In the present study, serum albumin was determined colorimetrically in three animal species and in man with the help of four currently used dye-reagents (protein binding reagents). The results were compared with those obtained from corresponding electrophoretic and biuret determinations, using Versatol as a standard-control throughout. Using 2-(4-hydroxyazobenzene)-benzoic acid and bromocresol purple as reagent for the albumin determinations, species-specific differences from the electrophoretic results were found. No such differences occurred with bromo-cresol green, except in rats. There was no significant difference in albumin concentrations between man, monkeys and rats. Total protein concentration was only similar in man and monkeys.
A new method is described for sampling and sample application in the ultra-trace analysis of air by gas chromatography-mass spectrometry. In this new technique, a preseparation occurs on the gas chromatographic column during sample application, since the atmospheric constituents that are co-condensed during sample application we call this method "Druckaufgabe" (pressure application). The method is not only of theoretical value. It has a potentially wide application to occupational and environmental problems.
After 14 days of 1 mug ethinyloestradiol per kg body weight per day, both sexes show highly significant increases in serum coeruloplasmin which are independent of the determination method. Expressed on a percentage basis, the concentrations of coeruloplasmin under oestrogen medication determined by the p-phenylendiamine oxidase reaction are significantly higher than those determined by the immunological-nephelometric method. This is because several coeruloplasmin components, mainly C and DO, take part in the determination with p-phenylendiamine oxidase, whereas the immunological method measures chiefly the coeruloplasmin DO component. The significant sex differences under oestrogen medication shown by the determination with p-phenylendiamine oxidase, can therefore be explained by the presence of C- and D-coeruloplasmins in women, while the serum of men contains almost exclusively D-coeruloplasmins. Under oestrogen medication, the increase occurs primarily in the DO- coeruloplasmin component, and women also show an increase in coeruloplasmin-C. The significant higher enzymatic concentrations in the serum of the women in contrast to the men are thus plausibly explained. With the immunological assay, the sex differences were only probably significant.
A simple potentiometric method for the determination of fluoirde ion in human bones is described. The bone samples were taken from a definite position in the iliac crest. The samples are prepared for analysis by ashing at 550 degrees C, dissolving in HNO3 (4 mol/1) and adjusting the pH with NaOH (4 mol/1) and Total Ion Strength Adjustment Buffer, Absolute values are derived from a calibration curve based on aqueous NaF-buffer solutions. The calibration curve is linear in the range 5.2-10(-3)-1.0-10(-5) mol/1F-. The minimum limit of measurement is 5.2 X 10(-6) mol/1F-. The method has an accuracy of 96.1 % or 92.3% with a series precision based on the Pearson variability coefficient of 1.68%. The normal mass fraction of fluoride in human bones was found to be between 69 and 1740 - 10(-6) F-/ash.
Initial experiments on the constant appearance of unesterified fatty acids and hydrocarbons in cereborspinal fluid are presented. With the present method of analysis, these two groups of substances can always be detected in cerebrospinal fluid. The constitution of the unesterified fatty acids as well as the hydrocarbons was determined using gaschromatography linked to mass spectrometry. There is no evidence that the unesterified fatty acids are derived from the blood, or that they are breakdown products of other lipid classes of the cerebrospinal fluid. The origin and significance of the hydrocarbons are not yet known, and remain the subject of further investigations.
The kappa-lambda light chain ratios, the presence of free light chains and the double ring formation, with antikappa and antilambda serum, in single radial immunodiffusion were investigated in serum and cerebrospinal fluid of patients with subacute sclerosing panencephalitis. Cerebrospinal fluid samples of several multiple sclerosis cases were considered simultaneously. The results obtained suggest special immunoglobulin synthesis in both diseases.
The incubation of urine with beta-glucuronidase/arylsuphatase from Helix pomatia results in the incomplete hydrolysis of urinary steroid conjugates, because inhibitors of these enzymes are present in urine; The inhibitors can be separated from the steroid conjugates by chromatography of the urine on a column of Amberlite XAD-2. By this method a complete enzymatic hydrolysis of urinary steroid conjugates is possible in 24 hours at 37 degrees C or in 2 hours at 55 degrees C.
The sensitivity of enzyme kinetic substrate determinations can be improved with the aid of competitive inhibitors. As an example, the determination of glucose dehydrogenase in the presence of potassium thiocyanate is described. The method has the advantage of rapid operation with satisfactory precision.
In patients with liver cirrhosis the concentrations of amino acids were measured by ion exchange chromatography in the serum of blood samples taken from various vessels during and after the performance of a porto-caval anastomosis. Statistical evaluation was carried out with a nonparametric test. In three female and eight male patients, amino acids were determined intra operationem in blood samples of the following vessels (n = number of blood samples): arm vein (n = 8), arteria femoralis (n =2), vena cava (n = 7), vena portae (n = p) and aorta abdominalis (n = 5). With exception of ornithine (aorta abdominalis versus vena cava), no statistically significant differences in the concentrations of amino acids were observed in the various blood vessels. One to three years after the introduction of the porto-caval shunt, amino acid concentrations were measured in blood from the arm vein and arteria femoralis in four female and five male patients. The concentrations of glutamic acid, phenylalanine and lysine were significantly higher in blood from the arm vein than in blood of the arteria femoralis. The concentrations of valine, leucine and isoleucine were markedly lower in patients with liver cirrhosis than in normal persons. On the basis of the present findings and of the results obtained with normal subjects, it may be concluded that porto-caval anastomosis does not exert a noticeable effect on the metabolism of amino acids in patients with liver cirrhosis.
Lactate measurements can be performed within 2-3 minutes after blood withdrawal from the patients by using an electrochemical enzymatic sensor for lactate. The values obtained reflect the actual state of the patient which is not the case with the slow classical method using lactate dehydrogenase and NAD. The sensor is reproducible and the influence of the main reducing substances found in the blood is small enough to be of no clinical significance. Drugs commonly used in intensive care stations have no influence on the sensor. In vitro lactate production of the blood cells has been studied under various conditions. 66 pairs of comparative measurements between the classical method and the lactate sensor resulted in a good correlation coefficient.
An automated method for the analysis of total urinary hydroxyproline using strong cation resin tablets of Hypronosticon is described for use in a clinical laboratory and the results are compared with those obtained by other methods. Even though good recoveries are obtained using the technique described in the present work by adding the internal standards either before or after hydrolysis of urine, the present method gave consistently lower values of urinary hydroxyproline compared with a manual and an automated method.
Desmosterol is found in various organs of rats that show signs of myotonia in their skeletal muscle as a result of treatment with 20.25-diazacholesterol. The amount of desmosterol depends on the time of treatment, and is different in different organs and different kinds of muscle. The increase in desmosterol is much lower and the rats do not show any signs of myotonia when fed a cholesterol rich diet in addition to treatment with 20.25-diazacholesterol. Treatment with triparanol also causes desmosterol accumulation but in these rats myotonia is rarely observed. Our results suggest that in the experimental animals myotonia becomes manifest when every second cholesterol molecule of the muscle cell membrane is replaced by desmosterol. This is easily achieved in animals fed with 20.25-diazacholesterol but rarely occurs with triparanol.
In a group of 15 cases the cortisol concentrations were determined in fetal and maternal plasma during labour and after delivery. In maternal plasma the levels were about twice as high as in fetal plasma and rose up to more than 1000 mug/l during labour (x = 639 +/- 222). After delivery the concentrations decreased. In fetal plasma, cortisol increased during labour from x = 173 +/- to x = 276 +/- 75 at the time of delivery and decreased to x = 106 +/- 36 within the first 23 hours. In a second group of 20 cases maternal and umbilical cord blood and blood of the newborn during the first 28 hours after delivery were analyzed. The values were in the same ranges as in the first group. Extremely high maternal levels were not correlated to higher fetal values. The cortisol levels of postmature children did not significantly differ from those of normal babies.
Article A Microelectrophoretic Method for the Separation of β-N-Acetylglucosaminidase A and B from Cultured Human Fibroblasts and Amniotic Cells with the Aid of Polyacrylamide Flat Disc Gels was published on January 1, 1975 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 13, issue 9).
The increasing number of determinations performed by radioimmunoassays necessitates rationalization of the procedures. An analyzer system has been developed in order to fully mechanize double antibody radioimmunoassays, which is essentially composed out of four independently working modules. The samples, in microliter vials, are carried in sample chains of up to 650 links. The first pipetting step is performed by syringes with displacement pistons. Additional reagents are rapidly added by an electronically controlled Hamilton repeating dispenser, which makes shaking procedures for mixing unnecessary. The bound/free separation is achieved discontinuously by use of Nuclepore-filters, which are carried in 3 inches distance (76 mm) by a 35 mm dark leader film. After covering the radioactive filter positions with an adhesive plastic foil from both sides, the film spool is directly inserted into a specially constructed gamma-counter. The results of the evaluation of the efficiency and of the precision of each module are presented in this paper.
A combined adsorption-gel filtration technique has been developed for the quantitation of the cortisol-binding capacity of transcortin: Endogenous steroids are removed from plasma by adsorption on uncoated charcoal. Saturation of the "stripped" binding sites of transcortin is accomplished by equilibrating the sample with a definite amount of labeled cortisol of low specific activity (0.1 muCi/mug). Transcortin-bound [4-14C]cortisol is isolated by gel filtration over Sephadex G-50 at 4 degrees C and measured by liquid scintillation counting. The cortisol-binding capacity of transcortin is calculated directly on the basis of the known specific activity of cortisol. The modification described eliminates methodological disadvantages associated with the original gel filtration procedures, i.e. the possible interference of various endogenous steroids with cortisol binding to transcortin, and the necessity of fluorometric or colorimetric determination of protein-bound cortisol. The values of the cortisol-binding capacity of transcortin in plasma obteined by this simplified assay are in close agreement with results reported in the literature (mean +/- S.D.): healthy males 261 +/- 23 mug/l) of transcortin-bound cortisol (n = 13), healthy nonpregnant females 255 +/- 31 mug/l (n = 15), and pregnant females prior to delivery 560 +/- 82 mug/l (n = 12).
The pyruvate and NADH concentrations and the buffer pH which gave maximal activity with LDH isoenzymes derived from human heart and liver tissue were established for the temperatures 25 degrees C, 30 degrees C, 35 degrees C, 37 degrees C,, 40 degrees C, 45 degrees C, and 50 degrees C. The velocities of the LDH isoenzymes using these maximal assay conditions were used to obtain Arrhenius plots, i.e. log initial velocity against inverse absolute temperature. The Arrhenius plots were linear with both isoenzyme preparations up to 45 degrees C. Between 45 degrees C and 50 degrees C it appeared that this linear relationship no longer held, particularly with the liver tissue. When the activation energies were calculated both isoenzyme preparations exhibited several points of inflexion, in each case occuring at the same temperatures. These inflexions represent a change in the reaction kinetics, possibly a conformational change in the enzyme. The results also indicate that the LDH 1 and 2 isoenzymes are more efficient than LDH 4 and 5.
The uptake of the shortest six fatty acids (acetic to octanoic) was studied in vitro, using everted segments of rat jejunum. The marked influence of medium-pH and fatty acid chain-length suggests that non-ionic diffusion through the lipoid membrane is quantitatively the most important way of transport, but ionic diffusion through the membrane as well as transport through hydrophilic pores also seem to play a role. Though fatt acids evidently are accumulated in the tissue-fluid, and saturation kinetics, competitive inhibition and sodium- as well as energy-dependence apparently are observed, the transport mechanism is assumed to involve solely passive diffusion, - the concept of a carrier-mediated transport for short and medium chain fatty acids seems improbable.
Assays for the determination of serum alpha-N-acetylglucosaminidase (EC 3.2.1.50) activity are described employing p-nitrophenyl-N-acetyl-alpha-D-glucosaminide, phenyl-N-acetyl-alpha-D-glucosaminide, and UDP-N-acetylglucosamine as substrates. A log normal distribution of the serum enzyme activity was found. The determination of serum alpha-N-acetylglucosaminidase activity proved to be a valuable tool for the recognition of homozygous and heterozygous carriers of the Sanfilippo B gene.
