Serum adenosine deaminase activities were determined for 307 healthy controls, 105 patients with various liver diseases including acute hepatitis, chronic hepatitis, hepatoma and liver cirrhosis, and34 patients with non-liver diseases. Determination of adenosine deaminase activity was effective for diagnosing all the liver diseases examined. The normal range of adenosine deaminase was 6.8 to 19.5 U/L, with the adenosine deaminase activity increasing in the order of acute hepatitis, chronic hepatitis, and liver cirrhosis. The validity (sensitivity+specificity) values of adenosine deaminase in acute hepatitis, hepatoma and chronic hepatitis were 1.00, 1.20 and 1.20, respectively. The highest validity (1.64) was found for liver cirrhosis when the cut-off level of adenosine deaminase activity was set at 40 U/L. Consecutive measurement of adenosine deaminase activity may be useful for diagnosing various liver diseases and the cut-off activity of 40 U/L seems to be especially important for predicting liver cirrhosis.
Enteric-coated capsules containing insulin (25 U/kg) and chymotrypsin inhibitor FK-401 (10 to 40 mg/kg) were administered orally to beagle dogs. FK-401 increased the intestinal absorption of insulin dose-dependently. When 40 mg/kg of FK-401 was co-administered with insulin, plasma IRI level reached to 366 μU/ml and plasma glucose fell to 59% compared with the level before. Such an effect was observed when insulin was co-administered with water-soluble chymotrypsin inhibitors FK-317 hydrochloride and FK-448, but was not when water-insoluble or slightly-soluble inhibitors chymostatin or FK-317 maleate was used. Some acids such as citric acid, lactic acid etc. enhanced the effect of chymotrypsin inhibitor on the absorption of insulin.
We describe a simple enzymatic method based on an enzymatic cycling reaction using D-amino acid as substrate for the determination of serum pyruvate. The enzymatic cycling reaction is as follows: pyruvate in serum and D-glutamate are transformed to Dalanine and 2-oxoglutarate by the catalysis of D-alanine aminotransferase (EC. 1. 4. 3. 3). The D-alanine produced is then converted to pyruvate with D-amino acid oxidase, producing hydrogen peroxide and ammonia. From this enzymatic cycling reaction, the hydrogen peroxide produced is determined colorimetrically with 4-aminoantipyrine and phenol by the catalysis of peroxidase. The standard curve was linear up to 4.0mg/dl and the mean recovery was 95.4%. The correlation coefficient between a method using deproteination and the present method was 0.955 (r) and the regression line was Y=1.12X-0.01 (mg/dl, Y: present method, X: deproteination method). The mean value of within-run reproducibility (C.V) was 4.9%. Thus the method based on enzymatic cycling using D-amino acid provides a simple, precise and accurate method. This method can be applied not only to the assay of pyruvate but also to that of other biological materials.
Human platelet associated IgG (PAIgG) levels were determined by competitive micro enzyme-linked immunosorbent assay (ELISA) with adequate reproducibility, recovery, and liniality for the sample dilution test. The washed platelet suspensions were prepared with the phosphate buffer saline containing 0.1% bovine serum albumin, and determined the IgG levels. The ELISA was done with 96 well microplate coating a purified human IgG. The horseradish peroxidase conjugated anti-human IgG antibody was incubated simultaneously with the sample, and colored with o-phenylenediamine. The normal PAIgG range (Mean ±SD) was 16.1±3.6ng/107P1 (n=69), and no difference was found in either sex. The levels were elevated in patients with idiopathic thrombocytopenic purpura (528.4±2132.6 ng/107) P1, n=63), aplastic anemia (243.4±262.4 ng/107 PI, n=23), iron deficiency anemia (19.0±7.4 ng/107 Pl, n=10), acute lymphatic leukemia (53.4±43.2 ng/107 Pl, n=6), acute myelogeneous leukemia (55.3±46.3 ng/107 Pl, n=8), chronic myelogeneous leukemia (61.6 ±41.4ng/107Pl, n=13), polycythemia vera (81.3±100.5ng/107Pl, n=9), and multiple myeloma (111.9±92.4 ng/107 Pl, n=10). After the storage of nine samples at 4°C for 6 days, the PAIgG level of each sample was elevated. Mean value of the nine samples was increased to 161% of that of samples stored at 4°C for a day. The mean platelet population was decreased to 50% of its beginning when the five platelet suspensions were stored at 4°C for 6 days.
We developed a kinetic method for measuring the activities of adenosine deaminase (EC 188.8.131.52, adenosine aminohydrolase: ADA) isozymes, based on their different Km values for adenosine as substrate. ADA activity was measured using two different concentrations of substrate; high (20 mmol/l) and low (0.5 mmol/l) concentration solutions of adenosine. This kinetic method dose not require gel filtration, and is thus a relatively simple and rapid procedure. Serum ADA is separated by gel filtration into two types of isozymes which have been called ADAi (small form; 35,000 daltons and large form; 280,000 daltons) and ADA2 (intermediate form; 100,000 daltons). ADA2 shows markedly different kinetic properties from those of ADA1; ADA2 has a higher Km value for adenosine and a lower deamination activity for 2'-deoxyadenosine than ADA1. Analytical recoveries of ADA isozymes by the kinetic method varied from 93.3 to 116.3%. Results of the present method correlated well with those of the gel filtration method in high-performance liquid chromatography. In clinical application, increased serum ADA2 activity was observed in chronic liver diseases, such as chronic hepatitis, liver cirrhosis, and hepato-celluler carcinoma. However, serum ADA1 activity did not increase significantly in only a few of many cases chronic liver disease. Studies on serial measurements of serum ADA isozymes in patients with liver disorders have demonstrated that the changes of ADA1 activity are similar to those of aspartate aminotransferase and alanine aminotransferase activity, but the changes of ADA2 activity differ. These results suggest that measurement of serum ADA isozymes activities by the present method is useful for testing liver function.
A high performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of bilirubin fractions using a column packed with octadecyl silane-bonded wide pore silica and a new elution system which consists of acidic methanol, dimethylsulfoxide and tetra-n-butylammonium hydrogen sulfate. This procedure does not need any prior treatment of serum unlike the method of Lauff et al., in which the deproteinization of globulin with sodium sulfate was required before chromatography. Human serum bilirubin was resolved into four fractions by linear gradient elution, in which the concentration of methanol was raised. The elution profile of serum bilirubin showed the diglucuronide bilirubin as the earliest eluting peak, followed in order by the monoglucuronide, serum albumin-bound bilirubin (delta fraction of bilirubin) and finally, unconjugated bilirubin. A single column has been used for more than 300 injections of serum samples without serious degradation of resolution or efficiency.
Two site immunoradiometric assay for human prolactin was developed by using 2 highly specific monoclonal antibodies. Although the procedure is simple one step sandwich assay with 3 hours of incubation time, there was no high-dose hook effect up to 84 μg/ml of prolactin. The intra- and interassay precision (CV%) were less than 6.4% ranging from 8.0 to 161 ng/ml and less than 3.4% ranging from 6.2 to 171 ng/ml, respectively. The good accuracy was also confirmed by recovery study (98.5±4.9%) and the good linearity on dilution study. The assay presented here showed a good correlation with a commercially avarable kit but 3 of 16 patients with hyperprolactinemia showed higher prolactin levels measured by the presented assay than by a commercial kit. From the results of Sephadex G-100 gel chromatography, the discrepancy of prolactin levels in those three patients could be explained by the increase of big-big and/or big prolactin.
Effects of heavy metals and alkaline earths on serum cholinesterase (pseudocholinesterase, pchE, EC 3. 1. 1.8) were studied. Eight metal ions (10-40 mM) of Ca++, Mg++, Ba++, Mn++, Co++, Cr+++, Cd++and Cu++ stimulated or inhibited the pchE activity. Changes in Ca++ and Mg++ around a physiological levels (2.0-5 mM) also influenced the pchE actvity. The influences (inhibition or stimulation) on the pchE activity by seven metal ions except of Cr+++ were relaxed by pooled human serum or serum protein such as albumin, α-globulin or γ-globulin. Ultrafiltration of the pchE solution which had been stimulated with Ca++ or Mg++ eliminated the metal ion and non-filtered pchE solution showed an original enzyme activity, suggesting a loose binding of metal ion with the pchE molecule and a reversible metal-induced influence. The present results suggest that changes in Ca++ or Mg++ level in the body fluid containing relatively poor proteins may influence the pchE activity in clinical examination.
When patients with chronic renal failure were treated with long-term hemodialysis, serum cadmium and lead levels were increased and chromium was decreased. The change in serum metal levels after a single treatment of hemodialysis was measured. The results are as follows: 1) Serum cadmium and lead values increased during a single hemodialysis but the levels per serum protein were unchanged. The change in serum cadmium and lead levels during the treatment depended on each metal concentration in dialyzing fluid. 2) When the dialyzing fluid was sampled periodically, cadmium and lead levels in the dialyzing fluid were shown to decrease throughout the treatment. These results show that serum cadmium and lead do not flow from the dialyzing fluid into the blood, but rather from the blood to the dialyzing fluid. 3) The chromium levels and chromium per protein ratio in serum tended to decrease during a single hemodialysis treatment. The chromium level in the dialyzing fluid increased after treatment, peaking 1 hour after the treatment. It is hypothesized that chromium was excreted from the serum to the dialyzing fluid. Serum chromium levels in the patients with the long-term hemodialysis treatment were similar to that in diabetic patients. Therefore, it is supposed that the metal levels in the dialyzing fluid control the serum metal levels and that cadmium and lead accumulate in the patients undergoing long-term ihemodialysis causing toxic symptoms.