Enzymes of lysosomal origin in human plasma and serum: assay conditions and parameters influencing the assay

The condition for maximal activity (pH, buffer, saturating substrate concentration, range of linear relationships between enzyme activity versus incubation time, and versus enzyme concentration) in the fluorimetric assay of several glycohydrolases of lysosomal origin in human plasma and serum have been established. The following enzymes were studied: alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-glucuronidase, alpha-mannosidase, alpha-fucosidase. All examined enzymes turned out to be more or less unstable upon storage at 37 degrees C, 4 degrees c, and -20 degrees C in both serum and plasma. The only exceptions were beta-glucuronidase, which was stable in plasma and serum, and alpha-fucosidase which was stable only in plasma. Generally the degree of instability was greater in serum than in plasma. The levels of some enzymes (alpha-galactosidase, beta-galactosidase, beta-N-acetyl glucosaminidase, beta=glucuronidase) were markedly higher in serum than in plasma; conversely the levels of the same enzymes in "platelet free" serum equalled those in plasma. This stresses the necessity to use freshly prepared plasma for lysosomal glycohydrolase assay. Under the procedural conditions recommended for the assay the methods for the determination of lysosomal glycohydrolases in plasma appeared to be simple, sensitive and reproducible.     

A quantitative assay of cortisol in human plasma by high performance liquid chromatography using a selective chemically bonded stationary phase

The extraction and subsequent liquid Chromatographie analysis of human plasma samples for cortisol is described.Extraction and chromatography are optimized, resulting in a recovery for cortisol of 96% and a detection limit of 1 μg cortisol in 100 ml plasma. The application of two chemically modified silicas has been evaluated.The specificity of the method was tested by field desorption — mass spectrometry experiments.     

Enzymatic determination of cholesterol in bile.

A simple, sensitive and rapid method for measuring the cholesterol concentration in bile is described. The method is based on the combination of an enzymatic technique and spectrophotometry. In the present study the results obtained with this method are compared with those obtained by the standard Liebermann-Burchard reaction on the same samples and are found to be in close agreement.     

Subarachnoid neurolytic block (L5-S1) and unilateral percutaneous cervical cordotomy in the treatment of pain secondary to pelvic malignant disease

The present study deals with the immediate and long-term results of subarachnoid neurolytic block (L5-S1) with 7.5%, 10% and 15% concentrations of phenol in glycerine and/or unilateral percutaneous cervical cordotomy in 73 patients (follow-up in 56 patients) suffering from perineal, perineopelvisacral or pelvisacral pain secondary to malignant diseases of the pelvic cavity. Subarachnoid neurolytic block (L5-S1) produced satisfactory, long-lasting relief of perineal pain when the higher concentrations of phenol (10 and 15%) were used. The only sequela reported was urinary retention. Percutaneous cervical cordotomy used for the treatment of pelvisacral or predominantly unilateral perineal pain gave complete pain relief until death in 76.7% of patients, either alone (36.7%) or in conjunction with pharmacological therapy (40%). Analysis of the data enabled us to establish the respective indications for the two procedures and to identify those cases in which their use may be complementary.     

Radioimmunological determination of apparent free cortisol concentration: Some physiological and clinical applications

The measurement of free cortisol would be preferable with respect to the total hormone content, since it yields more reliable information about the plasma levels of the biologically active steroid.The methods so far described for the measurement of free cortisol in plasma use radiolabelled cortisol for determination of the steroid free fraction, and are generally unsuitable for routine use.We have developed a new method for the determination of the apparent free plasma cortisol concentration by means of direct radioimmunological measurement of dialyzed cortisol. This method is characterized by a sufficient degree of reproducibility and high sensitivity.Apparent free cortisol concentration in 40 control subjects of both sexes (blood drawn at 8 a.m.) was 9.00 ± 4.6 ng/ml. The mean value of free cortisol concentration in blood samples drawn at 11--12 p.m. from 21 of these subjects was highly significantly different (2.3 ± 1.6 ng/ml, p < 0.001). In addition, in 13 of these subjects circadian variation of the apparent free cortisol concentration showed a pattern similar to that of total cortisol concentration. The mean free cortisol concentration found in a group of women during normal pregnancy was significant higher than in non-pregnant women.Patients with renal insufficiency do not show a significant difference in free cortisol plasma levels, whereas higher values were found in hepatic cyrrhosis.     

High performance liquid chromatographic analysis of the antiarrhythmic drugs procainamide, disopyramide, quinidine, propranolol and metabolites from serum extracts

We describe a method for the simultaneous high performance liquid chromatographic determination of several antiarrhythmic drugs and some of their metabolites after extraction from 2.5 mL of spiked pooled sera. The extracts were applied to a C8 reversed phase column. Nine compounds of interest were resolved within the 30 minute run. An initial mobile phase of 80% phosphate (25 mmol/L, pH 3.5), 20% organic (acteonitrile:methanol, 2:3) was maintained for 2 min at which time a linear gradient was used to change the mobile phase to 30% phosphate, 70% organic at 20 min after injection. This composition was maintained from an additional 5 min. Absorbance at 212 nm was used for detection. Peak area ratios of drug to internal standard (N-propionylprocainamide) were used for quantitation. The relative standard deviations (and mean solute concentrations) of daily duplicate determinations for 15 days are: procainamide, 5.1% (5.9 mg/L); N-acetylprocainamide, 9.3% (6.0 mg/L); Mono-N-dealkyldisopyramide, 3.7% (4.1 mg/L); disopyramide, 4.3% (4.0 mg/L); quinidine, 4.5% (6.5 mg/L); and propranolol, 5.1% (0.097 mg/L). Dihydroquinidine and 4-hydroxypropranolol were also resolved but not quantitated.     

An improved method for the determination of 5-phosphoribosyl 1-pyrophosphate.

A spectrophotometric method for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP) is presented which shows several advantages in comparison to the radiochemical techniques, such as a relatively simple, rapid and less expensive procedure. This technique has been used to evaluate PRPP content in erythrocytes, leukocytes and lymphocytes of normal subjects and individuals with partial hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) deficiency. The results obtained proved to be completely reliable in both groups of subjects examined, with values of PRPP similar to those observed by radiochemical techniques.     

Gas-liquid chromatographic and mass fragmentographic determination of 3-O-methylated catecholamines in human plasma.

Plasma 3-O-methylated catecholamines, i.e. 3-methoxytyramine, normetanephrine and metanephrine, were separated from catecholamines by passing through alumina and further purified by adsorbing on weakly acidic resin and Amberlite XAD-4. The amines were trifluoroacetylated and determined by gas chromatography or mass fragmentography. Tracer quantities of tritiated 3-MT, NMN or MN were used as internal standards for total recovery estimations. The contents of 3-O-methylated catecholamines in the plasma of normal persons and patients with hyperthyroidism, hypertension, neuroblastoma and pheochromocytoma were measured.     

Relevant and irrelevant anxiety in the reaction to pain

Despite its importance in pain perception, there is a paucity of research investigating the influence of anxiety. This study tested the proposition that anxiety can lead to the exacerbation of pain perception when the source of anxiety is related to the pain experience. When the source is related to something else, anxiety may even reduce the reaction to pain. Sources of anxiety were manipulated in the laboratory — anxiety related to pain and anxiety related to successful learning or the combination of anxiety related to both pain and learning. Verbal, physiological and behavioral differences were obtained showing that focus upon both the pain and the learning task yielded the strongest pain reactions, while focus upon the learning alone yielded the lowest pain reaction, but the largest learning errors. Focus upon pain was in-between. The theoretical implications of these data were discussed.     

3-Mercaptopyruvate sulfurtransferase (EC 2.8.1.2): a simple assay adapted to human blood cells

A product of cysteine catabolism, 3-mercaptopyruvate, is enzymatically degraded to sulfur and pyruvate by a sulfurtransferase (EC 2.8.1.2.) present in human red and white blood cells. A simple sulfurtransferase assay is reported which takes advantage of the fact that pyruvate is conveniently measured enzymatically by lactate dehydrogenase (LDH) after the elimination of 3-mercaptopyruvate, also a substrate of LDH, by addition of N-ethylmaleimide. Sulfite is employed as sulfur acceptor, and conditions for a reproducible assay including data for preparation and storage of reactants, their assay, and their optimal concentrations are given. The apparent K_m for sulfite is 6.5 · 10^?3 M and for 3-mercaptopyruvate is 1.9 · 10^?3 M. A reducing agent, in this assay dithiothreitol (Cleland's reagent), is essential for active transsulfuration. A normal metabolite, 3-mercaptopyruvate is reported elsewhere to have the capacity of producing polyploidy and chromosomal endoreduplication, a feature rendering it of interest in tumor metabolism.     

Automatic treatment of radioimmunoassay data: an experimental validation of the results.

A computational method for the automatic treatment of radioimmunoassay data has been developed and a computer program has been written in accordance. The main features of the approach used are the following: (a) a constant ratio between the bound fraction and its variance is assumed and estimated in each assay; (b) the points of the dose-response curve are fitted using the three-parameter function y' equals b1/(1 plus b2x(-b)3) where y' equals bound - nonspecific counts and x is the amount of hormone; the fitting is performed using the nonlinear, least-squares technique; (c) the values of the unknown samples are evaluated from the fitted standard curve; their confidence limits are computed taking into account both the variance of the bound replicates and the variance of the parameters of the dose-response curve. Experimental data that support the validity of the assumption on the variance of bound measurement and the suitability of the chosen function to fit the points of the standard curve are presented. A comparison between the confidence limits of the unknowns experimentally obtained and those computed by the program is reported and discussed.     

Characterization of human beta2-microglobulin by isoelectric focusing.

b2-Microglobulin (B2M) isolated from the urine of normal subjects and patients with cadaveric renal transplantation, showed 2 homologues by isoelectric focusing, one with a pI 5.3, the other with a pI 5.7. These proteins show identical molecular weights by dextran gel filtration and sodium dodecyl sulfate acrylamide gel electrophoresis. The immunogenic reactivity demonstrates partial identity using antiserum to human B2M from 2 different sources.     

Correlation of bile acid composition between liver tissue and bile

Bile acid composition of ten paired human livers and bile specimens were compared with gas chromatography-selected ion monitoring. The bile acid composition in liver and in bile was found to be similar but not identical. This difference seems to be a reflection of bile acid synthesis in liver tissue. It is suggested that analysis of bile acid composition in liver tissue is useful to evaluate abnormality of bile acid synthesis in several pathological states.     

Hormonal aspects of human gout — excretion of adrenal hormone derivatives in gouty patients

Fortyseven patients with gout, 28 of whom had not previously been treated with allopurinol, and 25 normal subjects, were examined for 24-h urinary excretion of the most important adrenal steroid derivatives.

Results were submitted to statistical analysis and several variables have been taken in consideration.

The untreated patients showed significantly higher values of uricemia, urinary uric acid, triglycerides, slightly higher values of androsterone, 11-oxo-androsterone + 11-oxo-etiocholanolone, dehydroepiandrosterone, and slightly lower values of 11-hydroxyandrosterone and pregnanetriol, in comparison to normal subjects.

The different hormonal pattern seems to discriminate between patients with gout and normal subjects.     

Analysis of antibodies to Aspergillus fumigatus antigens by class-specific enzyme-linked immunosorbent assay in patients with pulmonary aspergillosis

Antibodies to two commercial extracts of Aspergillus fumigatus were determined by a class-specific enzyme-linked immunosorbent assay (ELISA) in 203 serum samples from 139 patients with various pulmonary diseases. In 22 patients with aspergilloma immunoglobulin (Ig)M, IgA, and especially IgG antibodies were found, whereas in 50 patients with allergic alveolitis IgG antibody was most frequent, IgM occurring rarely. One patient with allergic bronchopulmonary aspergillosis demonstrated IgG and IgA antibodies. Of 20 cases with bronchial asthma, 10% reacted against A. fumigatus in immunodiffusion as well as in ELISA. Of 46 cases with carcinoma, tuberculosis, and miscellaneous pulmonary diseases, 17% were positive by immunodiffusion and 26% demonstrated antibodies usually IgG, by ELISA. Of 100 healthy blood donors, none had Aspergillus antibodies of the IgG class, whereas 3% were positive in the IgM and 3% in the IgA assay. The ELISA proved to be sensitive and useful in the follow-up of patients with aspergilloma after operation.     

Development and optimisation of a radioimmunoassay for plasma captopril

A specific and sensitive radioimmunoassay has been developed to monitor plasma levels of captopril, the first orally active angiotensin-converting enzyme inhibitor. Because of the reactive nature of the captopril thiol group, captopril was measured as the captopril-N-ethylmaleimide complex (captopril-NEM). Accuracy studies, using samples with known added concentrations of captopril, were satisfactory (r = 0.95, p less than 0.001, y = 0.97x - 2.02), and the radioimmunoassay results compared well with those determined by a gas chromatography/mass spectrometric method (r = 0.98, p less than 0.0005, y = 1.2x - 19). The minimum detection limit of the assay was 2 micrograms captopril/litre plasma.     

The application of high pressure liquid chromatography to the analysis of bile salts in human bile.

Two high pressure liquid chromatography (HPLC) systems have been evaluated for the separation of conjugated bile salts from human bile. Partisil-10 ODS with mobile phase of methanol/water pH 2 (55 : 45) at 200 nm, 0.1 AUF UV detection gave only partial separation of bile salts. However, a mu Bondapak fatty acid analysis column using isopropanol/8.8 mmol/l potassium phosphate pH 2.5 (32 : 68) as the mobile phase and 193 nm, 0.1 AUF UV detection separated all the six conjugated bile salts in bile. The limit of detection ranged from 0.1 microgram for sodium taurocholate to 0.2 microgram for sodium glycodeoxycholate. The reproducibility and the application of the method to the analysis of conjugated bile salts was demonstrated using bile from five patients. Its application to the studies of hepato-biliary disease is discussed.     

A rapid, new method for quantitative analysis of human amylase isozymes

Amylase isozymes of human serum and urine were separated by electrophoresis on a cellulose acetate membrane and visualized using a blue starchagar gel plate.The two isozymes separated, named S and P in order of their distance from the origin, corresponded to salivary amylase and pancreatic amylase, respectively. These two bands were measured quantitatively with a densitometer. The relative proportions of the isozymes were found to be of value in differentiation of hyperamylasemias due to disorders of the pancreas, parotid gland and other organs.