicle.
Data-base techniques for multiple two-dimensional polyacrylamide gel electrophoresis analyses.
Authors: Lipkin L E , Lemkin P F
Published: January 2020

Two-dimensional protein electrophoresis can benefit from a powerful set of computer-supported image processing and data structure/management procedures. Detection of quantitative differences is complicated by local inhomogeneities in the polyacrylamide base; biochemical changes and variations in temperature and preparative technique also make the between-gel density and x-y coordinate correspondences quite imprecise. The program presented here provides local alignment and computer-controlled variable “flicker” rates for multiple gels, with use of an interactive display system. Manual spot densitometry, referred to a National Bureau of Standards density wedge, can be complemented by a set of automatic densitometry routines for previously established lists of spots. The ability to establish a set of local landmarks, either by included standards or user identification, provides a basis for automatic n-way gel comparison for subsets or for the entire set of spots. Automatic segmentatin algorithms allow isolatin of spots and separation of touching and partially overlapping regions. Various analytical and statistical facilities are part of the user’s access to the interactively developed data base. The data-structure and image-manipulation techniques developed here allow for user-directed and heuristic comparisons with online presentation of intermediate and final results.
Multiparameter appraoch to the "fingerprinting" of fluorescent Pseudomonads.
Authors: Shelly D C , Warner I M , Quarles J M
Published: January 2020

A recent study has shown that an emission-excitation matrix could be used to provide a unique fingerprint for the selective identification and characterization of certain species of Pseudomonas. This paper describes the results of a systematic study of the variables that contribute to the uniqueness of the fingerprint. Additional insight concerning the nature of the fluorescent pigments of these species is gained by analysis with a high-performance liquid chromatograph/video fluorometer combination. While providing information about the analytical variables contributing to the analysis, this combination also added the variable of chromatographic retention time for unambiguous fingerprinting.
Microcolumn liquid chromatography: a tool of potential significance in biomedical research.
Authors: Novotny M
Published: January 2020

Recent progress in the area of microcolumn HPLC is reviewed with an emphasis on biomedically important directions. The potential of high efficiency separations, additional advantages of solvent economy, and the development of new detection and ancillary techniques of high-performance liquid chromatography are also discussed.
Enzymic determination of free fatty acids in serum.
Authors: Okabe H , Uji Y , Nagashima K , Noma A
Published: January 2020

We describe the enzymic determination of free fatty acids in serum with use of acyl-CoA synthetase (EC l.2.1.3), acyl-CoA oxidase (no EC no. assigned) and peroxidase (EC 1.11.1.7). The free fatty acids are activated by acyl-CoA synthetase in the presence of ATP and coenzyme A. The acyl-CoA formed is oxidized by acyl-CoA oxidase to enoyl-CoA with simultaneous production of hydrogen peroxide, which is oxidatively coupled with 4-amino-antipyrine and 2,4-dibromphenol in the presence of peroxidase to yield a product that absorbs maximally at 505 nm. Standard curves prepared with various kinds of fatty acids are linear to at least 2.0 mmol/L and are practically congruent. Ascorbic acid or bilirubin interferes slightly. Results by the present method correlate well with those by the colorimetric method involving 2-(2-thiozolylazo)-p-cresol (r = 0.98). Replicate analyses of standard solution and of two kinds of control sera demonstrated the following between-assay precision: mean absorbance 0.224 (SD 0.002), CV 0.85%; mean concentration, 326 (SD 7.3) and 1076 (SD 22.7) mumol/L, CV 2.24 and 2.11%, respectively.
Antigen--antibody reaction investiaged with use of a chemically modified electrode.
Authors: Yamamoto N , Nagasawa Y , Shuto S , Tsubomura H , Sawai M , Okumura H
Published: January 2020

The antigen-antibody reaction of human choriogenadotropin has been potentiometrically investigated by use of a cyanogen bromide-treated electrode coated with the corresponding anti-serum. The potential of the modified electrode shifts in the positive direction upon contact with a solution of choriogonadotropin. The rate of the reaction at the interface between the electrode and the solution is estimated from the potentiometric measurement to be of the order of 10(4) mol-1 s-1 in diethyl barbiturate buffer, depending to some extent on the rotation speed of the stirrer. The change in potential is almost proportional to choriogonadotropin concentration. It is also pH dependent, the maximum response being at pH 8.7. The technique, applied to samples of human urine, has shown a specific response to choriogonadotropin.
Improved analysis for urinary polyamines by use of high-voltage electrophoresis on paper.
Authors: Fujita K , Nagatsu T , Shinpo K , Maruta K , Teradaira R , Nakamura M
Published: January 2020

This method for assay of polyamines (putrescine, spermidine, and spermine) in large numbers of urine samples is based on preliminary analyte isolation by use of a Dowex 50W column, separation by high-voltage paper electrophoresis, reaction with ninhydrin to detect the separated amines, and subsequent direct assay with a dual-wave-length thin-layer chromatography scanner by the zigzag scanning method. By optimizing the conditions for the Dowex 50W column and high-voltage paper electrophoresis, putrescine, cadaverine, spermidine, and spermine were completely separated. The major technical improvements are room-temperature collection of urine with toluene, shorter hydrolysis time, complete separation of putrescine from cadaverine and histamine, and elimination of interference with spermine measurement. Polyamine concentrations as measured by this method agreed with those obtained by use of an amino acid analyzer. Reference intervals by this method were similar to those reported by Russell (Clin. Chem. 23: 22, 1977) with an automated amino acid analyzer. Putrescine, spermidine, and spermine excretion in urine from patients with blood cancers and solid cancers was significantly increased; information on concentrations of putrescine and spermidine were especially useful for following the clinical efficacy of cancer treatments.
Radioimmunoassay of progesterone in unextracted serum.
Authors: Haynes S P , Corcoran J M , Eastman C J , Doy F A
Published: January 2020

We describe a rapid, precise radiommunoassay for progesterone in 25 muL of unextracted serum. Progesterone is released from its binding protein by adding an optimal amount of cortisol, which binds to the same protein (cortisol binding globulin) as progesterone. The amount of cortisol required does not cross react with the specific progesterone antibody used. This approach considerably shortens assay time and removes a tedious and imprecise stage in the conventional assay of serum progesterone. Results correlated well (r = 0.97) with a method involving organic solvent extraction of progesterone from serum. During the two years we have used this method in a busy diagnostic endocrine laboratory, the between-assay precision (CV) for low-, medium-, and high-concentration quality control sera was 12, 7, and 9%, respectively. Data from participation in an independent external quality-control program verified the adequacies of the method.
Kinetic properties of gamma-glutamyltransferase from human liver.
Authors: PetitClerc C , Shiele F , Bagrel D , Mahassen A , Siest G
Published: January 2020

We studied the kinetic properties of purified "heavy" form of human liver gamma-glutamyltransferase (EC 2.3.2.2) in the presence and absence of the acceptor substrate glycylglycine under Vmax conditions and as a function of pH. gamma-Glutamyl carboxynitroanilide was used as the donor substrate. Our data suggest that hydrolysis of donor substrate is the major pathway in the absence of acceptor. Autotransfer, if it occurs, is not important. Hydrolysis and transfer reactions have different pH profiles both for Vmax and Km. The pH dependency of Vmax and Km for both the hydrolase and the transferase reactions most probably reflects a change in the rate-limiting step: deacylation of the enzyme at acidic pH and acylation at alkaline pH. Negative cooperativity, observed for both donor and acceptor substrates near neutral pH, is interpreted in terms of more than one active site per dimer for each substrate.
Fluorometry of ethylene glycol in serum.
Authors: Meola J M , Rosano T G , Swift T A
Published: January 2020

We describe an assay for ethylene glycol in serum, which is based on the Hantzsch condensation reaction. Ethylene glycol is dehydrogenated by sodium periodate to form two molecules of formaldehyde, which is then combined with ammonium ion and acetylacetone to form a fluorescent diacetyllutidine derivative. The detection limit of this method is 50 mg/L. The procedure is sensitive, requires only 100 micro L of serum, and the standard curve is linear over a working range of 50 to 250 mg/L. Average analytical recovery ranged from 99 to 102% and within-run precision studies showed CVs of < 5%. Reagents are easily prepared and available.
Dilution as a source of error: implications for preparation and calibration of laboratory standards and for quality control of radioimmunoassays.
Authors: Gaines Das R E
Published: January 2020

Although dilution of materials is generally accepted to involve some error, workers often assume that this error may be discounted. However, comparing solutions of two identical materials diluted in the same way shows that dilution errors can cause a considerable departure from the expected identity. I describe this effect and its dependence on the errors at various stages of the dilution process.
Effect of detergent on kinetic Jaffé-method assay of creatinine.
Authors: Bergman A , Ohman G
Published: January 2020

Using isotope dilution-mass spectrometry as reference method, we show that the kinetic Jaffé method for determination of serum creatinine sometimes produces results that are much too high. Such falsely increased values were obtained in about 10% of the sera collected for routine determination of creatinine, and were most common when certain types of collecting tubes were used. The interferences could be overcome by use of detergent in the reaction mixture.
Determination of 8-methoxypsoralen in suction-blister fluid and serum by liquid chromatography.
Authors: Herfst M J , Edelbroek P M , de Wolff F A
Published: January 2020

A method is described for determination of 8-methoxypsoralen to 0.2 mL of suction-blister fluid or 1 mL of serum from psoriatic patients being treated with this drug. The drug is extracted from the biological matrix at pH 9.0 with a mixture of dichloromethane and light petroleum ether. 5-Methoxypsoralen is used as internal standard. Separation and quantitation are performed on a “high-performance” liquid chromatograph with use of an RP 18 reversed-phase column and detection at 245 nm. Accuracy and precision are good. Some benzodiazepines and their metabolites interfere. The lowest detectable concentration is 10 microgram/L, which means that the method is sufficiently sensitive to measure the drug concentrations in serum and suction-blister fluid for pharmacokinetic studies in patients being treated with a therapeutic dosage.
Heparin--magnesium chloride--albumin method for enzymic measurement of cholesterol in high-density lipoprotein.
Authors: Tallet F , Raichvarg D , Canal J
Published: January 2020

We describe a new method for measuring high-density-lipoprotein cholesterol in sera, with use of heparin, MgCl2, and albumin to precipitate very-low- and low-density lipoproteins, Lipoprotein electrophoresis and immunochemical methods show that high-density lipoprotein is completely and specifically isolated in the supernate, the cholesterol concentration of which is estimated by the enzymic method of Allain et al. (Clin. Chem. 20, 470, 1974). The precision of the method is excellent (CV = 1%) and the response is linear. Values so obtained correlate well with those obtained with the heparin-Mn2+ (r = 0.98) or the phosphotungstate-Mg2+ (r = 0.98) procedures. The technique is appropriate for routine clinical laboratory use.
Gas-chromatographic method of analysis for urinary organic acids. I. Retention indices of 155 metabolically important compounds.
Authors: Tanaka K , Hine D G , West-Dull A , Lynn T B
Published: January 2020

Gas-chromatographic retention indices are given, in terms of methylene units, for 155 metabolically important compounds (mostly organic acids) as trimethylsilyl derivatives on 10% OV-1 and 10% OV-17 columns. Comprehensive references on metabolic diseases that can be diagnosed by detection of these metabolites are cross-indexed to facilitate the use of the methylene-unit list. With the data presented here, it is now possible to diagnose more than 25 well-defined organic acidurias by use of gas chromatography alone.
Age-, sex-, and race-related differences in myoglobin concentrations in the serum of healthy persons.
Authors: Chen I W , David R , Maxon H R , Sperling M , Stein E A
Published: January 2020

A radioimmunoassay was used to study the relation of race, sex, and age to serum myoglobin concentrations in ostensibly healthy individuals: 75 white men, 76 white women, 75 black men, and 66 black women, ranging in age from 20 to 85 years. Mean serum myoglobin values were significantly higher in men than in women in both races (35 vs 31 microgram/L for whites and 44 vs 29 microgram/L for blacks). Black men had higher values than white men, but no corresponding difference was observed in the female population. Except for the group of black men, which consistently had the highest values for serum myoglobin, values were always higher for the older groups (greater than or equal to 50 years) than the younger, irrespective of race or sex. Serum myoglobin and age were significantly and directly correlated only among white men (r = 0.3408 p < 0.01, n = 75). If results were expressed as a myoglobin/creatinine ratio, the distinctions by race, sex, and age were partly eliminated. Reference intervals for serum myoglobin, expressed both ways, are given for different race, sex, and age groups.
A 57Co-soap assay for plasma total non-esterified fatty acids compared with a gas-liquid chromatographic method.
Authors: Turnell D C , Price C P , France M M
Published: January 2020

We describe a relatively rapid radiochemical assay for determining plasma non-esterified fatty acids, by use of a Dole-type (J. Clin. Invest. 35: 150, 1956) extraction and formation of 57Co soaps. Results correlated well (r = 0.957) with those by gas-liquid chromatography. The between-batch CV for 1.12 mmol of non-esterified fatty acids per liter was 7%; the sensitivity is estimated to be 22 mumol/L.
Measurement of urinary constituents with the Astra-8 Multichannel Analyzer.
Authors: Blumenfeld T A , Griffith B
Published: January 2020

The Beckman Astra-8 is a computerized, discrete-microsample, multiple-test clinical chemistry analyzer. We have examined its capability to measure urine Na+, K+, Cl-, urea nitrogen, glucose, and creatinine and found that (a) the Astra measurements are linear, accurate, and precise in concentration ranges of urine analytes; (b) urine analyte concentrations measured with the Astra correlate well with those by comparison micromethods (p < 0.001); and (c) the Astra has no significant carryover from adjacent urine samples or from adjacent urine and serum samples.
Creatine kinase isoenzyme patterns in neoplasms of peripheral nerve.
Authors: Balaton J , Hoag G N , Franks C R , Smith C R , DeCoteau W E
Published: January 2020

Normal peripheral nerve and neoplastic lesions of peripheral nerve varied in their creatine kinase (CK; EC 2.7.3.2) isoenzyme pattern, as assessed both with electrophoresis and with column chromatography. All three isoenzymes were seen in normal peripheral nerve, but the peripheral nerve tumors, neurofibroma and neurilemmoma, demonstrated predominantly CK-1 isoenzyme activity, with a trace amount of CK-3. No CK-2 activity was demonstrated in these tumors. In contrast, malignant schwannoma tissue contained all three isoenzymes, but in a different proportion than in normal peripheral nerve.
Pattern of urinary proteins and peptides in patients with rheumatoid arthritis investigated with the Iso-Dalt technique.
Authors: Clark P M , Kricka L J , Whitehead T P
Published: January 2020

Characteristic differences in the pattern of urinary proteins and peptides have been found in patients with rheumatoid arthritis, compared with patterns from healthy controls. These differences have been demonstrated with a two-dimensional gel electrophoretic technique (Iso-Dalt) involving isoelectric focusing in the first dimension, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Using simple photographic techniques, one can obtain a composite pattern of the individual protein-stained gels for each group. Comparison of the composite patterns from the rheumatoid arthritis group and the control group revealed several proteins in the urine of the rheumatoid arthritis patients not found in the control group. Two groupings of these proteins were identified: acidic, high-Mr proteins and more basic, low-Mr proteins.
Improved electrothermal determination of aluminum in serum by atomic absorption spectroscopy.
Authors: Alderman F R , Gitelman H J
Published: January 2020

We describe an improved method for determination of aluminum in serum by atomic absorption spectrophotometry with a graphite furnace. Interferences have been controlled and total combustion of the samples achieved by the use of new diluent in a molybdenum-treated graphite tube. The detection limit of this method is 2 microgram/L.
3-hydroxydecanedioic acid and related homologues: urinary metabolites in ketoacidosis.
Authors: Greter J , Lindstedt S , Seeman H , Steen G
Published: January 2020

Urine from patients with ketoacidosis was found to contain a number of aliphatic 3-hydroxy dicarboxylic acids. The acids were purified by silicic acid chromatography and their structures determined by gas chromatography-mass spectrometry of different derivatives. The major compound was 3-hydroxydecanedioic acid. Minor compounds were 3-hydroxyoctanedioic acid, 3-hydroxyoctenedioic acid, 3-hydroxydecenedioic acid, 3-hydroxydodecanedioic acid, 3-hydroxydodecenedioic acid, 3-hydroxytetradecenedioic acid, and 3-hydroxytetradecadienedioic acid. The excretion of 3-hydroxydecanedioic acid correlated positively with the excretion of hexanedioic acid, another metabolite constantly found in ketoacidosis (Pettersen et al., Clin. Chim. Acta 38: 17-24, 1972). We suggest that the 3-hydroxy dicarboxylic acids are formed from fatty acids by a combination of omega-oxidation and incomplete beta-oxidation.
Gas-chromatographic determination of amantadine in human urine.
Authors: Stumph M J , Noall M W , Knight V
Published: January 2020

We describe a gas--liquid-chromatographic method for determining the concentration of amantadine hydrochloride in urine with beta-phenylethylamine as internal standard. The urine sample is made alkaline and extracted with 0.5 mL of chloroform. After centrifugation the aqueous layer is aspirated, and an aliquot of the organic layer is injected directly into the gas chromatograph. Concentration and instrument response are linearly related between 2 and 125 mg/L. The limit of detection was 0.5 mg/L. Mean analytical recovery was calculated to be 97%.
Comparison of radial immunodiffusion and laser nephelometry for quantitating some serum proteins.
Authors: Alexander R L
Published: January 2020

After quantitating immunoglobulins G, A, and M and complement C3c and C4 in serum by using a laser nephelometer coupled with a data processor, I compared these results with values obtained by an early-readout radial immunodiffusion method. Day-to-day precision was better for nephelometry than for radial immunodiffusion for all proteins analyzed. The average coefficient of variation was 6.0% for nephelometry and 9.9% for radial immunodiffusion. Comparison of these methods gave ranked correlation coefficients of 0.945, 0.981, 0.932, 0.803, and 0.792 for IgG, IgA, IgM, C3c, and C4, respectively. Nephelometry gave significantly higher values than radial immunodiffusion for IgG, IgA, IgM, and C3c, and significantly lower values for C4 (p less than 0.001). Part of this bias was found to be due to the equation programmed in the data processor for calculating the standard curves. Within 95% limits, nephelometry gave higher normal ranges than radial immunodiffusion for IgG, IgA, and IgM. Other possible factors that can produce this bias are discussed.
Room temperature radioassay for B12 with oyster toadfish (Opsanus tau) serum as binder.
Authors: Ithakissios D S , Kubiatowicz D O , Wicks J H
Published: January 2020

We describe a vitamin B12 radioassay with oyster toadfish (Opsanus tau) serum in which endogenous serum B12 is released from its protein complexes at room temperature with NaOH at pH 13.6. The radioassay is reproducible (CV 4.9% within assay, 8.4% between assays) and sensitive (10 ng/L) and requires minimum manipulations. Assay accuracy is unaffected by serum protein.
Amniotic fluid phospholipids measured by continuous-development thin-layer chromatography.
Authors: Kolins M D , Epstein E , Civin W H , Weiner S
Published: January 2020

We describe a one-dimensional thin-layer chromatographic system for separation of amniotic fluid phosphatidylcholine, sphingomyelin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine in amniotic fluid. We utilize short-bed continuous development and "high performance" thin-layer chromatography. Phospholipids are detected with an antimony molybdate staining reagent and quantitated by transmittance densitometry. This system is more sensitive to changes in lecithin/sphingomyelin ratios than are planimetric evaluations.
Kinetic serum creatinine assays I. The role of various factors in determining specificity.
Authors: Bowers L D
Published: January 2020

We studied the specificity of kinetic methods of analysis, with emphasis on creatinine determination. The error contributed by an interferent that reacts with the analytical reagent and absorbs at the wavelength of analysis is shown to be related to the ratio of the equilibrium absorbances of the analyte and interferent, the ratio of the rate constants, the extent of the analytical reaction, and the duration of the measurement. To assess the potential diminution of the interference with kinetic methods, we determined the rate constants of several alpha-keto acids in the Jaffé reaction. Evaluation of the advantages of various measuring techniques with respect to specificity showed the main factors to be the extent of the analytical reaction and the relative value of the rate constants. Total specificity for creatine appears to be unattainable with currently used kinetic techniques.
Kinetic serum creatinine assays. II. A critical evaluation and review.
Authors: Bowers L D , Wong E T
Published: January 2020

We have evaluated the Technicon AutoAnalyzer method, the Beckman Creatinine Analyzer II, DuPont aca, Electro-Nucleonics GEMSAEC, and Instrumentation Laboratory Model 916 kinetic methods for determination of serum creatinine. These methods, which involve the Jaffé reaction, all showed a statistically significant proportional error relative to an ion-exchange pretreatment method for creatinine in 92 serum samples; the GEMSAEC method also showed a bias. The positive interference of several acid metabolites was quantitated in each of the methods. The evidence indicates that bilirubin, although associated with a negative creatinine interference, is not the sole interferent. We determined the magnitude of this interference and studied several methods for minimizing it.
125I-labeled gonadoliberin and high specific activity and immunoreactivity: method of iodination and rapid separation.
Authors: Sarda A K , Barnes M A , Nair R M
Published: January 2020

We describe optimum conditions for iodinating gonadoliberin with use of relatively large proportions of Na 125I. Products of the iodination are separated on an anion-exchange resin (Amberlite IRA-400). The 125I-labeled gonadoliberin thus obtained has a high specific activity (1400 to 1590 Ci/g); because of the conditions of iodination, we believe that the predominant species of the labeled decapeptide is the mono-iodinated one. Our separation and purification of the labeled substance on ion-exchange resin is rapid, economical, and less cumbersome than the use of a Biogel P-2 column. There is no adsorption of the labeled hormone onto the resin, as evidenced by analytical recovery studies with tritium-labeled gonadoliberin. Paper-strip chromatoelectrophoresis showed no free Na 125I or radiolabeled damaged peptide fragments after purification on the resin. When antiserum was used at a concentration 32-fold that used in the regular assay procedure, only 4% of the radioactivity remained in the free form, indicating the high immunoreactivity of the labeled hormone.
Effects of sample aging on total cholesterol values determined by the automated ferric chloride-sulfuric acid and Liebermann-Burchard procedures.
Authors: Wood P D , Bachorik P S , Albers J J , Stewart C C , Winn C , Lippel K
Published: January 2020

To investigate the comparability of three commonly used methods for determination of total cholesterol in plasma in several studies, we used fresh plasma samples as well as plasmas and reference sera that had been stored frozen at −15 degrees C for as long as several years. Duplicate determinations by the manual method of Abell et al. (J. Biol. Chem. 195: 357, 1952) were compared with estimates from one to five continuous-flow analyzers by the ferric chloride-sulfuric acid procedure and also with estimates from five to 13 continuous-flow analyzers by the Liebermann-Burchard procedure with calibrator, as part of the laboratory standardization activities of the Lipid Research Clinics. The agreement among all three procedures was generally within acceptable limits (within 5% of the manual method) when plasmas or sera were fresh or had been frozen for less than one month. Results by the manual method of Abell et al. agreed well with those by the automated Liebermann-Burchard method for samples that had been stored at −15 degrees C for as long as two years. However, the automated ferric chloride-sulfuric acid procedure often showed unacceptably high values (as compared with those from the manual method) for samples that had been stored frozen for a year or more. With the ferric chloride-sulfuric acid method, measured cholesterol concentration increased about 2.5% per year of storage for at least two years. We conclude that reference sera of plasmas that have been kept in long-term frozen storage (−15 degrees C) are not suitable for ongoing standardization of the automated ferric chloride-sulfuric acid assay for cholesterol.
Purification of an alkaline phosphatase-lipoprotein-X complex.
Authors: Weijers R N , Kruijswijk H , Baak J M
Published: January 2020

An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.
Improved separation of creatine kinase isoenzymes by use of DEAE-Sepharose Cl-6B.
Authors: Bondar R J , Shevchik D G , Hsu M Y , Kohler M M
Published: January 2020

We describe here a simple, rapid chromatographic procedure for quantitatively separating serum creatine kinase isoenzymes (EC 2.7.3.2), with diethylaminoethyl (DEAE)-Sepharose CL-6B as the anion-exchanger. We established the column bed height and the elution parameters by use of a simplex procedure. DEAE-Sepharose CL-6B equilibrated in tris(hydroxymethyl)aminomethane (50 mmol/L, pH 7.5, and containing 30 mmol of NaCl per liter) is packed into a miniature polystyrene column with bed dimensions of 0.7 x 1.5 cm. The DEAE-Sepharose column system was evaluated and compared with a DEAE-Sephadex A-50 column system. The results indicate that the Sepharose column yields MM, MB, and BB isoenzymes uniquely, without cross contamination. Coefficients of variation (CV’s) for 10 replicate column assays were 2.8, 5.9, and 15.2% for 569 U of MM per liter, 82.3 U of MB per liter, and 9.0 U of BB per liter, respectively. The serum sample was enriched with baboon heart extract. Day-to-day reproducibility for a serum control assayed on 10 days yielded CV’s of 4.6, 9.9, and 40.3% for 391, 45.3 and 1.9 U of isoenzymes MM, MB, and BB per liter, respectively. A reference interval for each isoenzymes was derived from data on 81 men and 63 women.
Microencapsulated antibodies in radioimmunoassay. III. Determination of cortisol.
Authors: Bordens R W , Halpern E P
Published: January 2020

We describe a method for directly measuring total plasma cortisol by use of a microencapsulated antibody. The antibody microcapsules were incubated with plasma and 125I-labeled cortisol in a competitive reaction at 37 degrees C for 15 min, then separated by centrifugation; the radioactivity of the pellet was counted for 1 min. Analytical recovery of cortisol from three plasma pools was 97.6% (SD 8.5%) and was unaffected by triglyceridemia, hemolysis, or icterus. The standard curve was linear to 500 microgram/L and the sensitivity was 10 microgram/l. Recovery of cortisol added as the hemisuccinate was 102% (SD 10.6%), whereas that of the conjugate cortisol hemisuccinate/bovine serum albumin was 4.3% (SD 3.5%). This confirmed that the microcapsule excludes large molecules. The within-day CV for two control pools was 9.8 and 12.8%, the day-to-day variation 13.4 and 13.8%.
Lot-to-lot variation in three thyroid-related radioimmunoassay kits.
Authors: Zweig M H , Robertson E A
Published: January 2020

We examined lot-to-lot variation in triiodothyronine, thyroxine, and thyrotropin radioimmunoassay kits over 14–20 months. For the thyroxine assay, less than 20% of the overall variation was attributable to lot-to-lot changes. In contrast, for the triiodothyronine and thyrotropin assays half to two-thirds of the overall variation was attributable to lot-to-lot differences. The shifts seen in control results were also observed in results for patients. These shifts produced disturbing deviations on quality-control charts but did not appear to compromise the clinical utility of the results. We conclude that lot-to-lot variation can account for a major part of the long term variability of radioimmunoassay kit results and should be considered in evaluating kits and monitoring their performance.
Evaluation of standard curves in the graded hemagglutination-inhibition method applied to quantitation of human immunoglobulins.
Authors: Wolk M
Published: January 2020

For accurate quantitation of human immunoglobulins A and B by graded hemagglutination inhibition, I have established standard curves. For IgA, plotting the inner diameter of the ring formed by sedimented erythrocytes vs the log of the antigen concentrations gives a straight line. Plotting the same parameters for IgG gives a hyperbolic line. These ratios between antigen concentration and ring diameter were constant for at least one year. Both undiluted and 10,000-fold diluted human serum may be quantitated by this method, depending only on immunoglobulin concentrations. Precision (CV) of the system, assessed in 35 patients’ sera over the period of one year, ranged from 0.5 to 15.2% (mean 10%) for IgA and from 1.4 to 14.9% (mean 7.3%) for IgG.
Bilirubin binding capacity of albumin isolated from cord-blood serum is less than that from serum of adults.
Authors: Alayoff A , Kapitulnik J , Konijn A , Kaufmann N A , Blondheim S H
Published: January 2020

As estimated by Sephadex gel filtration, the bilirubin binding capacity of albumin isolated from cord-blood serum by ion-exchange chromatography is less than that of albumin isolated from serum of adults. Albumin isolated from cord-blood serum showed increased bilirubin binding as compared with the albumin in the native serum. These findings suggest that the lower bilirubin binding capacity of serum from newborns as compared with serum from adults is a result of both an intrinsic deficiency in binding capacity of neonatal albumin and the presence of substances in neonatal serum that interfere with bilirubin binding.
Problems in radioimmunoassay of human lutropin with commercially available reagents.
Authors: Hammond J E , Phillips J C , Straight C B , Hammond M G
Published: January 2020

To evaluate five commercially available reagent sets supplied for the radioimmunoassay of lutropin, we determined whether there was parallelism between the curve given by dilutions of the standards supplied by the manufacturers, by dilutions of a serum pool, and by dilutions of a standard preparation from human pituitaries, LER-907. These studies demonstrated significant analytical problems with three of the five sets. We conclude that each user should carefully evaluate all commercially available radioimmunoassays for lutropin (and, by inference, for other peptide hormones) before use.
Decreasing sample volume used in the “Sequential Multichannel Analyzer Computerized” (SMAC).
Authors: Stamper R , Robertshaw D M
Published: January 2020

We describe modifications to two cartridges, those for alkaline phosphatase and creatinine, on an 18-channel SMAC (Sequential Multichannel Analyzer Computerized), which allow all 18 tests to be run with a commonly prediluted sample. This decreases the required sample size and eliminates the problem of fibrin clots in the stream-splitter on the main sample line. The modifications can be made rapidly and require few new components. Accuracy, precision, linearity, and carryover are acceptable after these modifications, as judged by comparison with premodification data. Some financial savings ensue from the decreased consumption of reagents and the fewer repeat analyses necessitated by clotting problems.
Total serum cholesterol by isotope dilution/mass spectrometry: a candidate definitive method.
Authors: Cohen A , Hertz H S , Mandel J , Paule R C , Schaffer R , Sniegoski L T , Sun T , Welch M J , White E
Published: January 2020

We describe a highly accurate and precise method for determination of total cholesterol in serum by isotope dilution/mass spectrometry. The method was developed for a Study Group of the Committee on Standards of the American Association for Clinical Chemistry, for use in establishing the accuracy of a candidate reference method for total cholesterol, and fulfills their criteria for a definitive method. Cholesterol-d7 is added to serum, with the weight ratio of cholesterol-d7 to total serum cholesterol kept near to 1:1. The esters are hydrolyzed and the cholesterol is separated and converted into the trimethylsilyl ether derivative for measurement by combined gas chromatography/mass spectrometry. The intensity ratio of the molecular ions at m/z 465 and 458 is measured for each sample and for two calibration mixtures, according to a prescribed bracketing protocol. A weight ratio for the sample is obtained by linear interpolation of the ion-intensity ratios, and the total cholesterol is then calculated. The method was applied four times over several weeks to each of five serum pools. Statistical analysis involving consideration of both replication error and variability between weeks gave a coefficient of variation for a single measurement of 0.36%. The absence of interferences in the method was demonstrated by measurements at several other masses.
Liquid-chromatographic separation of urinary 5-hydroxy-3-indoleacetic acid, with measurement at 254 nm.
Authors: Draganac P S , Steindel S J , Trawick W G
Published: January 2020

A "high-performance" liquid-chromatographic procedure for 5-hydroxy-3-indoleacetic acid is described and compared with a colorimetric method in which 1-nitroso-2-naphthol is used. The analyte and an internal standard, p-nitrobenzoic acid, were extracted into diethyl ether from urine at pH 4.0 (acidified with HCl) to which sodium chloride had been added, and the ether was back-extracted with acetate buffer, pH 9.2. Aliquots of this extract were injected into a reversed-phase liquid-chromatographic column and eluted with pH 3.5 acetate buffer/methanol (95/5 by vol); the effluent was monitored at 254 nm. The precision (CV) of the method was 11.8% at 1.8 mg/L, 5.5% at 92 mg/L. Analytical recovery averaged 84%. The colorimetric method gave higher values for the analyte than did the chromatographic method for all patients' urines.
Use of soluble and Staphylococcus aureus-immobilized second antibody compared in a radioimmunoassay for human alpha-fetoprotein.
Authors: Ruch F E , Knight G J
Published: January 2020

Staphylococcus aureus cells coated with rabbit anti-goat immunoglobulin G were compared with soluble second antibody in a radioimmunoassay for human serum alphafetoprotein. Recovery of primary immune complexes with use of the bacterial immunoadsorbant (a) allowed second-antibody incubation time to be shortened from 3 h to 20 min at 37 degrees C, (b) decreased by 80% the amount of second antibody required per assay, thereby significantly reducing assay costs, and (c) provided a convenient and reproducible method for separation of free and bound antigen. The ease of preparation and the uniformity and stability of the S. aureus second-antibody reagent are additional advantages that may be useful in other radioimmunoassay procedures.