Use of the Romanian product Silimarina in the treatment of chronic liver diseases.

The Romanian product Silimarina (synonym Legalon) was administered in a randomized double-blind trial, to a group of 180 patients with chronic persistent hepatitis (CPH), chronic active hepatitis (CAH) and hepatic cirrhosis (HC). The trial lasted for 40 days. The results showed favourable effects similar with those obtained with other preparations produced by foreign drug industries. The Romanian product proved to have no toxic effect. The authors discuss the present possibilities of estimating the evolution of chronic liver disease.

Variations of zinc, calcium and magnesium in normal subjects and in patients with neoplasias.

The serum zinc, calcium and magnesium concentration as well as the possible relationship between these elements was determined by means of atomic absorption spectrophotometry in 150 patients with prostatic, lung, liver and breast neoplasms as well as with chronic leukemia, and in 80 normal subjects. These determinations showed that zinc concentration is increased in almost all the forms of cancer except prostatic and lung cancer in which the zinc values were below the lowest normal limits. From this study it also resulted that high zinc values favour the development of cancer. The antagonic relationship between zinc and calcium or magnesium in neoplasias proves the protective role played by the latter elements in the forms of cancer in such cases.

Study of the relationship between the granulocyte LDH, alkaline phosphatase and Zn at the level of the leukocyte in patients with chronic myeloid leukemia.

Mixed leukocyte suspensions were prepared from heparinized blood collected from healthy subjects and from patients with chronic myeloid leukemia (CML). In all the suspensions determinations were made for: zinc, by atomic absorption; granulocyte alkaline phosphatase (GAP), using the method with p-nitrophenylphosphatase; granulocyte LDH, by means of the enzymatic autoanalyser LKD 8,600. In the patients with CML, the values of zinc and of granulocyte alkaline phosphatase activity were very low while the granulocyte LDH values were higher than normal. The chromatogram of the granulocyte LDH isoenzymes on DEAE-Sephadex A50 minicolumn (0.5 X 12 cm) showed an "alpha type abnormality" revealed by the increased activity of the isoenzymes with high electrophoretic mobility LDH2 and LDH1 specific for tissues with intense oxidative phosphorylation. In the normal subjects the chromatogram of the leukocyte LDH isoenzymes showed a type M (skeletal muscle) prevalence denoting intense anaerobic glycolysis. Therefore the low zinc concentrations (0.55 micrograms mg N2 as compared with the normal 1.24 micrograms mg N2) in these patients cause the decrease of GAP activity by the lack of zinc in the active center of the enzyme and the decrease of cellular permeability thus allowing the extracellular release of granulocyte LDH.

The metabolic behaviour of the cellular populations in the pleural or peritoneal effusions from patients with malignant diseases.

Complex morphologic, biochemical and cyto-enzymochemical investigations have been performed in the pleural or peritoneal effusions which occurred in 80 patients with various neoplastic diseases (pleural, pulmonary, hepatic, genital or breast cancers). As shown by the morphologic and biochemical tests, the proportion of fluids with a malignant or benignant character varied widely in the different forms of neoplasias. In the same effusions, the authors have studied the metabolic behaviour of acid phosphatase activity, the glycogen and nucleolar RNA amounts in the lymphocytes, in the malignant cells and in the mesothelial ones. High values of these cyto-enzymochemical components were found in the lymphocytes and the malignant cells from fluids with a positive malignant character, although similar changes were also observed in many cells of the effusions with morphologically negative reactions, and in a lesser proportion in those from patients with adenocirrhosis. It is concluded that such investigations could contribute to a better understanding of the metabolic behaviour of the cells from neoplastic effusions thus increasing the possibility of detecting the malignant character of the cells in the pleural or peritoneal effusions with a morphologically negative character.

The metabolic behaviour of the lymphocytes from serous effusions in various benign internal diseases.

The functional status of the lymphocytes from pleural or peritoneal effusions occurring in 148 patients with various internal diseases such as tuberculosis, liver cirrhosis, heart failure and pneumonias was studied by laboratory investigations including morphologic examination, cytoenzymatic, cytochemical and biochemical tests for determining the scores of acid phosphatase activity and of endolymphocytic glycogen, as well as the presence of nucleolar RNA. These values were found increased in the fluids from tb patients and in some cases of liver cirrhosis, heart failure and metapneumonic pleurisies. The variations of these parameters are assumed to provide information on the metabolic behaviour of the lymphocytes present in these fluids and on their participation to the immune, inflammatory processes which occur in the course of some of the diseases investigated.

Alkaline phosphatase activity improved by the addition of zinc in granulocytes from leukemic subjects (in vitro study).

Granulocyte alkaline phosphatase (GAP) was extracted from the leukocyte suspensions of 15 subjects with chronic myeloid leukemia (CML), determining the Zn content by atomic absorption spectrophotometry. The purified enzymatic preparation was dialyzed against a ZnCl2 solution (.01 mg%) in TRIS-HCl 0.1 M, pH 7.5, for 48 hours. After dialysis Zn concentration and GAP activity reached normal values. This suggests that the GAP molecule isolated from CML subjects is not altered as it is able to receive at most 2 Zn moles responsible for the enzymatic function.

Aspect of the evolution of viral hepatitis two years after the acute phase.

A group of 214 nonselected former patients with viral hepatitis of which 15 HBsAg negative and 62 HBsAg positive, was studied prospectively by following the evolutive variations between the first and second year after the acute phase of disease. In both groups the percentage of cured cases increased moderately but the evolutive process toward the chronic stage was observed to reappear in some of the cases apparently cured thus proving that the process of becoming chronic is not completed within 1 year after the acute phase. The percentage of chronic cases after HBsAg positive hepatitis is clearly higher than after HBsAg negative hepatitis the difference being more evident in active chronic hepatitis. The risk of becoming chronic is greater after HBsAg positive viral hepatitis with prolonged clinical forms and the HB antigen also persists longer in the serum. In conclusion the necessity of clinical and biologic control of all former patients with viral hepatitis for at least 2 years after the acute phase is strongly emphasized.

The relationship zinc--alkaline phosphatase activity in normal and pathologic granulocytes.

To study the relationship granulocyte alkaline phosphatase (AP)--zinc (Zn) in mixed leukocyte suspensions, the enzyme from 51 mixed leukocyte suspensions was separated and purified. In the pure extract, determinations were carried out for enzymatic activity--using the autoanalyzer LKB 8600--and for Zn content--using atomic absorption spectrophotometry. From the data obtained it resulted that, in agreement with the results in the literature Zn concentration in the mixed leukocyte suspensions from chronic myeloid leukemia, (CML) patients is about half that in the normal controls (0.65 +/- 0.15 as compared with 1.24 +/- 0.15 micrograms/mg total nitrogen). Granulocyte AP separated from normal leukocyte suspensions was found to bind about 95% of the total Zn content, i.e., 1.21 +/- 0.15 micrograms/mg total nitrogen and its enzymatic activity was about 16 U/g total nitrogen. In CML patients the enzyme bound a mean Zn concentration of 0.55 +/- 0.15 micrograms/mg total nitrogen and its mean activity was 2.25 U/g total nitrogen, i.e., about 86% of the normal values found in the controls. The enzymatic activity decreased to zero after only 20 min dialysis with 8-hydroxyquinoleine-5-sulfonic acid (8HC5S) as compared with the 60-80 min in the controls. It can be concluded that the leukemic leukocyte is unable to form the metal-enzyme complex owing to the deficit of the Zn involved in the enzymatic activity.

Separation of creatine-kinase isoenzymes on ion-exchange mini-columns for diagnostic purposes.

For diagnostic purposes, creatine-kinase (CK) isoenzymes were separated on mini-columns of DEAE Sephadex A50 from the serum of 55 patients with myocardial infarction or cerebral disturbances and from 45 normal subjects. The optimum conditions of separation were determined by separation of CK isoenzymes (CK--MM, CK--MB, CK--BB) from human organ homogenates (skeletal muscle, myocardium, brain). In the patients with myocardial infarction the CK-MB isoenzyme activity amounted to more than 6% of the total enzyme value. The chromatogram in these cases was similar to that of human myocardium homogenate. The CK-BB isoenzyme was found only in patients with severe cerebral disturbances (cerebral hemorrhage). In such cases the chromatogram was similar to that of human brain homogenate. Therefore, by associating the assay of serum CK total activity with the separation of CK isoenzymes it was proved that the CK--MB and CK--BB isoenzymes can supply a useful test in differential diagnosis.

Separation and purification of LDH and alkaline phosphatase isoenzymes for the improvement of organo-specific diagnosis.

The LDH isoenzymes from mixed granulocytic populations and from homogenates of different organs, as well as alkaline phosphatase from human liver and intestine, were separated and purified by means of chromatography using ion exchangers (DEAE-cellulose and DEAE-Sephadex A50). Comparison of purified LDH isoenzyme chromatograms with the zymograms in agar gel has allowed the determination of the isoenzymatic pattern of the respective tissues and its correlation with the predominant energetic processes. The purified extracts of alkaline phosphate have lead to the characterization of the main molecular forms of the enzyme thus enabling a more precise organo-specific diagnosis.

Circadian variations of certain blood components currently investigated.

The circadian variations of serum components such as: cholesterol, LDH, GOT, alkaline phosphatase, proteins, bilirubin, ferrous ions and creatinine were investigated. Blood collection was made two times in 24 hrs.: 5-6 p.m. and 7-8 a.m. on the next day. The data obtained allowed the classification of the subjects into two groups: stable and variable. The most interesting observations were: evening decrease of cholesterol, evening increases of LDH with a tendency of intensification of the LDH5 fraction in the zymogram and evening decreases of ferrous ion.

Study of serum intestinal alkaline phosphatase isoenzyme in correlation with the ABO blood group system and secretory status in ulcer patients.

Serum alkaline phosphatase (AP) electrophoretic phenotype and the level of its activity inhibited by l-phenylalanine 5 mM (the stereospecific inhibitor of the intestinal AP isoenzyme) were investigated in 312 subjects (132 healthy controls, 89 patients with duodenal ulcer, 31 with gastric ulcer and 60 family members of the duodenal ulcer patients) in correlation with the ABO blood group system and secretory status. In the control subjects, those with A(II) blood group showed a predominance of the p degrees electrophoretic phenotype while phenotypes p+ and p++ were more frequent in the controls with O(I) and B(III) blood groups and in the secretory ones. In the patients with duodenal ulcer and their family members the frequency of phenotypes p+ and p+ was significantly higher than in the controls. The same distribution of frequencies was observed for the level of AP activity inhibited by l-phenylalanine. The results obtained are discussed in the light of a possible genetic linkage.

Attempts to improve the early enzymatic diagnosis of coronary heart disease.

Based on the hypothesis that beta lipoproteins have the ability to trap certain enzymes, an attempt was made to release these enzymes from the eventual beta lipoprotein blockade, by subjecting the serum to different processings such as: freezing-thawing (3 times), ultrasonation or treatment with Triton X100 or sodium desoxycholate. After the first two procedures an increase of serum LDH and alkaline phosphatase activities was observed in about half the sera investigated (normal and pathologic). In few cases of coronary heart disease (two out of 60), LDH activity reached a clearly pathologic level.

Platelet energetic enzymes.

The LDH isoenzymic pattern was studied in platelet cytolysates from man and rabbits, and the two forms of ATP-ase-the Mg++ and the Ca++ activated ones--were assayed in human platelet cytolysates. In man, the platelet LDH zymogram was of the intermediary type. Some of the zymograms presented 4 fractions others 5; in all patterns the H/M subunits ratio was above 1. In rabbits, the platelet LDH zymogram was of the anodal type with only 4 bands; the H/M subunits ratio showed the same variations as in man. Of the two forms of ATP-ase, the Mg++ activated one was dominant in the platelet cytolysates studied.