Determination of total 3 alpha-hydroxy bile acids in serum.

The usual techniques for determination to total 3 alpha-hydroxy bile acids in serum involving liquid-solid extraction of the bile acids with the adsorbent XAD-2 and fluorimetric measurement of NADH generated from the reaction with a NAD-linked 3 alpha-hydroxysteroid dehydrogenase are evaluated and improved. The influence of different types of enzyme preparations on the results is examined. The results with the improved technique are compared to the results obtained with another method, avoiding extraction of the bile acids before the enzymatic reaction which is followed by fluorimetric measurement of resorufin, produced by transfer of the hydrogen of the generated NADH by diaphorase to resazurin. No significant difference between the results with the two types of methods was found. The concentration of total 3 alpha-hydroxy bile acids in serum of 46 fasting 'healthy' individuals aged 17 to 82 years is estimated. 30 were females, of whom 10 were taking estrogen-containing oral contraceptives, and 16 were males. Mean +/- standard deviation in all the females was 3.0 +/- 1.1 micromol/l, and in the males 4.0 +/- 1.9 micromol/l. There was no significant difference between any of the groups.     

Determination of gold in plasma and plasma fractions by atomic absorption spectrometry and by neutron activation analysis.

Three techniques of gold analysis, flame and electrothermal atomic absorption spectrometry and neutron activation analysis, have been compared, using plasma and plasma fractions (derived by gel chromatography) from rheumatoid patients receiving aurothiomalate therapy and from plasma samples incubated with aurothiomalate in vitro. The three methods correlated well in the analysis of gold in whole plasma, but only neutron activation analysis was suitable for the assay of all the plasma fractions. The susceptibility of the two atomic absorption methods to interference by sodium chloride was investigated.     

Determination of serum urea by mass fragmentography.

A mass fragmentographic method of high accuracy for determination of serum urea is described. A fixed amount of [15N2]urea is added to a fixed amount of serum, then the urea is converted into 5,5-diallyl barbituric acid by coupling with diallyl malonic acid diethyl ester. The barbiturate is then transferred from an alkaline water phase into an organic phase containing methyl iodine by ion-pair extraction using tetrabutyl ammonium as the positive counterion. The amount of urea is determined from the ratio between the recordings at m/e 236 and m/e 238 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with an MID-unit (multiple-ion detector). The two ions used correspond to the molecular peak in the mass spectrum of the methyl derivative of unlabeled and labeled 5,5-diallyl barbituric acid, respectively. The relative standard deviation of the method was 3.6%. A comparison between the mass fragmentographic method and a routine method for determination of serum urea based on the urease-Berthelot reaction gave a high correlation (r = 0.99) and a regression coefficient of 0.95.     

Isoenzymes of human phosphofructokinase

Human liver phosphofructokinase has been isolated in order to obtain antibodies against human liver phosphofructokinase. With the antiserum it could be shown in human liver that two types of phosphofructokinase exist with no subunits in common. Leucocytes, erythrocytes and kidney also possess the liver (L-) type subunit and a small amount of L-type is present in brain. There is no L-type of phosphofructokinase present in muscle and heart tissue.     

Human alpha2-macroglobulin and its antitrypsic and antithrombin activities in serum and plasma.

alpha2-Macroglobulin level, trypsin protein esterase and progressive antithrombin activities were measured in normal and nephrotic sera and plasma. Trypsin protein esterase activity was proportional to the alpha2-macroglobulin concentration in serum and plasma from both normal and nephrotic patients. The results were different, however, with progressive antithrombin activity: in normal plasma, antithrombin III is the main thrombin inhibitor, then alpha2-macroglobulin and alpha1-antitrypsin, whereas in nephrotic syndrome patients, alpha2-macroglobulin is the main thrombin inhibitor.     

Determination of bile acids in serum by capillary gas-liquid chromatography.

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete chromatographic separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter x 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20,000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr.125, 471--485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181--187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 mumol/l in males, and 1.0, 0.8, 1.4 and 0.2 mumol/l in females.     

Enzyme immunoassay for total oestrogens in pregnancy plasma or serum.

We have developed an enzyme immunoassay for total oestrogens in pregnancy serum of plasma, using horseradish peroxidase (HRP) as the marker enzyme. A test combination consisting of an antiserum against oestriol-16/17-monosuccinyl-albumin and oestriol-16/17-monosuccinyl-HRP yielded a sensitive system, which reacted to approximately the same extent with oestrone, oestradiol, oestriol and their 16- and 17-conjugates. Samples had to be diluted 1 to 10 to avoid interference of plasma factors with the immune reaction. Bound/free separation was achieved with the double antibody solid phase (DASP) method. The HRP activity of the bound fraction was measured, after washing, to eliminate plasma factors disturbing the HRP reaction. The detection limit of the assay system was approx. 0.1 pmol/tube, while the index of precision lambda ranged from 0.02 to 0.06. To measure total oestrogens, including the 3-conjugated ones, we used an enzymatic hydrolysis with an extract of Helix pomatia. Hydrolysis was found to be optimal after 1 h at 50 degrees C and pH 5.0. The method was used on serum samples from normal pregnancies. The results showed a very good correlation (r=0.98) with those obtained by radioimmunoassay. Normal values for total oestrogens during pregnancy were determined in a multicentre clinical trial.     

Determination of deuterium-labeled phenylalanine and tyrosine in human plasma with high pressure liquid chromatography and mass spectrometry.

A method is presented for the recovery of deuterated phenylalanine and tyrosine from human plasma. Phenylthiohydantoine derivatives are formed (Edman reaction) which are separated and isolated by high pressure liquid chromatography. The relative concentration of the deuterated amino acid is determined by mass spectrometry. The results obtained from a healthy person after oral loading with 40% monodeuterated L-phenylalanine are presented. The method appears to be suitable for in vivo studies of phenylalanine metabolism in humans.     

Determination of serum triglycerides by mass fragmentography

A mass fragmentographic reference method for determination of serum triglycerides is described. A fixed amount of a mixture of [1,1,2,2,3,3-²H₅]glycerol tripalmitate and [1,1,2,2,3,3-²H₅]glycerol trioleate (500 nmol) is added to a fixed amount of serum (250 μ1) and extracted with Chloroform/methanol (2:1, v/v). The triglycerides are isolated by means of thin-layer chromatography. The glycerol obtained after acid hydrolysis is converted into the tri-trimethylsilyl derivative and the amount of unlabeled glycerol is determined from the ratio between the recordings at m/e 218 and m/e 222, obtained after analysis with a gas chromatograph-mass spectrometer equipped with an MID (multiple ion detector). The two ions correspond to the peak at M?90 and M?91 in the mass spectrum of the tri-trimethylsilyl derivative of unlabeled and (1,1,2,3,3-²H₅)-labeled glycerol. The relative standard deviation of the method in the range 0.4–5.8 mm 01/1 was 2.4%. A fully enzymatic routine method for determination of triglycerides was compared with the mass fragmentographic reference method. There was a good correlation between the two methods (r = 0.995) and the regression coefficient was 1.19.     

An enzymatic assay for available zinc in plasma and serum

A convenient enzymatic assay for available zinc in plasma and serum was developed. The assay is based upon the reactivation of the zinc metalloenzyme alkaline phosphatase which had been previously inactivated by nitrilotriacetic acid. The enzymatic assay was applied to serum samples from human subjects in a study of zinc utilization in pregnancy and to plasma samples from rats in a zinc deficiency study. Most plasma samples with low zinc levels (less than 70 μg/dl) reactivated the enzyme to less than 30% of control activity. Most samples with normal zinc levels (70–120 μg/dl) reactivated the enzyme 30–65%. Samples with elevated zinc levels (> 120 μg/dl) reactivated the enzyme over 70%. The assay is quick, convenient, and in principle measures functionally available zinc.     

Determination of pregnancy zone protein in serum using an automated immunoprecipitin reaction method.

Determination of pregnancy zone protein in serum by means of an automated immunoprecipitin reaction method and pretreatment with polyethylene glycol for reducing the serum blanks is described. Using this procedure the sensitivity of the method was greatly improved, from originally 100 mg/l to 1 mg/l. The precision "between days" was 9.9%. A positive and significant correlation to electroimmuno assay is demonstrated. In apparently healthy controls the median serum pregnancy zone protein in females was 38 mg/l (2--91 mg/l) and in males 2 mg/l (0--20 mg/l). No correlation between serum pregnancy zone protein and age could be demonstrated. In malignant diseases our results seem to confirm a relationship between increased pregnancy zone protein and spreading of the tumours. Serum pregnancy zone protein in disseminated malignancies are increased significantly compared to controls. In males 75% with disseminated tumours have elevated serum pregnancy zone protein.     

Determination of oxalates in plasma and urine using gas chromatography

A gas-chromatographic procedure for the analysis of oxalic acid is described. The procedure requires relatively small quantities of urine (1 ml) or plasma (5 ml). The procedure consists of three steps: (1) extraction of oxalic acid by acidified diethyl ether; (2) esterification by isopropanol; and (3) final analysis by gas chromatography.The oxalic acid was found to have an elution temperature of 107°C and a retention time of 14 min. The standard curve is linear up to 800 nmol. In the described condition the lower limit of detection is 20 nmol.The mean normal plasma and urine oxalate levels ($\text{x}\text{?}\text{± 2}\text{S.D.}$) were found to be 20 μmol/l ± 17.5 and 275 μmol/24 h ± 200 respectively.     

The origin of a cathode-migrating creatine kinase found in serum from a cancer patient

The origin of an atypical creatine kinase (CK, ATP:creatine W-phosphotransferase, EC 2.7.3.2) migrating cathodic to the MM position found in the serum of a cancer patient was studied. The electrophoretic mobility of the atypical CK is similar to that of the fast-moving cathodal mitochondrial CK. The relative molecular mass was estimated to be approximately 350000, and was similar to that of the fast-moving cathodal mitochondrial CK. The atypical CK reacted with anti-human mitochondrial CK antibody. It is therefore suggested that the atypical CK is of mitochondrial origin.After incubation in 2 mol/l urea, the enzyme was converted into a new form migrating to the MM position. The conversion was observed in liver mitochondrial CK but not heart mitochondrial CK. The residual CK activity after heating at 56°C for 60 s was 77%, and the apparent K_m value for creatine phosphate at 30°C was about 0.27 mmol for the atypical CK. These characteristics were very similar to those of the liver mitochondrial CK, because the data from the enzyme determined at the same time were 75% for residual enzyme activity to heat, and 0.24 mmol for apparent K_m value. Therefore liver mitochondria are suggested to be the source of the atypical CK.     

Determination of plasma 3-methoxy-4-hydroxyphenyl-glycol by pulsed electron capture gas chromatography.

An improved method is described for determining picogram quantities of 3-methoxy-4-hydroxyphenylglycol (MHPG) in plasma of humans and of other species. The method makes use of gas-liquid chromatography and electron capture detection. Low level nonlinearity of detector response was corrected by operating the detector in the pulsed rather then the customary steady state mode. Detector overloading was prevented by heat coagulation of plasma proteins and subsequent ultrafiltration. Sensitivity was significantly enhanced by utilizing a derivatizing agent carrying a higher number of electrophores. Baseline conditions are described and control values for plasma MHPG of human volunteers, Rhesus monkeys and rats are presented.     

Determination of total bile acids in serum. A comparison of a radioimmunoassay with an enzymatic-fluorimetric method

A solid phase radioimmunoassay kit method for total conjugated bile acids has been compared to an enzymatic fluorimetric method for total serum bile acids. The methods were compared with respect to: precision, cross-reactivity (molar equivalence) of different bile salts, recovery of different bile salts from serum, the reference range for a healthy population, linearity, coefficient of correlation, diagnostic effectiveness, cost and ease of assay.Both assays seemed equally capable of predicting the presence or absence of liver disease. Radioimmunoassay had little advantage over the enzymatic-fluorimetric method. Its relative ease was far outweighed by its greater cost and poorer analytical performance.     

The origin of plasma creatine kinase 1, detected in patients during and following open heart surgery

Plasma creatine kinase 1 (CK-1) was detected intra-operatively in 6 out of 6 patients and postoperatively in 15 out of 22 patients, undergoing cardiac surgery. A transient increase in plasma levels of creatine kinase 2 (CK-2) and total creatine kinase (CK-tot.) activity was observed in all patients. The disappearance rates for the 2 isoenzymes in the circulation were CK-1: Kd = 4.7 X 10(-3) min-1, and CK-2: Kd = 0.60 X 10(-1) min-3. Analysis of vessel and heart tissue showed that the saphenous vein contained mainly CK-1; high activities of all three isoenzymes were found in the parts of the heart investigated. Most probably, both CK-1 and CK-2 are liberated from injured cardiac tissue.     

An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.