bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of serum desipramine and 2-hydroxydesipramine for pharmacokinetic applications by HPLC with ultraviolet detection.
Authors: Kenney J T , Orsulak P J , Kolodner R M , Burton M E
Published: January 2020

This procedure for measuring desipramine and its 2-hydroxy metabolite in serum at a minimum concentration of 1 micrograms/L involves high-performance liquid chromatography (HPLC), with ultraviolet detection at 214 nm. After desipramine and 2-hydroxydesipramine are extracted from alkalinized serum by a single-step solvent extraction, they are separated by HPLC and quantified with amitriptyline as the internal standard. Desipramine, 2-hydroxydesipramine, and amitriptyline are separated in 6 min. The standard curve is linear (r = 1.000) for both desipramine and 2-hydroxydesipramine concentrations over the range of 1 to 100 micrograms/L, and the assay demonstrates an excellent precision profile, even at low concentrations. Between-run CVs for 20 and 60 micrograms/L controls (n = 20) were 3.9% and 3.6% for desipramine and 3.4% and 3.8% for 2-hydroxydesipramine, respectively. In a pharmacokinetic evaluation of patients with depression, we examined single-dose elimination curves before and after a four-week regimen of desipramine treatment. The results showed that the method's simplicity and high precision render it ideal for pharmacokinetic studies of desipramine.
Biochemical contribution to diagnosis and study of a new case of D-glyceric acidemia/aciduria.
Authors: Fontaine M , Porchet N , Largilliere C , Marrakchi S , Lhermitte M , Aubert J P , Degand P
Published: January 2020

During organic acid screening by gas chromatography/mass spectrometry, we detected a large peak corresponding to glyceric acid in a patient's urine sample. The D(+) configuration was demonstrated by a polarimetric method and by enzymatic stereospecificity of D-glycerate dehydrogenase (EC 1.1.1.29). We biochemically investigated this fifth reported case of D-glyceric acidemia. In our patient, loading tests with L-serine and fructose led to an increase of D-glyceric acid in both plasma and urine. Determination of other metabolites involved in D-glycerate metabolism revealed no abnormality in any sample examined. After comparing all our results with those of the preceding observations described in the literature, we suggest a possible enzymatic defect located on one of the metabolic pathways shared by fructose and L-serine, possibly at the level of hepatic D-glycerate kinase (EC 2.7.1.31). Nevertheless, a primary defect of L-serine catabolism cannot be entirely excluded.
Plasma glutathione S-transferase and F protein are more sensitive than alanine aminotransferase as markers of paracetamol (acetaminophen)-induced liver damage.
Authors: Beckett G J , Foster G R , Hussey A J , Oliveira D B , Donovan J W , Prescott L F , Proudfoot A T
Published: January 2020

Concentrations of glutathione S-transferase (GST; glutathione transferase; EC 2.5.1.18) B1 subunits, F protein, and the activity of alanine aminotransferase (ALT; EC 2.6.1.2) were measured in sequential plasma samples taken from nine patients with self-administered paracetamol (acetaminophen) poisoning. GST exceeded the reference interval in all patients at the time of admission, and F protein was increased in seven. In contrast, abnormal activities of ALT in plasma were found in only one of the nine on admission, a patient admitted 12 h after poisoning. Subsequent to admission nine, eight, and five patients, respectively, had abnormal concentrations of GST, F protein, and ALT. When expressed as multiples of the upper reference limit, the highest values for GST measured in each patient always far exceeded the greatest abnormalities in ALT; this was true for F protein in only five patients. Patients in whom the concentration of GST exceeded 10 micrograms/L on admission subsequently went on to develop moderate or severe liver damage, despite treatment with N-acetylcysteine. F protein and ALT measurements on admission were not as efficient as GST at predicting the clinical outcome of the patients. We conclude that GST and F protein offer clear advantages over ALT for detecting minor degrees of acute liver dysfunction, particularly when only centrilobular damage may be involved.
Effect of ingestion of hemoproteins on fecal excretion of hemes and porphyrins.
Authors: Rose I S , Young G P , St John D J , Deacon M C , Blake D , Henderson R W
Published: January 2020

Stools from asymptomatic volunteers on diets containing red meat, whole blood, or high fiber were analyzed for their content of hemes and dicarboxylic (heme-derived) porphyrins by the "HemoQuant" assay, the "Hemoccult" test, and "high-performance" liquid chromatography (HPLC). In 49 subjects, ingestion of red meat increased HemoQuant-determined combined fecal heme plus dicarboxylic porphyrins by an average 375%; the contribution of heme-derived porphyrins to total fecal porphyrins increased from 37% to 78%. Of subjects on a red-meat diet, 27% passed stools with a porphyrin content suggestive of a porphyria, compared with only 4% on a red-meat-free diet. These increases were due largely to protoporphyrin and its derivatives pemptoporphyrin and deuteroporphyrin, all of which were present in feces as iron-free porphyrins and iron-ligated (heme) forms. Ingestion of blood had an effect similar to that of red meat, but ingestion of fiber had no effect. These effects of dietary and endogenous hemoproteins must be considered when such methods are used to test feces for occult blood or to test for excess fecal porphyrins as an indicator of a porphyria.
Computerized quantitative analysis of coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis.
Authors: Burgess-Cassler A , Johansen J J , Santek D A , Ide J R , Kendrick N C
Published: January 2020

Two-dimensional electrophoresis in combination with Coomassie Blue staining was refined for use as a quantitative method. Microcomputer software was developed for use with the IBM AT and compatible computers for analyzing the gels. To test the refined method to determine its usefulness in simultaneous measurements of 28 human serum proteins, we measured each protein relative to a single standard (bovine serum albumin) polymerized at different concentrations in a calibration scale, rather than using 28 individual standards. All samples were analyzed in triplicate. We evaluated calibration, linearity of response, recoveries, units, within-run CV, and between-run CV. The five isoforms of apolipoprotein A-I were analyzed in samples from 16 healthy donors and the isoform ratios determined. The method as presented here should prove useful for diagnosis of non-urgent disease states and for analysis for protein isoforms in relation to disease; it should also be applicable to assays of proteins in other fluids and tissues.
Relative importance of estrogen and progesterone receptor assays as prognostic indicators in primary breast cancer: a short-term study.
Authors: Brocklehurst D , Wilde C E , Finbow J A , Brett R , Champion A E , Dewhurst D G
Published: January 2020

This short-term study of the relative importance of estrogen and progesterone receptors shows that progesterone receptor correlates better than estrogen receptor with tumor recurrence regardless of lymph-node status. Life-table analysis has effectively identified only two groups of patients that may be classified by progesterone receptor status alone. Progesterone-receptor negativity correlated well with tumors of histological Grade III; estrogen-receptor positivity correlated with Grade I and II tumors. The earlier recurrence of Grade III breast tumors may explain why progesterone receptor is a better prognostic indicator than estrogen receptor in short-term studies.
Solid-phase sample extraction for rapid determination of methylmalonic acid in serum and urine by a stable-isotope-dilution method.
Authors: Rasmussen K
Published: January 2020

Methylmalonic acid concentrations are increased in serum in vitamin B12 (cobalamin) deficiency. Here I demonstrate the successful use of anion-exchange extraction for improving newly developed analytical procedures and describe well-documented, reliable performance of this method for rapid determination of methylmalonic acid. The sorbent counter ion is formate, and the elution solvent is formic acid. The dicyclohexyl derivative is measured by selected ion monitoring. For serum, the assay curve is linear from 0.026 to 200 mumol/L. The normal reference interval is 0.08 to 0.56 mumol/L. Added methylmalonic acid is accurately quantified. The sensitivity and the precision exceed those of the current method by three orders of magnitude. The total and within-day CVs are 4.6% to 7.9% and 2.6% to 4.7%, respectively. Similar figures were obtained for urine. This convenient method is useful for evaluation of cobalamin deficiency, especially in patients with normal or moderately depressed cobalamin concentrations in serum.
How sensitive are immunometric assays for thyrotropin?
Authors: McConway M G , Chapman R S , Beastall G H , Brown E , Tillman J , Bonar J A , Hutchison A , Allison T , Finlayson J , Weston R
Published: January 2020

The usual method for calculation of the "sensitivity" of thyrotropin immunometric assays is multireplicate analysis of the zero analyte standard. Although this is a statistically valid estimate of the scatter likely to be found in the response variable, it is unrelated to normal analytical practice (usually analysis in duplicate) and estimates intra-assay errors only. This study was designed to assess the analytical performance of 10 immunometric assays used routinely for measurement of thyrotropin in human serum. Response data from each assay were accumulated to provide (a) an estimate of "sensitivity" from multireplicate analysis and (b) an estimate of "minimum detection limit," relating directly to errors associated with routine performance and derived from a minimum of 500 duplicate analyses. We conclude that the "minimum detection limit" should be promoted as a more meaningful measure of assay performance at low analyte concentrations than the "sensitivity" derived from multireplicate analysis of the zero-analyte standard.
Automated enhanced latex agglutination assay for rheumatoid factors in serum.
Authors: Winkles J W , Lunec J , Gray L
Published: January 2020

This improved assay of rheumatoid factors in serum, described here for use with the Baker "Encore" centrifugal analyzer, is efficient, with 250-sample throughput per hour; reproducible, with between-batch CV = 5% and within-batch CV = 2% (mid-assay range); and results correlate well (r = 0.9) with those by other methods. The method is fully quantitative and automated, involves no predilution steps, and can be adapted for use in a wide range of systems. It has a sensitivity of 96% and specificity of 80% in diagnosing rheumatoid arthritis.
Concentrations of carotenoids, retinol, and tocopherol in plasma, in response to ingestion of a meal.
Authors: Brown E D , Rose A , Craft N , Seidel K E , Smith J C
Published: January 2020

Field studies and epidemiological surveys may necessitate obtaining a blood sample from a nonfasted subject for nutritional assessments. We measured the effect of a standardized test meal, eaten after an overnight fast, on the concentrations of seven carotenoid fractions, retinol, and tocopherol in plasma of eight healthy adults. The 790-calorie test meal did not alter the measured concentrations. We conclude that blood sampled up to 4 h after breakfast can be validly used for these measurements.
Differences between capillary and venous blood-alcohol concentrations as a function of time after drinking, with emphasis on sampling variations in left vs right arm.
Authors: Jones A W , Jönsson K A , Jorfeldt L
Published: January 2020

Twelve healthy men drank 0.8 g of ethanol per kilogram of body weight during 30 min after an overnight (10 h) fast. At nine exactly timed intervals (30-390 min after the start of drinking), blood was sampled through indwelling catheters in cubital veins on the left and right arms. Immediately thereafter, capillary blood was sampled from fingertips on the left and right hands. The blood ethanol concentration (BAC) was determined by headspace gas chromatography. The SD for alcohol determinations in venous blood, including the left vs right arm sampling variation, was 30 mg/L (range 8.3-83 mg/L), whereas for capillary blood the SD was 35 mg/L (range 11-60 mg/L). This difference much exceeded the purely analytical errors: SD = 2.67 mg/L for venous blood and 14.2 mg/L for fingertip blood. During the first 60 min after the subjects started to drink, capillary BAC exceeded venous BAC, the mean difference at 30 min being 136 mg/L (range 36-216 mg/L). In the postabsorptive state later than 60 min after drinking, venous BAC exceeded capillary BAC [mean difference 58 mg/L (range 0.0-170 mg/L]), the values for venous and capillary BAC crossing 37 min (range 6-77 min) after the end of drinking. Apparently, the source of blood analyzed, venous or capillary, must be considered in clinical pharmacokinetic studies of ethanol.
Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies.
Authors: Piechaczyk M , Baldet L , Pau B , Bastide J M
Published: January 2020

A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs.
Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants.
Authors: Paleari R , Arcelloni C , Paroni R , Fermo I , Mosca A
Published: January 2020

We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb Köln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.
Atomic absorption spectrometric determination of selenium in human blood components.
Authors: Hansson L , Pettersson J , Eriksson L , Olin A
Published: January 2020

We separated blood from five healthy blood donors into plasma, erythrocytes, platelets, and leukocytes; counted the number of cells in each fraction; and determined the selenium content of each component by hydride generation atomic absorption spectrometry. The mean (+/- SD) selenium concentrations and amounts measured were as follows: whole blood 102.3 +/- 16.1 micrograms/L, plasma 76.9 +/- 10.6 micrograms/L, erythrocytes 13.7 +/- 2.8 ag per cell, platelets 4.8 +/- 1.1 ag per cell, and leukocytes 99 +/- 26 ag per cell.
Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay.
Authors: Barnard G , Kohen F , Mikola H , Lövgren T
Published: January 2020

We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.
Biological variation of urinary N-acetyl-beta-D-glucosaminidase: practical and clinical implications.
Authors: Stolarek I , Howey J E , Fraser C G
Published: January 2020

The analytical, within-subject, and between-subject components of variation of N-acetyl-beta-D-glucosaminidase (NAG) were estimated from duplicate assays of 10 timed first morning and 10 untimed urine specimens collected from each of 15 ostensibly healthy individuals. Results were expressed in terms of activity, NAG/creatinine ratio, and excretion rate. Current analytical methods can achieve desirable performance standards. NAG has little individuality, and conventional population-based reference intervals are therefore useful. In view of the practicability and relatively low within-subject variation, for routine clinical purposes we prefer assay of NAG in first morning urine, expressing the results in terms of activity. Using the results of assays of different specimens, we found that the correlation between urinary albumin and NAG varied considerably, owing to the large intrinsic variability of both analytes; this might explain previous conflicting results.
A new immunoradiometric assay for corticotropin evaluated in normal subjects and patients with Cushing's syndrome.
Authors: Raff H , Findling J W
Published: January 2020

We evaluated a new, commercially available two-site immunoradiometric assay (IRMA) for corticotropin (ACTH) in human plasma. The precision and detection limit were an improvement over radioimmunoassay (RIA). Addition of ACTH 1-24 or ACTH 18-39 to plasma containing ACTH 1-39 resulted in a decrease in measured ACTH. Results by both IRMA and RIA are reported and compared for normal subjects, patients with Cushing's disease, and patients with ectopic ACTH. Effects of administering dexamethasone, metyrapone, ACTH 1-24, and corticoliberin were evaluated. The present assay is more sensitive, specific, and reproducible than RIA. The presence of fragments of ACTH or "big" ACTH that are reactive in RIA may lead to artifactually low ACTH concentrations by IRMA. Therefore, RIA may still be necessary for differential diagnosis of ectopic ACTH syndromes. However, the IRMA is an improvement for evaluating pituitary--adrenal function in patients with low concentrations of ACTH in plasma.
Rapid determination of toxic cyanide concentrations in blood.
Authors: Lundquist P , Sörbo B
Published: January 2020

This method for the rapid colorimetry of cyanide in blood, applicable to patients exposed to toxic levels of cyanide, is based on the König reaction, which produces a chromophore from cyanide as well as thiocyanate. The latter compound, normally present in blood, is confined to plasma. Thus, its interference with determination of cyanide was eliminated by performing the assay on the erythrocytes, which contain most of the blood cyanide. Furthermore, cyanide was trapped in the erythrocytes and stabilized during the initial washing steps by conversion of hemoglobin to methemoglobin with inorganic nitrite.
Three methods compared for estimating the fraction of testosterone and estradiol not bound to sex-hormone-binding globulin.
Authors: Pearce S , Dowsett M , Jeffcoate S L
Published: January 2020

Here we propose and validate, both theoretically and experimentally, a new technique for measuring the percentage of non-sex-hormone-binding globulin (SHBG) bound to testosterone and estradiol. In this method, SHBG is saturated with 1 mumol of 5 alpha-dihydrotestosterone per liter and the non-protein-bound fraction is subsequently measured in treated and untreated samples by centrifugal ultrafiltration dialysis (CUFD). We compared this technique with two previously published methods for measuring the percentage of albumin-bound steroid--ammonium sulfate precipitation and heat denaturation of SHBG followed by CUFD--and also with previously published computer predictions. The ammonium sulfate precipitation gave results closest to the computer-predicted percentage of albumin-bound testosterone and estradiol. Of the two techniques involving CUFD, the 5 alpha-dihydrotestosterone saturation method was the more reproducible. Results by both CUFD methods were higher than the computer-based predictions for albumin-bound steroid in pregnancy serum, in part perhaps because the CUFD measurements represent the non-SHBG-bound fraction rather than that which is albumin-bound alone.
Mechanism of false-negative urine cannabinoid immunoassay screens by Visine eyedrops.
Authors: Pearson S D , Ash K O , Urry F M
Published: January 2020

To drug-free urine specimens, we added the following drugs of abuse to give concentrations twice the cutoff value for positive test results: 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (9-carboxy-THC), oxazepam, secobarbital, morphine, benzoylecgonine, amphetamine, or phencyclidine (PCP). Visine was then added. Although measured concentrations of several drugs were decreased in the presence of Visine, false-negative results were obtained only for 9-carboxy-THC for the EMIT-d.a.u. and TDx urine cannabinoid assays. Visine also decreased 9-carboxy-THC as measured by the Abuscreen assay. At low concentrations of Visine, false-negative cannabinoid results were attributable to the benzalkonium chloride ingredient of Visine. The added Visine was not detectable by routine urine analysis and had no effect on the activity of the glucose-6-phosphate dehydrogenase-drug conjugate used in the EMIT-d.a.u. assays. Moreover, analysis by gas chromatography/mass spectrometry showed no chemical modification or loss of 9-carboxy-THC in the Visine-adulterated urine specimens. However, Visine did increase the adhesion of 9-carboxy-THC to the borosilicate glass specimen containers. Results of ultrafiltration studies with Visine suggest that 9-carboxy-THC partitions between the aqueous solvent and the hydrophobic interior of benzalkonium chloride micelles, thereby reducing the availability of 9-carboxy-THC in antibody-based assays.
Enzymic urea assay: a new colorimetric method based on hydrogen peroxide measurement.
Authors: Lespinas F , Dupuy G , Revol F , Aubry C
Published: January 2020

We describe a new enzymic colorimetric method in which urea is measured in serum by use of a single reagent mixture. Ammonia produced by urea hydrolysis, catalyzed by urease, reacts with glutamate and ATP in the presence of glutamine synthetase. The ADP so produced is assayed in reactions catalyzed sequentially by pyruvate kinase and pyruvate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 500 or 550 nm in a reaction catalyzed by horseradish peroxidase, with phenol/4-aminophenazone as the chromogen. The reaction is complete in 15 min at 37 degrees C. The standard curve is linear up to a urea concentration of 40 mmol/L. Precision is good; CVs ranged from 2.5% to 3.1%. Results by the present method compared well with those by a candidate Reference Method and are not subject to interferences from commonly used drugs and anticoagulants.
Separation and identification of urinary proteins and stone-matrix proteins by mini-slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Authors: Fraij B M
Published: January 2020

Mini-slab electrophoresis on sodium dodecyl sulfate/polyacrylamide gel can be used to detect protein in unconcentrated urines containing more than 100 mg of protein per liter. Several urinary proteins--including albumin, transferrin, and the Tamm-Horsfall mucoprotein--precipitate, partly or totally, if urine samples are stored at 4 degrees C for one or two days. The present procedure permits identification of the albumin and transferrin in unconcentrated urines, thus avoiding their partial loss. Stone-matrix proteins in 10 mg or more of urinary calculi also could be detected. Albumin, transferrin, and Tamm-Horsfall mucoprotein accounted for greater than 40% of the total matrix protein in most of the urinary stones analyzed. The precipitation properties of these proteins and their high concentrations in stone matrix relative to those in urine suggest that they may play a role in stone formation and growth.
Immunocatalytic assay of pancreatic alpha-amylase in serum and urine with a specific monoclonal antibody.
Authors: Svens E , Käpyaho K , Tanner P , Weber T H
Published: January 2020

In this immunocatalytic assay for alpha-amylase (EC 3.2.1.1) of pancreatic origin, a highly specific monoclonal antibody coupled to plastic beads is used to extract pancreatic amylase from samples, leaving salivary amylase in solution. The catalytic activity of the bound pancreatic amylase is then determined with blocked p-nitrophenyl maltoheptaoside as substrate. The method shows no cross-reactivity with salivary amylase, analytical recovery is 89-109% for pancreatic amylase, and interassay imprecision is 7.1-7.7%. We used the method to determine pancreatic amylase in serum and urine from healthy controls and different patient groups. The reference intervals for 34 supposedly healthy controls were: serum, 10-48 U/L (mean 27 U/L); urine, less than 20-435 U/L (mean 104 U/L). Results by the present assay correlated well with a salivary amylase inhibition assay (Boehringer Mannheim). We conclude that the described immunocatalytic assay is clinically useful for detecting increased activities of pancreatic amylase in serum and urine.
Biological variability of 26 clinical chemistry analytes in elderly people.
Authors: Fraser C G , Cummings S T , Wilkinson S P , Neville R G , Knox J D , Ho O , MacWalter R S
Published: January 2020

Analytical, within-subject, and between-subject components of variation were estimated for 26 clinical chemistry analytes from duplicate analyses of 10 specimens collected from 27 healthy elderly subjects over a period of 20 weeks. Within-subject variations were similar to those generated previously by us in younger subjects. We conclude, therefore, that homeostasis is not compromised by age alone, and biological variability does not increase simply with age. All analytes except serum water had marked individuality, showing that conventional population-based reference values are of limited utility. The critical differences required for two results to be significantly (P less than or equal to 0.05) changed are not the same as those that prompt action by clinicians. Although heterogeneity of within-subject variation does exist, we believe that the critical differences generated will be useful in routine clinical decision making.
Immunoradiometric assay of corticotropin with use of avidin-biotin separation.
Authors: Zahradnik R , Brennan G , Hutchison J S , Odell W D
Published: January 2020

We have developed a "sandwich"-type immunoradiometric assay for corticotropin (ACTH), with a detection limit of 2 ng/L. Two antibodies are used: a mouse monoclonal antibody directed against ACTH[1-17] and labeled with 125I; and a purified polyclonal goat antibody directed against ACTH[34-39] and conjugated to biotin. We could separate 125I-labeled antibody bound to ACTH from 125I-labeled antibody not bound to ACTH by using an avidin-biotin bridge, with avidin bound to a polystyrene ball. This assay reacts with ACTH[1-39] but shows no reaction with ACTH fragments [1-24], [1-17], or [34-39], or with melanotropin, endorphins, or lipotropin. This assay is sensitive enough to detect ACTH in plasma of all normal adults. Concentrations measured in 94 adults between 0800 and 1000 hours were normally distributed on a log scale, with a mean of 19.5 ng/L and a 95% range of 7.1 to 53.8 ng/L. Dexamethasone given at 2300 hours to 14 adults suppressed ACTH to less than 4 ng/L in 13 of the subjects and to 8 ng/L in the 14th. Metyrapone given to 13 adults at 2300 hours increased ACTH to 245.3 ng/L (95% range, 90.1 to 667.7 ng/L). This assay accurately classified patients with disorders of the adrenal system.
Determination of the alkyl lysophospholipid derivative ET-18-OCH3, a new antineoplastic drug, in plasma.
Authors: Schaefer H G , Rohdewald P
Published: January 2020

We describe a sensitive method for measuring the concentration of the new antineoplastic drug ET-18-OCH3 in plasma. After plasma lipids are extracted, ET-18-OCH3 is separated from the excess of endogenous lipids by thin-layer chromatography and specific enzymatic hydrolysis of sphingomyelin by the action of sphingomyelinase. Analytical recovery after the complete isolation was 73.5% (CV = 9.8%, n = 15). [3H]-ET-18-OCH3 is used as internal standard. A densitometric method in which 8-anilino-1-naphthalenesulfonate, Mg salt, is used as fluorescent agent (excitation at 367 nm and emission greater than 390 nm) allows the sensitive determination of ET-18-OCH3 down to 0.1 mg/L (CV greater than 30%). The day-to-day CV is 25% for concentrations of 0.15 to 0.625 mg/L, 12% for 1.5 to 5.0 mg/L. Preliminary pharmacokinetic data reveal gastrointestinal absorption of ET-18-OCH3 after multiple oral administration.
Unreliable visual estimation of the incidence and amount of turbidity, hemolysis, and icterus in serum from hospitalized patients.
Authors: Glick M R , Ryder K W , Glick S J , Woods J R
Published: January 2020

We examined the frequency of occurrence for turbidity, hemolysis, or icterus in 2599 serum samples submitted for chemistry testing in an acute-care general hospital. Each specimen was compared visually with full-color photographs of adulterated serum, and designated as either "0" (containing no interferent), or trace, 1+, 2+, 3+, 4+, or 5+. Visible interferents (1+ or greater) were thought to be present in 838 (31%) of the specimens (icterus, 525; hemolysis, 244; lipemia, 69). To assess the accuracy of such visual grading, we determined the concentration of triglycerides, hemoglobin, or bilirubin in the specimens considered to be contaminated. There was little agreement between the actual concentration of each interferent and the assigned grade of turbidity, hemolysis, or icterus, confirming the unreliability of human visual estimation of these potentially interfering substances.
Correction of instrument- and reagent-based differences in determination of the International Normalized Ratio (INR) for monitoring anticoagulant therapy.
Authors: van Rijn J L , Schmidt N A , Rutten W P
Published: January 2020

As recommended by the World Health Organization, standardization of prothrombin time assays involves conversion of prothrombin times into International Normalized Ratios (INR). We investigated the effect of two different methods (Nycomed's Thrombotest, and Instrumentation Laboratory's PT-fibrinogen) and three coagulation instruments (Schnitger & Gross, KC-10, and ACL) on calculations of INR. The INR plots showed considerable scatter of individual values around the regression lines when the two different methods were compared. Systematic differences in the outcome of INR calculation were related to the use of the different coagulation instruments. Prothrombin times obtained with the different instruments were linearly correlated. We used the bias of these lines to correct results for both the patients' samples and the reference samples. This correction yielded INR values from the different instruments that agreed well.
Fluorescence polarization immunoassay of gentamicin or netilmicin in blood spotted on filter paper.
Authors: Fujimoto T , Tsuda Y , Tawa R , Hirose S
Published: January 2020

In this improved simple method for determination of aminoglycoside antibiotics in dried-blood spots on filter paper, gentamicin or netilmicin is recovered from the blood spot most effectively by incubation for 60 min in an ultrafiltration tube containing 500 microL of 0.5 mol/L Na2HPO4 buffer. The eluates from the paper are centrifuged, then transferred to an Abbott TDx cartridge for measurement of gentamicin or netilmicin by fluorescence polarization immunoassay. The dried sample on paper is stable for about eight days at ambient temperature. Intra-assay CVs for gentamicin and netilmicin are less than 8.5% and less than 6.1%, respectively. Analytical recovery of gentamicin and netilmicin from the paper exceeded 90%. This method permits simple blood collection and monitoring of the therapeutic concentration of gentamicin or netilmicin in serum, particularly that of newborn infants and small children.
Determination of trans-phylloquinone in children's serum.
Authors: Moussa F , Dufour L , Didry J R , Aymard P
Published: January 2020

By optimizing the conditions for determining trans-phylloquinone and its metabolite, K-2,3-epoxide, in serum through a two-step HPLC process combined with fluorometric detection after coulometric reduction, we have been able to develop a method applicable to small volumes of serum (200 to 500 microL). The limit of detection (signal-to-noise ratio of 3) was 15 ng/L for trans-phylloquinone, 30 ng/L for K-2,3-epoxide. The trans-phylloquinone concentrations measured by this method in serum from 82 children, ages one to six years, whose results were normal for overall coagulation tests, ranged from 40 to 880 ng/L (median 175 ng/L). We discuss these findings and compare them with vitamin K1(20) values reported for adults.
Indirect measurement of desferrioxamine and its chelated compounds aluminoxamine and ferrioxamine by Zeeman atomic absorption spectrometry.
Authors: D'Haese P C , Lamberts L V , De Broe M E
Published: January 2020

This indirect method for determination of desferrioxamine (DFO, deferoxamine, Desferal) and its chelated compounds ferrioxamine (FO) and aluminoxamine (AIO) in biological fluids involves metal chelate formation and solvent extraction, the metal being determined by Zeeman atomic absorption spectrometry (Zeeman-AAS). Special attention was paid to the pH and recovery of extraction, iron-aluminum interactions, extraction times and volumes, centrifugation rates, and instrument settings. Precautions were taken to avoid contamination with aluminum and iron and to prevent co-extraction of iron and aluminum not chelated to DFO. The addition calibration technique was used for standardization. Mean +/- SD recoveries of AIO and FO after extraction were respectively 98.3% +/- 3.7% and 101% +/- 3.7%. Sensitivity was 45 pg/0.0044 A.s for aluminum and 10 pg/0.0044 A.s for iron. Detection limits were 0.22 mumol/L for AIO, 0.06 mumol/L for DFO and FO.
Arginine pharmacokinetics in humans assessed with an enzymatic assay adapted to a centrifugal analyzer.
Authors: van Haeften T W , Konings C H
Published: January 2020

Arginine is used in supra-physiological concentrations as an insulin secretagogue, in both in vitro and in vivo studies. To investigate the pharmacokinetics of arginine in humans, we have developed a rapid, automated assay of arginine in serum, based on our manual enzymatic method (Clin Chim Acta 1988, 176; 185-94). The limit of linearity of the automated assay was an arginine concentration of 3 mmol/L. Within-run CVs for Ortho control sera with added arginine were 5.5%, 0.8%, and 0.7% at concentrations of 0.16, 1.30, and 2.50 mmol/L, respectively. After 30 min of primed continuous infusions with arginine at infusion rates of 3, 9, 15, and 21 mg/kg per minute, mean (+/- SEM) arginine concentrations in serum from eight volunteers were 1.17 +/- 0.08, 3.44 +/- 0.21, 6.84 +/- 0.58, and 9.25 +/- 0.39 mmol/L, respectively, well within the range of arginine concentrations shown (in vitro) to stimulate insulin secretion. Metabolic clearance of arginine was approximately 11 mL/kg body wt per minute. For the lowest three infusion rates, the half-life (t1/2) of arginine was approximately 15 min and the volume of distribution (Vd) was approximately 290 mL/kg. At the highest infusion rate, t1/2 was significantly increased (27.3 +/- 3.1 min), owing to an increased Vd (446 +/- 83 mL/kg).
Effects of in vivo and in vitro production of lactic acid on ionized, protein-bound, and complex-bound calcium in blood.
Authors: Toffaletti J , Abrams B
Published: January 2020

We have studied, both in vitro and in vivo, the quantitative effects of lactic acid production on concentrations of ionized calcium, bound calcium, pH, bicarbonate, and albumin. To do so, we examined the effects of addition of aqueous solutions of either hydrochloric acid, lactic acid, or lithium lactate to blood; we studied in vitro accumulation by storing blood sealed in tubes at room temperature for 5 h, then exposing the blood to air; and we induced in vivo production of lactic acid in healthy individuals who climbed stairs for 10 min. Lactic acid evidently affects the ionized, protein-bound, and complex-bound calcium concentrations in the following ways: (a) hydrogen ions from lactic acid bind to protein, which decreases protein-bound calcium; (b) lactate chelates calcium ions from free ionized calcium and protein-bound calcium about equally; and (c) the loss of a millimole of bicarbonate, either by exposure of blood to air or by respiratory alkalosis, results in the release of about 7 mumol of calcium ions, which re-equilibrate with both the protein-bound and ionized calcium. Because lactate apparently removes calcium ions directly from albumin, our study indicates that protein-bound calcium readily provides calcium ions that buffer changes in the concentration of ionized calcium.
Measurements of glutathione S-transferase B1 in plasma after birth asphyxia: an early indication of hepatocellular damage.
Authors: Beckett G J , Hussey A J , Laing I , Howie A F , Hayes J D , Strange R C , Faulder C G , Hume R
Published: January 2020

Concentrations of glutathione S-transferase (glutathione transferase; EC 2.5.1.18) B1 and B2 subunits (B1 and B2) and activity of alanine aminotransferase (ALT; EC 2.6.1.2) were measured in sequential plasma samples taken from 14 infants with birth asphyxia. Within 6 h of asphyxia, abnormal concentrations of B1 were found in 11 infants, whereas only seven infants showed abnormal ALT activities at this time. In plasma sampled 24 h after birth, values for ALT were abnormal in 10, whereas values for B1 were abnormal in six. Abnormal concentrations of B2 were found in relatively few of these infants, apparently because this monomer is poorly expressed in liver samples obtained up to 41 weeks after conception. We conclude that measurement of B1 may provide a useful index of hepatic impairment in birth-asphyxiated infants.
High-performance liquid chromatography/mass spectrometry: state of the art for the drug analysis laboratory.
Authors: Bowers L D
Published: January 2020

The combination of HPLC and mass spectrometry has great promise for the toxicology laboratory. In the past five years, significant progress has been made toward producing a reliable interface for these techniques. Thermospray, liquid ion evaporation, ion-spray, and the particle beam separator are all viable "second generation" approaches with significant advantages and disadvantages. I review the operation of these interfaces with an orientation to their use in the drug analysis laboratory. Thermospray, ion-spray, and liquid ion evaporation primarily supply information about molecular mass. The benefits and limitations of using tandem mass spectrometry to obtain additional structural information will also be discussed.
Effect of counting errors on immunoassay precision.
Authors: Klee G G , Post G
Published: January 2020

Using mathematical analysis and computer simulation, we studied the effect of gamma scintillation counting error on two radioimmunoassays (RIAs) and an immunoradiometric assay (IRMA). To analyze the propagation of the counting errors into the estimation of analyte concentration, we empirically derived parameters for logit-log data-reduction models for assays of digoxin and triiodothyronine (RIAs) and ferritin (IRMA). The component of the analytical error attributable to counting variability, when expressed as a CV of the analyte concentration, decreased approximately linearly with the inverse of the square root of the maximum counts bound. Larger counting-error CVs were found at lower concentrations for both RIAs and the IRMA. Substantially smaller CVs for overall assay were found when the maximum counts bound progressively increased from 500 to 10,000 counts, but further increases in maximum bound counts resulted in little decrease in overall assay CV except when very low concentrations of analyte were being measured. Therefore, RIA and IRMA systems based in duplicate determinations having at least 10,000 maximum counts bound should have adequate precision, except possibly at very low concentrations.
The between-run component of variation in internal quality control.
Authors: Linnet K
Published: January 2020

Design of control charts for the mean, the within-run component of variance, and the ratio of between-run to within-run components of variance is outlined. The between-run component of variation is the main source of imprecision for analytes determined by an enzymo- or radioimmunoassay principle; accordingly, explicit control of this component is especially relevant for these types of analytes. Power curves for typical situations are presented. I also show that a between-run component of variation puts an upper limit on the achievable power towards systematic errors. Therefore, when the between-run component of variation exceeds the within-run component, use of no more than about four controls per run is reasonable at a given concentration.
Influence of matrix on concentrations of somatotropin measured in serum with commercial immunoradiometric assays.
Authors: Felder R A , Holl R W , Martha P , Bauler G , Hellman P , Wills M R , Thorner M O
Published: January 2020

Using immunoradiometric assays (IRMAs) from Hybritech Inc. (H) and Nichols Institute Diagnostics (ND), we measured somatotropin (human growth hormone, hGH) in serum samples obtained every 20 min for 24 h from 10 prepubertal subjects with short stature. Results obtained with the ND reagents were 2.74 times greater than those obtained with the H reagents (P = 0.00001, r = 0.94, SEE = 3.9, n = 720). We therefore compared the IRMAs with the standard hGH RIA from the National Institutes of Health (NIH) National Hormone and Pituitary Program, using the genetically engineered hGH preparations (from Genentech Inc.) 22-kDa hGH and methionated 20-kDa hGH. We also assayed human pituitary hGH (NIH, lot no. AFP-4793B). Each hGH preparation was diluted in three diluent buffer systems: horse serum from H and from ND, and human serum. The RIA and H-IRMA gave superimposable standard curves for all hGH preparations in each diluent. The methionated 20-kDa hGH was not detected in the H-assay. Use of human serum matrix in the ND-IRMA shifted the standard curve as compared with the horse-serum matrix, giving equivalent binding at lower concentrations; i.e., serum hGH was overestimated in samples assayed against standards diluted in horse serum. Quality-control materials (Ciba-Corning) yielded disparate results in all three assays, yet human serum pools containing hGH gave similar results in the H and the NIH assays, and higher values in ND. When a human serum standard was used in the ND assay, both IRMAs gave similar results to the RIA assay for human serum samples. Reference intervals for hGH should be determined by each analytical laboratory, to prevent misdiagnosis of patients. Furthermore, quality-control material should be of human origin, because commercially supplied quality-control material does not react the same as human serum in some hGH assays.
Cobalamin-binding capacity of haptocorrin and transcobalamin: age-correlated reference intervals and values from patients.
Authors: Gimsing P , Nexø E
Published: January 2020

Unsaturated cobalamin-binding capacity in plasma (P-UBBC) is determined by use of a silica gel (QUSO G32) to separate haptocorrin (P-ApoHC) and transcobalamin (P-ApoTC). The method is sensitive and precise: detection limit 13 pmol/L, interassay coefficients of variation 3% for P-UBBC (mean = 1080 pmol/L), 4% for P-ApoTC (mean = 700 pmol/L) (n = 30). Values for P-UBBC, P-ApoHC, P-ApoTC, and P-TBBC (P-UBBC plus P-cobalamin) determined in a population study of 228 individuals, ages 21-87 years, did not differ by sex. These values increased with age, whereas the cobalamin saturation (P-cobalamin as percentage of P-TBBC) decreased with age. However, these changes were statistically significant but marginal and thus not clinically important. We therefore suggest using combined reference intervals (central 95 percentiles) for all age groups: 500-1200 pmol/L for P-UBBC, 90-275 pmol/L for P-ApoHC, 400-930 pmol/L for P-ApoTC, 850-1600 pmol/L for P-TBBC, and 20-50% for cobalamin saturation. Results for 277 inpatients show high P-ApoHC in myeloproliferative disorders or acute nonlymphatic leukemia, whereas P-ApoTC concentrations are high in some patients with lymphoproliferative disorders or autoimmune diseases.
Neopterin measured in serum and tissue culture supernates by a competitive enzyme-linked immunosorbant assay.
Authors: Barak M , Merzbach D , Gruener N
Published: January 2020

In this solid-phase competitive enzyme-linked immunosorbant assay for neopterin (a product of activated macrophages) in serum or supernates of cell cultures, we incubate antiserum to neopterin with standards or samples in the presence of solid-phase-bound conjugate of carrier-neopterin. Incubation with second antibody labeled with peroxidase then follows, before reaction with substrate. Analytical recovery, precision, and sensitivity of this method are similar to those of other immunoassays. The assay range is between 0.4 and 100 micrograms of neopterin per liter. Results obtained by this method compared with those from a conventional radioimmunoassay gave a correlation coefficient of 0.974.
Radioimmunoassay of beta-microseminoprotein, a prostatic-secreted protein present in sera of both men and women.
Authors: Abrahamsson P A , Andersson C , Björk T , Fernlund P , Lilja H , Murne A , Weiber H
Published: January 2020

We describe a simple radioimmunoassay of beta-microseminoprotein, one of the three most abundant secretory proteins of the prostate gland. The detection limit of the assay is 1 microgram/L, and its precision, expressed as the total coefficient of variation, is less than 10% for values between 10 and 150 micrograms/L. Using this assay, we found that beta-microseminoprotein immunoreactivity was present in sera from both sexes at about the same concentration. The protein detected had the same molecular size on gel chromatography as the protein isolated from seminal plasma, and dilution curves for the sera paralleled that for the pure protein. The findings suggest that beta-microseminoprotein is present in serum of healthy subjects of both sexes and that it originates in tissue other than the prostate gland. The range of the serum concentration was 0-10.6 micrograms/L (median 4.1) for 51 healthy adult women and 1.1-14.7 micrograms/L (median 6.2) for 35 healthy adult men not older than 40 years. In males with prostatic cancer the concentration in serum was highly variable and often greatly increased. The concentration of beta-microseminoprotein was correlated with that of creatinine in serum, suggesting that the protein is eliminated--at least partly--from the circulation by glomerular filtration. Little of the protein was present in the urine of women. In urine from men the concentration was high and variable, probably because of local contribution from the prostate gland to the urethral urine.