Mercury monitor for ambient air.

An isotope-shift, Zeeman-effect atomic absorption spectrometer has been demonstrated to have sufficient sensitivity to continuously monitor the total mercury content of ambient air. At present, the minimum total mercury concentration detectable with this device is 0.2 microgram per cubic meter of air-one fifth of the proposed federal guideline. This is the first technique which responds to both mercury vapor and mercury in particulates available for continuous monitoring at this concentration.

Phosphatidylglycerol in amniotic fluid. Comparison of an "ultrasensitive" immunologic assay with TLC and enzymatic assay.

Phosphatidylglycerol (PG) in amniotic fluid is recognized as a good indicator of fetal lung maturity and is unaffected by moderate amounts of blood or meconium contamination. A rapid immunologic agglutination assay, Ultrasensitive AmnioStat-FLM (FLM), was compared with two-dimensional thin-layer chromatography (TLC) and an enzymic, colorimetric procedure (E-PG). Eighty amniotic fluid specimens were analyzed. FLM results were reported as high (H), intermediate (I), or low positive (L). TLC was compared with FLM:H (n = 27), mean 0.14 (fraction of total phospholipids); I (n = 7), mean 0.11; L (n = 9), mean 0.03; negative results had no detectable PG by TLC. In 33 cases E-PG was compared with FLM:H (n = 9), mean 7.0 mumol/L; I (n = 5), mean 8.1 mumol/L; L (n = 3), mean 3.0 mumol/L; negative (n = 16), mean 3.2 mumol/L. Records were reviewed in 70 cases. Thirty cases were excluded: sample to delivery time was greater than 72 hours; steroids were given or sepsis was documented. Fetal lung immaturity was clinically present in six cases: respiratory distress syndrome in three cases and transient tachypnea of the newborn (TTN) in three cases. One false positive result was identified (TTN, FLM:H). FLM sensitivity for fetal lung maturity was 85.3%, specificity was 83.3%, and the positive predictive value for fetal lung maturity was 96.7%. FLM is a fast, reliable indicator of fetal lung maturity.

Application of a sensitive and specific reagent for the determination of serum iron to the Bayer DAX48.

We describe a modification of a previously described serum iron procedure applied to the Bayer DAX48 (Bayer Diagnostics, Tarrytown, NY) automated chemistry analyzer. The iron-ligand used in this assay, 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamine) phenol (nitro-PAPS), has a molar absorptivity of 94,000 L mol(-1) cm(-1), which is three to four times more sensitive than the more commonly used ligands. The increased sensitivity of the iron-ligand complex facilitates modification of a Ferene S method that requires a smaller sample volume while it maintains the precision of the assay. Because the reagent does not contain ascorbate, the "onboard" stability has been increased to more than 4 weeks. The reagent seems to be quite insensitive to icterus and hemolysis. Furthermore, the interference of turbidity caused by triglycerides, abnormal proteins, or fibrinogen, present in samples from patients undergoing anticoagulant therapy, seems to have been eliminated.

A liquid-stable reagent for lactic acid levels. Application to the Hitachi 911 and Beckman CX7.

We evaluated the use of a new lactate oxidase-based reagent for the determination of serum and plasma lactic acid levels with the Hitachi 911 (Roche Diagnostics, Indianapolis, IN) and the Beckman CX7 (Beckman Instruments, Brea, CA). Evaluation studies demonstrated on-board stability of at least 3 months and a calibration stability of more than 5 months. Within- and between-day imprecision of this reagent was less than 2% for both applications. The reagent is free of the deleterious effects of triglyceride up to levels of 1,400 mg/dL (15.8 mmol/L), bilirubin to concentrations of 24.6 mg/dL (420 mumol/L), and hemoglobin, from lysed erythrocytes, to levels of more than 0.3 g/dL (3.0 g/L). When used on the Hitachi 911 for the determination of plasma lactate concentrations, the reagent correlates with the Dade aca III (Dade International, Deerfield, IL). When applied to the Beckman CX7 for the determination of serum lactate levels, the method correlates with the Beckman method.

Spectrophotometric study of total protein-albumin methods applied to cerebrospinal fluid.

A spectrophotometric study was carried out for three proteins assays when modification of their serum procedures using bromcresol green, bromcresol purple and biuret reagents were applied to the determinations of total proteins and albumin in cerebrospinal fluids. A novel concentration device wherein the sample itself was used as the primary diluent for the three reagents concentrated to contain the proper amounts of chemicals in smaller volumes than suggested in their serum procedures allowed reasonable absorbance signals to be obtained. Low molecular weight molecules were separated from the albumin and globulins of the fluids by centrifugal ultrafiltration using a 25K cutoff and spectra were obtained for both high and low molecular weight fractions. Some materials were obtained in the separated ultrafiltrates which gave reactions with all three reagents, reactions which either overlapped the spectra of the albumin reactions or superimposed the spectra obtained with the total protein reaction. A screening procedure for cerebrospinal fluid total proteins or centrifugally ultrafiltered albumin appears reasonable as an inference from studies made, although further elucidation of the low molecular weight fractions in needed as a confirmation device.

A method for the sequential colorimetric determination of serum triglycerides and cholesterol.

A simple spectrophotometric method for the sequential determination of triglycerides and cholesterol from a single serum sample was developed. In this two-stage procedure, the triglycerides and cholesterol esters are first hydrolysed to glycerol and free cholesterol respectively, with simultaneous scavenging of the liberated free fatty acids, a technique that ensures clarity of the sample. The glycerol is subsequently reacted to result in an intense red chromogen with a peak absorption maximum at 510 nm following a series of enzymic reactions. In the second stage, addition of cholesterol oxidase leads to oxidation of free cholesterol generated from the cholesterol esters in the first stage and the free cholesterol normally present in the sample, yielding in a similar fashion the identical red chromogen whose absorbance is also measured at 510 nm. Results obtained with the proposed method demonstrate good correlation with established individual procedures for triglycerides and cholesterol.

On the use of a sensitive indicator reaction for the automated glucose oxidase-peroxidase coupled reaction.

A study into the substitution of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate for phenol in the indicator reaction for an automated glucose procedure is presented. Apart from having physical properties that are more conducive to easy handling than phenol, the former material affords considerably more sensitivity than does the latter. Results obtained with either of these glucose oxidase-coupled systems demonstrate good correlation not only with each other but also with the glucose oxidase procedure of the Beckman Astra 8.

Spectrophotometric study of several sensitive reagents for serum iron.

A comparison of three sensitive ligands for iron is described. All show varying degrees of enhancement of values by copper, an element that is the primary interference encountered in serum assays for iron. Two of three tested reagents, 2,4-bis(5,6-diphenyl-1,2,4-triazin-3-yl) pyridine tetrasulfonate and Ferene S are not current clinical chemistry choices whereas ferrozine is widely used. The described study presents spectral characterization of the copper interference with all three ligands. The virtual complete removal of this interference, by the masking actions of either thioglycolic acid, neocuproine or thiourea, is also described . Evidence will be presented that contradicts a previously proposed mechanism for the action of thioglycolic acid which suggests that copper is prevented from dissociating from its protein-binding site by this compound. The preparation of a protein-free filtrate as the matrix in which to generate the color reaction is described, and alternative procedures discussed in brief.

Investigation of reaction intermediates of the urea-diacetylmonoxime reaction.

1. An investigation of the intermediates of the urea-diacetylmonoxime reaction and the isolation of the protochromogen has been described. A comparative spectral study of the protochromogen with that of the literature values suggested the structure of the protochromogen to be a diureide. 2. The diureides of 1-phenyl-1,2-propanedione-monoxime and 2,3-pentanedione were synthesized and their visible, U.V., I.R., NMR, and mass desorption spectrometry data were analyzed. The study suggests that the structure of each of these compound is that of a diureide. When these diureides were redissolved in acid media, they gave the color expected of the corresponding chromogens found from the respective diketones and urea. 3. The 3a-methyl-6a-phenyl-glycoluril was synthesized by a base-catalyzed reaction. When this glycoluril was dissolved in concentrated HCl, a pink color was obtained, similar to that of the corresponding diureide. These studies indicate that both the diureide and the glycoluril (if formed at all in acid-catalyzed reaction) are converted to the same chromogen, possibly via a rearrangement. 4. When 2,3-butanedionemonoximethiosemicarbazone was reacted with urea in acid medium, characteristic spectral data were obtained which were very similar to those of the urea-diacetylmonoximethiosemicarbazide reaction. This tends to indicate that the possible structures of the protochromogens are similar and of the open chain diureide type.

Differential spectrophotometry for the determination of serum methemalbumin.

A method for the direct determination of methemalbumin in serum by means of difference scanning spectrophotometry is described. The advantages of this quantification technique resides in the ability to obviate unequal background characteristics such as turbidity in one treated sample portion, the avoidance of subtractive measurement on the sides of steep spectra, and a qualitative observation simultaneously derived from the single difference scan. A measure of the stability of the prepared standards is described along with a minimized use of sample and reagent volumes if the latter is desired.

A procedure for the kinetic colorimetric determination of serum cholinesterase activity.

A choline oxidase-peroxidase coupled enzyme procedures is proposed for the determination of cholinesterase activity in human serum. This system is not only kinetic and colorimetric but is also relatively quick and simple to perform. The initial comparisons suggest that this method correlates well with a commonly used propionylthiocholine-dinitrobis-(nitro-benzoic acid) technique. Large amounts of bilirubin in the sample appear to have only minor deleterious effects on the assay. Since there are only two reagents that may be premixed, the procedure appears to be amenable to automation. The use of a mixture of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate and 4-aminoantipyrene in the peroxidase catalyzed indicator reaction provides for a marked increase in sensitivity over previously reported 4-aminoantipyrene-phenol systems. This augmented sensitivity provides for a relatively large reagent to sample ratio. In addition, the reagents lend themselves toward lyophilization or "dry-fill".

The turbid specimen as an analytical medium: hemoglobin determination as a model.

The quantitation of chemical constituents in lipemic samples is a major problem confronting the clinical laboratory. Currently, a number of cumbersome and time-consuming methods are used to clarify samples before analysis. However, the use of enzymic hydrolysis of triglycerides along with efficient chemical removal of the formed non-esterified fatty acids is exemplified here as an excellent alternative to the current methods of clarification such as ultracentrifugation, extraction or chemical precipitation of low density and very low density lipoproteins. This method of clarifying milky serum has been used by us to assay hemoglobin in severely lipemic blood samples as an analytical model.

On-line clarification for the measurement of serum glucose in hyperlipidemic specimens.

The optical aberrations due to the presence of the turbidity caused by hyperlipidemia has been eliminated in two serum glucose procedures. This has been accomplished by incorporating lipase and alpha-cyclodextrin into the two glucose reagents. The hydrolytic action of the lipase generates water soluble glycerol and insoluble fatty acids. By including the chemical scavenger alpha-cyclodextrin into the reagent the fatty acids are solubilized and thus the production of a second turbidity is avoided. Because the clearing reagents are incorporated into the glucose reagents, this is an on-line process and no additional labor is required to clear the sample-reagent mixture. Furthermore, no additional time is required as the clearing occurs in the same period of time that it takes for the indicator reactions to reach equilibrium.

Potential problems in serum protein electrophoresis.

Potential problems are described that one could encounter in carrying out an electrophoretic procedure including its ancillary phases of visualization (staining) and quantification (densitometry). Endpoint-like measurements for separated isoenzymes may provide artifactual kinetic values as well, because stain measurement is fixed at a single time whereas reagent blanking in the electrophoretic medium is substituted for the conventional serum initial absorbance readings of test-tube determinations. Truncation of separated electrophoretic zones or opacity of an electrophoretic anti-convection medium such as uncleared cellulose acetate may also interfere with absolute quantification procedures.

The clinical use of alkaline phosphatase enzymes.

The enzyme alkaline phosphatase is an important serum analyte and its elevation in serum is correlated with the pressure of bone, liver, and other diseases. The analysis of the isoenzymes of alkaline phosphatase is an aid in diagnosing liver and/or bone disease, especially the high molecular weight isoenzymes that appear in cholestatic liver disease.

Cholesterol methodology for human studies.

A classification and review of the methodology involved in the determination of serum cholesterol for human (or animal) studies are presented. The purpose of both is to enable selection of a technique appropriate for the assay intended with a reasonable understanding of its advantages, disadvantages and limitations. The various methods discussed include direct reaction systems, partial isolation systems and complete isolation systems, as well as screening, reference and definitive procedures. The interferences that could occur are considered, especially those caused by hemoglobin, the turbidity in lipidemia, and bilirubin, as well as interferences caused by optical aberrations and chemical reactants. The various instrumental methods used to determine cholesterol or a substitute determinand such as hydrogem peroxide are discussed, including spectrophotometry, electrochemistry and densitometry of electrophoretically separated proteins.

Use of alkaline phosphatase isoenzyme analysis in the evaluation of cholestatic liver disease.

A useful laboratory test for the differentiation of liver, bone, and intestinal alkaline phosphatase (ALP) isoenzymes in serum is presented. Electrophoresis in polyacrylamide gel is performed with untreated serum as well as with serum incubated at 56 degrees C for 10 min. The heating step denatures bone isoenzyme which may obscure the liver ALP band when present in large amounts. Visualization of ALP activity is accomplished by the use of buffered p-toluidinium 5-bromo-4-chloro-indolyl phosphate and magnesium ions. In serum of patients with cholestatic liver disease, the occurrence of large molecular weight liver cell membrane fragments which contain ALP activity is postulated. These ALP-containing fragments occur at the origin of the electrophoretogram, unable to penetrate the small pore separation gel. Abnormalities involving ALP isoenzymes, such as bone isoenzyme arising from increased osteoblastic activity, may be detected. Intestinal isoenzyme, normally present in small amounts in some subjects of blood groups B or O, may be elevated in certain liver diseases, such as cirrhosis. By the use of this method the routine question of whether an ALP found to be increased in a screening procedure is due to liver or bone abnormality may be answered. In addition, the occurrence of abnormal ALP bands arising from cholestatic conditions and the occurrence of abnormal amounts of intestinal isoenzyme may also be detected.

Some aspects of bilirubin determination in the newborn using dimethyl sulfoxide.

Although serum bilirubin determination is a common procedure in most clinical laboratories, the test seems to be of particular importance in the screening of neonates, especially the premature or the erythroblastotic infant. However, bilirubin determination is subject to interferences, particularly in the presence of hemolysis and lipemia, which may seriously affect the results. It is, therefore, of paramount importance to the pediatrician to be aware of these potential shortcomings of the test. To improve the reliability of bilirubin determination, a method using dimethyl sulfoxide is proposed which is simple, rapid, and ideally-suited for small amounts of blood such as are commonly encountered in pediatrics. The effect of hymolysis and moderate lipemia are minimal, and the use of a sample blank can usually be eliminated in most circumstances.

Detergent altered alkaline phosphatase patterns of liver disease.

A procedure has been described whereby the high molecular weight alkaline phosphatase (slow-moving) isoenzyme may be studied by means of polyacrylamide gel electrophoresis. By treatment of sera containing this isoenzyme with some detergents of the nonionic Triton octylphenoxyethanol series, the high molecular weight alkaline phosphatase isoenzyme is altered so that its electrophoretic mobility more closely resembles that of the usual alklaine phosphatase isoenzymes. The high molecular weight isoenzyme is thought to be associated with phosphatidyl choline and/or liproproteins. The detergent action is to dissociated the alkaline phosphatase from its lipid carrier. It is thought that these lipid-alkaline phosphatase complexes are associated with liver cell fragments. The detergent altered slow-moving alkaline phosphatase may migrate as a single band, from two to four new bands, or as several new bands. Liver, bone and intestinal alkaline phosphatase isoenzymes are unaffected by detergent action.

Review of phosphate methodologies.

A description of the extensive literature on the methodologies for the determination of serum phosphate has been reviewed. The evolution of the various phases of the analytical techniques developed in the last century leading to the simplified, sensitive and accurate procedures of the present have been presented in some detail. A procedure involving a simple direct reaction for the determination of inorganic phosphate in serum using ascorbic acid as a reducing agent and a citrate-arsenite mixture as a stabilizing-sensitizing reagent, all in a semi-aqueous medium containing dimethysulfoxide and the detergent Teepol 610 is recommended. Jaundice and mild hemolysis are noninterferencts and turbidity from severe lipemia is easily overcome by reversing the sequence in which reagents are added because citrate binds molybdate in preference to phosphate. Thus, the serum blank and the reacted serum are identical in makeup thereby yielding an idealized correction for irrelevant absorption. Reaction characteristics and potential errors are included in the discussion of the procedure.