Iron and ferritin concentrations in exhaled breath condensate of children with asthma.

Maintenance of iron homeostasis is of utmost importance for the respiratory system physiology and pathophysiology. Local iron deficiency or accumulation may result in particular respiratory function impairment. The aim of the present study was to find out whether iron and ferritin could be determined in exhaled breath condensate (EBC) of healthy children and children with asthma. Oxidative stress was verified by determination of EBC superoxide dismutase (SOD) activity, and the airway inflammatory process by determination of exhaled nitric oxide (F(E)NO). EBC was collected from 39 children (22 healthy children as a control group and 17 asthmatics) using an EcoScreen condenser. Iron, ferritin, and SOD were determined on optimization and validation for low concentrations. In comparison with a control group, asthma patients had a statistically significantly lower iron concentration (p = 0.0001) and higher SOD catalytic activity (p = 0.0160), with no significant difference in ferritin levels (p = 0.5252), although percentile values indicated elevated ferritin concentration in about half of asthma patients. F(E)NO values were significantly higher in the asthma group (p = 0.0047). This preliminary study demonstrated the possibility of determining iron and ferritin concentrations and SOD activity in EBC, and a significant difference in EBC iron and SOD between asthma patients and healthy children.

Superoxide dismutase, copper and zinc concentrations in platelet-rich plasma of pneumonia patients.

BACKGROUND: The aim of this study was to analyse platelet superoxide dismutase (SOD) activities (total SOD, manganese SOD and copper zinc SOD) and copper (Cu) and zinc (Zn) concentrations during the course of community-acquired pneumonia (CAP), and to compare them between patients with normal platelet count and those who have developed reactive thrombocytosis (RT). METHODS: Platelet count, SOD activities and Cu and Zn concentrations in platelet-rich plasma were measured in patients with CAP on admission and at discharge. RESULTS: Post-therapeutic platelet count increased significantly from the value recorded on admission. By the end of treatment, 42% of patients developed RT. All platelet SOD activities as well as Cu concentration were significantly lower in CAP patients than in control subjects. The initial Zn concentration was greater in CAP patients compared with controls and showed a decrease at discharge. On admission, there was no difference in all SOD activities between either subgroup with normal platelet count or subgroup with RT. At discharge all SOD activities were significantly lower in patients with RT. Also, catalytic activities of those enzymes were significantly lower in both subgroups in comparison with the initial values. Post-therapeutic Cu value was lower in patients with RT in comparison with patients having normal platelet count. Zn concentration decreased significantly at discharge when compared with the initial values only in patients with RT. CONCLUSION: The pattern of changes might be indicative of a certain role of platelets in antioxidant response during treatment in CAP patients.

Transient hyperphosphatasemia in an infant with bronchiolitis and pneumonia.

We present a case of benign transient hyperphosphatasemia in a 4-month-old infant with acute bronchiolitis and pneumonia. During hospitalization the infant had an increased catalytic activity of alkaline phosphatase (ALP): day 2, 5074 U/L; day 3, 5622-U/L; and day 8, 3129 U/L. The x-ray, leukocytosis, and C-reactive protein findings pointed to bacterial etiology of the respiratory disorder. Electrophoretic separation revealed an atypical isoenzyme profile: fast anodal, near-cathodal and bone fractions. ALP levels normalized within 54 days, and control electrophoresis indicated normal liver, placental/placental-like, intestinal and bone isoenzymes. The appearance of atypical fast anodal and near-cathodal fractions of ALP in this infant during the course of acute lower respiratory tract infection and rapid return to the reference intervals pointed to benign transient hyperphosphatasemia.

COPD: magnesium in the plasma and polymorphonuclear cells of patients during a stable phase.

Magnesium is one of the most important factors for regulation of inflammatory response as well as muscle function, and COPD is a multicomponent disease characterized by abnormal inflammatory response of the lungs with systemic muscle dysfunction. Because polymorphonuclear (PMN) cells are significantly represented in the pathogenesis of COPD, concentrations of total (tMg) and ionised magnesium (iMg) were determined in plasma and isolated PMN cells in 46 patients in stable phase of COPD (past smokers, current smokers, and non-smokers), 24 healthy smokers and 37 healthy non-smokers. In the same samples concentrations of total (tCa) and ionised calcium (iCa) were determined, due to the antagonism of magnesium towards calcium. We found decreased biological active iMg in PMN compared to the group of healthy non-smokers (5.42, 1.98-17.31 micromol/10(9) cells vs. 7.50, 3.27-15.15 micromol/10(9) cells, p < 0.05). In the plasma and isolated PMN of the patients the ratio of total calcium/total magnesium (tCa/tMg) was significantly increased (2.89, 2.15-3.86 and 1.19, 0.07-9.87) compared to the group of healthy non-smokers (2.65, 2.19-3.44 and 0.67, 0.14-2.40, p < 0.05) and to the group of healthy smokers (2.58, 2.26-3.24 and 0.66, 0.14-2.85, p < 0.05). In the group of patients the concentration of tCa was significantly increased in all samples compared to the healthy group of non-smokers and healthy smokers. The results of univariant logistic regression analysis for smoking, concentration of tCa and ratio of tCa/tMg in PMN showed high odds ratio for COPD status. These results raise a possibility that intracellular polymorphonuclear value of magnesium could be a distinctive marker for COPD risk disclosure among smokers.

Magnesium concentration in plasma, leukocytes and urine of children with intermittent asthma.

BACKGROUND: Magnesium (Mg) is involved in numerous physiological functions, including protein folding, intracellular signalling and enzyme catalysis. It acts as a smooth muscle relaxant. We decided to test changes of total Mg concentration in plasma, leukocytes and urine of 16 healthy children and 26 patients with intermittent asthma aged 3 to 14 years. METHODS: Samples were taken on the first day of broncho-obstruction and five days after the acute attack. During this period, patients were under salbutamol therapy. RESULTS: Plasma Mg concentration in patients increased by about 40% (first day 0.58+/-0.05 mmol Mg/l and five days later 0.64+/-0.04 mmol Mg/l) compared with healthy children (0.42+/-0.04 mmol Mg/l). Leukocyte Mg concentrations showed significant changes. On the first day of broncho-obstruction, Mg in leukocytes significantly decreased by about 60% (1.16+/-0.31 mmol Mg/g protein) compared to healthy children (3.04+/-0.68 mmol Mg/g protein). Five days later, Mg values significantly increased (3.28+/-1.09 mmol Mg/g protein) and almost reached the values of the healthy group. Mg concentration in urine statistically decreased by about 30% (0.55+/-0.06 mmol Mg/mmol creatinine) on the first day of broncho-obstruction compared to healthy children (0.75+/-0.05 mmol Mg/mmol creatinine). Five days after the acute attack, Mg concentration in patients' urine (0.73+/-0.07 mmol Mg/mmol creatinine) was close to values of healthy children. CONCLUSION: The results obtained indicate that the intracellular measurement of Mg concentration is relevant for estimation of magnesium concentration in the human organism. Hence, determination of Mg concentration in leukocytes may be used in evaluation of asthmatic pathology.

Studies of ochratoxin A-induced inhibition of phenylalanine hydroxylase and its reversal by phenylalanine.

Ochratoxin A (OTA) is a nephrotoxic, hepatotoxic, and teratogenic mycotoxin produced by storage molds on a variety of foodstuffs. Its chemical structure is composed of an isocumarin part linked to l-phenylalanine. Inhibition of phenylalanine hydroxylase and other enzymes that use phenylalanine as substrate is based on this structural homology. We have examined the effects of low doses of ochratoxin A on the activity of phenylalanine hydroxylase in kidney and in liver of experimental animals. Daily administration of ochratoxin A (50 microg/kg body wt, for 10 and 35 days, respectively) caused a significant reduction in the phenylalanine hydroxylase activity. Inhibition was more pronounced in liver than in kidney, although actual ochratoxin A concentration was higher in the kidney tissue. We observed an apparent increase in the affinity of phenylalanine hydroxylase for substrate following OTA administration to animals. However, simple competitive inhibition was observed for both tissues in vitro (K(i liver) = 0.0119 +/- 0.002 mM and K(i kidney) = 0.13 +/- 0.026 mM). Simultaneous application of ochratoxin A with phenylalanine could reduce inhibition of phenylalanine hydroxylase, in particular in liver. Enzyme activity was almost completely preserved after 35 days of combined treatment. The results obtained suggest that daily administration of ochratoxin A in low doses produced an inhibitory effect that could be diminished by competitive action of l-phenylalanine.

Bisalbuminemia in two Croatian families.

BACKGROUND: Bisalbuminemia is a dysproteinemia characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis on cellulose acetate sheets. Bisalbuminemia may occur as a hereditary trait or as analytical interference with some drugs, especially penicillin. METHODS: Two patients with the finding of bisalbuminemia are presented. Both patients (patient 1 was a 4-1/2-month-old male infant, and patient 2 was a 15-year-old boy) were admitted for respiratory infection. RESULTS: Bisalbuminemia was detected by serum protein electrophoresis and confirmed by isoelectric focusing in pH gradient gel (pH range 4.0-6.5). This finding was supported by simultaneous detection of abnormal albumin in the mother of patient 1, while the father had normal albumin. The abnormal fast albumin in both patients had an increased relative mobility of 1.08 when measured from the sample application position. CONCLUSIONS: The results presented are the first description of albumin mutations in Croatia (that according to the CISMEL group could be classified as ZC/HZ), and present the first step in identification prior to determination of structural change and amino acid sequence in the albumin molecule.

External quality assessment in clinical chemistry: review of the situation in Croatia with particular reference to equipment.

The effect of the use of new flame photometers (Ciba Corning 480, Chiron, Salzburg, Austria) and multichannel analyzers (BM Hitachi 904 or BM Hitachi 911, both Boehringer GmbH, Mannheim, Germany) on analytical quality was assessed in a number of medical biochemical laboratories (n=58) at health centers and general hospitals that provide primary health care services for the respective catchment areas. The laboratories were supplied during 1996 and 1997 with new equipment, as part of the First Croatian Health Project, Primary Health Care Subproject, carried out by the Croatian Ministry of Health and Croatian Institute of Health Insurance. We evaluated analytical performance of these laboratories for 19 analytes, according to the results reported in the Croatian External Quality Assessment Program for medical biochemical laboratories, performed four times per year. Changes of percentages of particular methods used by the participating laboratories were observed for inorganic phosphate, total bilirubin, urea and creatinine. Such changes were even more pronounced for enzymes, where changes in the measurement procedure such as incubation temperature and buffers were observed in the methods used. Evaluation of method performance revealed that after the introduction of new equipment interlaboratory variation decreased, so that an increasing proportion of laboratories included in the Project produced results within the target limits.

Valproate and carbamazepine comedication changes hepatic enzyme activities in sera of epileptic children.

Previous observation that valproic acid (VPA) and carbamazepine (CBZ) caused hepatic damage prompted us to investigate the effects of VPA or CBZ monotherapy and VPA + CBZ comedication on the number of hepatic enzyme activities in sera of epileptic children. This study compares alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) activities in sera of children treated with VPA (n=42), or CBZ (n=36) taken as a monotherapy, with VPA + CBZ combined therapy (n=36). The effect of VPA alone is greater on the activity of AST than on other enzymes, while CBZ therapy changes primarily the activities of GGT. The mean catalytic activity of AST was significantly elevated in groups on VPA, CBZ and VPA + CBZ treatment (2.02-, 1.49- and 1.45-fold increase, respectively) as compared to the control values. Changes in the ALT activity followed different patterns. The maximal increase was observed in the CBZ group with a smaller increase in the group on VPA + CBZ polytherapy, whereas only 15% of patients receiving VPA showed an average 1.38-fold increase of the mean enzyme activity. Increase in the catalytic activity of GGT probably reflects the induction produced by the CBZ treatment, either alone or in combination. Children on CBZ monotherapy showed an increase of mean catalytic activity of about twofold in 56% of patients. Children on VPA + CBZ comedication showed a similar behaviour, while VPA alone produced a moderate (1.44-fold) increase in 23% of children. However, concentrations of VPA and CBZ in sera of patients receiving monotherapy were within the expected therapeutic limits, whereas subtherapeutic levels of VPA were found in 30% of children on VPA + CBZ comedication. We propose that individual dosage adjustment in VPA + CBZ polytherapy should be combined with monitoring of relevant enzyme activities in serum.

Influence of ochratoxin A treatment on the activity of membrane bound enzymes in rat brain regions.

Ochratoxin A is a mycotoxin produced by Aspergillus ochraceus and is a natural contaminant of mouldy food. We examined the neuroactive potential of ochratoxin A by measuring the changes in the activities of several membrane bound, cytoplasmic and lysosomal enzymes in the brain of adult female rats, following subchronic application of ochratoxin A. The activities of both soluble and membrane bound fractions of ecto-5'nucleotidase, ecto-Ca2+/Mg2+ATPase, alanine aminopeptidase, gamma-glutamyl transferase, as well as activities of lactate dehydrogenase and of N-acetyl-beta-D-glucosaminidase were followed. Biochemical effects were examined in cerebral cortex, cerebellum and hippocampus. The results obtained showed physiologically significant alterations in the activity of enzymes tested. The changes were found to be time-dependent and regionally selective. Compared to controls, statistically significant increases in gamma-glutamyl transferase were observed in all three brain regions, while in the case of alanine aminopeptidase activities differed with regard to region, the highest increase being observed in hippocampus. Ecto-Ca2+/Mg2+ATPase and ecto-5'nucleotidase showed distinct changes lasting for 20 days of treatment, while increase in the activities of N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase were visible only at the beginning of the treatment. By the end of the trial the activities of almost all enzymes returned back to normal values.

Effect of sodium valproate on renal cell brush-border enzymes in rats.

The effect of sodium valproate (200 mg/kg body weight) on renal cells was investigated during a 10-day trial, by determining the catalytic activities of alanine aminopeptidase, gamma-glutamyltransferase and alkaline phosphatase in the membrane cell brush-border of the renal proximal tubules. Four days after the administration of sodium valproate, a significant increase was observed in the volume of urine (9.2 +/- 4.2 ml/18-h volume; control group, 3.06 +/- 1.8 ml/18-h volume), and in the catalytic activities of gamma-glutamyltransferase (2.69 times that measured in the control group) and alkaline phosphatase (3.02 times that measured in the control group) in urine. After prolonged treatment the urine became alkaline (pH = 10.5 on day 10), thereby excluding alanine aminopeptidase as a useful indicator of renal cell changes following the administration of sodium valproate. The activities of alkaline phosphatase and gamma-glutamyltransferase in isolated vesicles of renal brush-border membrane cells were significantly increased (p < 0.05) and decreased, respectively. On the basis of the results obtained, we believe that the determination of the catalytic activities of gamma-glutamyltransferase and alkaline phosphatase in urine might prove useful for the follow-up of the renal cell state during therapy with sodium valproate.

Ochratoxin A impairs activity of the membrane bound enzymes in rat pancreas.

Ochratoxin A is a mycotoxin produced by Aspergillus ochraceus and is a natural contaminant of moldly food. Ochratoxin A has a number of toxic effects, some of which may be related to the changes in the cell membrane. We measured the activities of 5 pancreatic, membrane bound enzymes in female Fisher rats that were given low oral doses of ochratoxin A (120 micrograms/kg body weight per day) during 20-35 days. The amount of toxin corresponds to 1.5 mg/kg in the feed, daily. These doses are in the range of natural contamination found in feed. The enzymes studied were alanine aminopeptidase, alkaline phosphatase, ecto-Ca2+/Mg(2+)-ATPase, gamma-glutamyl transferase and ecto-5'-nucleotidase. Treatment lasting 20 days caused a strong decrease in the activity of alanine aminopeptidase, Ca2+/Mg(2+)-ATPase and alkaline phosphatase to 0.76 +/- 0.04, 0.53 +/- 0.03 and 0.30 +/- 0.02 of the control values, respectively (p < 0.05). No significant changes in the activity of gamma-glutamyl transferase and 5'-nucleotidase were observed. However, activity of alanine aminopeptidase returned to normal values after 35 days of treatment, suggesting an adaptation of the organism, or a substitution of a released enzyme. Activities of alkaline phosphatase and Ca2+/Mg(2+)-ATPase remained significantly reduced to 0.42 +/- 0.03 and 0.52 +/- 0.04, respectively (p < 0.01). We conclude that treatment of rats with low doses of ochratoxin A resulted in reduction of the activities of the membrane bound enzymes, most probably by inducing their release, as a result of the impairment of the functional integrity of cell membranes.

Arginase, a new marker of mammary carcinoma.

Activities of arginase, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were determined in sera obtained in a group of healthy women, women with verified carcinoma of the breast, benign mastopathy, a group of patients with carcinoma of various organs and a group of patients with acute viral hepatitis. Preoperative values of serum arginase activity in patients with breast carcinoma were up to 4-fold those found in healthy women. Sensitivity of the test was 86%. After the surgery, the activity decreased abruptly during the first week and normalised within 15-30 days. In benign diseases of the breast, the activity of arginase was normal. Serum arginase activity is raised in both benign and malignant liver diseases, however, the quotients alanine aminotransferase/arginase, aspartate aminotransferase/arginase and alkaline phosphatase/arginase differ significantly. Thus, use of alanine aminotransferase/arginase quotient implies a high degree of confidence in differentiating between increased arginase activity in mammary carcinoma (alanine aminotransferase/arginase = 0.572 +/- 0.278) and high arginase activity in hepatitis (alanine aminotransferase/arginase = 12.226 +/- 1.822).

Effect of ochratoxin A on brush border enzymes of rat kidney.

Ochratoxin A was given orally at 60 microgram/kg body weight in neutral olive oil to Fischer rats for 30 days, at which time they were killed. Clinical state, weights of animals and of their organs and urea and creatinine concentrations were not affected during the exposure period. Significant increases in the activity of enzymes in urine were found: 60% increase in alanine aminopeptidase, 45% increase in gamma-glutamyl-transferase and 90% increase in alkaline phosphatase. These changes indicate early pathological changes in the kidney. Relatively small amounts of the toxin thus affect kidney membrane cells.

Multiple forms of gamma-glutamyltransferase and lipoproteins.

The gamma-glutamyltransferase isoenzyme patterns originating from human serum and homogenates of liver, kidney, pancreas and intestine in the presence and in absence of isolated lipoproteins has been studied. On the basis of these results one can conclude that the distribution of a variety of gamma-glutamyltransferase activities obtained by the electrophoresis of blood serum is not a consequence of an existence of a large number of true isoenzymes, but of increased concentrations of lipoproteins which bind to the enzyme thus causing the appearance of gamma-glutamyltransferase in the region of the appropriate lipoproteins.

Changes of activities of some transferases, alkaline phosphatase and cholinesterase in the blood of women using oral contraceptives and in vitro influence of these agents on tissular enzyme levels in rat liver.

Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, and alkaline phosphatase activities in the blood serum of women taking the oral contraceptive preparation Microgynon through extended periods were raised; the activity of cholinesterase was simultaneously reduced. In rats liver homogenates ethynylestradiol, one of the active components of Microgynon, acted as an inducer of gamma-glutamyltransferase and alkaline phosphatase while leaving aspartate aminotransferase and alanine aminotransferase unaffected, but reduced the level of cholinesterase. Norgestrel, the other active component of the preparation, suppressed the biosynthesis of gamma-glutamyltransferase and alkaline phosphatase while leaving aspartate aminotransferase, alanine aminotransferase and cholinesterase levels unaffected. A mixture of ethynylestradiol plus norgestrel in the mass proportion occurring in Microgynon produced the same effects upon gamma-glutamyltransferase and alkaline phosphatase as ethynylestradiol alone. Estradiol, the parent hormone of ethynylestradiol, lacked the inducing capability of the latter while ethynylpropargyl chloride induced gamma-glutamyltransferase and alkaline phosphatase so it was concluded the inducing effect of ethynylestradiol must be ascribed to the ethynyl radical. Progesterone, the parent of norgestrel, shared the latter's suppressive activity for gamma-glutamyltransferase and alkaline phosphatase biosynthesis, and behaved like its derivative towards the other enzymes.

Effects of neuroleptic phenothiazines on the activities of aminotransferases and gamma-glutamyltransferase in serum and liver.

Serum alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase activities were monitored in psychiatric patients receiving normal doses of phenothiazine neuroleptics over a 30-day period. The first two enzymes showed slight initial increases and a subsequent return to normal, while the third showed a slight increase. In rats, dosage levels exceeding those used in human therapy produced much larger increases in the catalytic concentrations of all three enzymes in serum (1.4, 0.7 and 0.5 above the control value, respectively), and somewhat smaller increases in the liver homogenates of these animals.