Enzyme Activity In Leukocytes Research Articles

Biochemical studies on leukocyte and fibroblast human β-galactosidase

Objectives: Some biochemical characteristics of the human leukocyte and fibroblast β-galactosidase were studied. Design and Methods: Leukocyte and fibroblast enzyme activity was determined fluorometricaly using 4-methylumbelliferyl-β-D-galactoside as artificial substrate. Optimum pH, K m, V max and thermostability of the enzyme at 42 °C were determined. Results: The leukocyte and fibroblast enzyme has an optimum pH at 4.2, which is in agreement with the lysosomal origin of the enzyme. The K m of the enzyme was 0.62 in leukocytes and 0.67 in fibroblasts, and V max was 289.9 nmol/h/mg of protein and 1779.2 nmol/h/mg of protein in the two tissues, respectively. When fibroblast or leukocyte β-galactosidase was pre-incubated at 42 °C, it did not retain its activity because the residual activity after 80 minutes of pre-incubation at this temperature was lower than 30% of the initial activity both in leukocytes and fibroblasts. Conclusions: This was the first study of K m, V max and thermostability of β-galactosidase performed on leukocytes and provided data for a better characterization of the enzyme β-galactosidase, allowing the improvement of the analytical conditions.

Thymidylate synthase activity in leukocytes from patients with chronic myelocytic leukemia and acute lymphocytic leukemia and its inhibition by phenanthroindolizidine alkaloids pergularinine and tylophorinidine

Thymidylate synthase (TS) (EC 2.1.1.45) provides precursors for DNA biosynthesis through a de novo pathway and is a key target enzyme for cancer chemotherapy. TS levels of human leukemic leukocytes from patients with chronic myelocytic leukemia (CML) and acute lymphocytic leukemia (ALL) were observed to be highly elevated (66- and 33-fold for CML and ALL, respectively) compared to the usual low level of basal activity in normal healthy controls. In vitro inhibition studies on the human leukemic leukocyte TS with the phenanthroindolizidine alkaloids pergularinine (PGL) and tylophorinidine (TPD) (isolated from the Indian medicinal herb Pergularia pallida) were conducted for the preliminary screening tests for their antitumor activity. The leukemic leukocyte enzyme activity was potently inhibited by PGL and TPD (IC 50=50 μM) in both types of leukemias. These alkaloids were assessed for biological evaluation for the first time as potential antileukemic agents.

Gaucher's disease and pregnancy.

Gaucher's disease is an autosomal recessive lysosomal storage disease, resulting from a deficiency of the enzyme glucocerebrosidase, which is required for the lysosomal degradation of glycolipids. The clinical manifestations of the disease show a large heterogeneity, including hepatosplenomegaly, "bone crisis" and fracture, anemia, thrombocytopenia and, in the rarest types II and III, neurological decompensation. Type I, the most common form, usually presents with less severe symptoms and at a more advanced age. More than 30 mutations within the glucocerebrosidase gene have been recognized, and certain mutations seem to be related with a particular phenotype expression of the disease. Modern diagnosis of Gaucher's disease is performed by either determining the enzyme activity in peripheral blood leukocytes or through DNA-based analysis. Pregnancy concurrent with Gaucher's disease has several risks, including an increased severity of anemia and thrombocytopenia that can potentiate postpartum bleeding, and increased risk of infection and possibly an increased spontaneous abortion rate. Nevertheless, the majority of these pregnancies seem to proceed to term without significant complications. The effects that pregnancy might have on the course of the disease are still unresolved. Enzyme replacement therapy with alglucerase is the treatment of choice for patients with Gaucher's disease, but it is yet to be shown whether alglucerase reduces the risk of these complications during pregnancy and whether its use has any adverse effect on fetal development. We present an extensive review of the current literature regarding Gaucher's disease with special emphasis on pregnancies coexistent with this disease and, an analysis of the genetics, relevant prenatal diagnostic issues, and current treatment modalities.

Lysosomal enzyme activities in serum and leukocytes from patients with carbohydrate‐deficient glycoprotein syndrome type IA (phosphomannomutase deficiency)

From 10 patients with carbohydrate-deficient glycoprotein (CDG) syndrome due to phosphomannomutase (PMM) deficiency, out of 10 lysosomal enzymes, 7 enzyme activities were measured in serum and 9 in leukocytes. In serum there was a 2-fold to 4-fold increase in activity of beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, and arylsulphatase A. In leukocytes, however, several enzymes had reduced activity, particularly alpha-fucosidase, beta-glucuronidase and alpha-mannosidase. These abnormalities could result from missorting, defective reuptake and/or reduced stability of the enzymes due to the defective glycosylation.

Plasma polymorphonuclear leukocyte elastase levels and its relation to disease activity in psoriasis

Although some increased enzyme activities in PMN leukocytes isolated from the circulation of psoriatic patients have been previously described, none of these enzymes had been studied in plasma for determination of disease activity. The purpose of this study was to evaluate whether plasma PMN elastase levels might be a good marker for determination of disease activity of psoriatic patients. Plasma PMN elastase and some acute phase reactants including alpha-1 antitrypsin, alpha-2 macroglobulin, fibrinogen, erythrocyte sedimentation rate(ESR), and transferrin were studied in thirty-nine patients with psoriasis, including 16 controlled patients, and forty healthy control subjects. PMN elastase and the acute phase reactants except transferrin were found to be significantly higher in the psoriatic patients than in control subjects. The PMN elastase level in the patients was about 6-fold higher than that of the control group. The PMN elastase levels in the patients in the inactive period was two-fold higher than in the control subjects. PMN elastase was found to correlate significantly with PMN leukocyte count and alpha-1 antitrypsin in active and inactive periods of the disease, but with fibrinogen and psoriasis area and severity index (PASI) only in the active period of the disease. We conclude that plasma PMN elastase level may be a more specific and sensitive inflammatory marker than alpha-1 antitrypsin, alpha-2 macroglobulin, ESR, and may be a good marker for diagnosis and follow up of the disease activity of the psoriatic patients.

Glycolytic Enzyme Activities in Leukocytes of Thoroughbreds Undergoing Training Exercise.

Glycolytic enzyme activities and expression of glucokinase (GK) were investigated in leukocytes (WBC) from Thoroughbreds, racehorses undergoing continuous training exercise and riding horses. Glycolytic enzyme activities in WBC from racehorses were higher than those from riding horses. Especially activities of GK, rate-limiting enzyme of glycolysis, were significantly higher in WBC from racehorses than in those from riding horses. Analysis of reverse transcription-polymerase chain reaction (RT-PCR) with specific primer for GK showed that expression of mRNA of GK was remarkably increased in WBC from racehorses. This suggested that activities and gene expression of some kinds of glycolytic enzymes including GK in WBC are possibly a good indicator for assessing glucose metabolism in Thoroughbreds undergoing training exercise.

Type 4 Phosphodiesterase Inhibitors Have Clinical and In Vitro Anti-inflammatory Effects in Atopic Dermatitis

Increased cyclic AMP-phosphodiesterase activity in peripheral blood leukocytes is associated with the immune and inflammatory hyperreactivity that characterizes atopic dermatitis. Atopic phosphodiesterase has high sensitivity to a variety of enzyme inhibitors, suggesting an increased therapeutic advantage. The objective of this study was to use in vitro assays to identify a potent phosphodiesterase inhibitor and then to investigate its effectiveness in treating atopic dermatitis. Leukocyte enzyme activity was measured by radioenzyme assay, whereas prostaglandin E2 and interleukins 10 (IL-10) and 4 (IL-4) were measured in 24-h culture supernatants of mononuclear leukocytes by immunoassays. The effect of a topical phosphodiesterase inhibitor on atopic dermatitis lesional skin was assessed by double-blind, paired comparisons of active drug and placebo ointments applied to symmetrically involved sites over a 28-d period. Using in vitro, assays, we demonstrated the ability of selective high-potency phosphodiesterase inhibitors to reduce prostaglandin E2, IL-10, and IL-4 production in atopic mononuclear leukocyte cultures. We selected the Type 4 phosphodiesterase inhibitor, CP80,633, based on its inhibitory potency, for clinical testing by topical, bilateral paired comparisons in 20 patients with atopic dermatitis and demonstrated significant reductions of all inflammatory parameters. Phosphodiesterase inhibitors modulate several pathways contributing to the exaggerated immune and inflammatory responses, which characterize atopic dermatitis. This in vivo demonstration of anti-inflammatory efficacy may provide a useful alternative to the over-reliance on corticosteroid therapy in atopic disease.

Lysosomal enzyme activities in skeletal muscle of patients with neuromuscular diseases

Activities of nine lysosomal enzymes and pH-dependent isozyme patterns of alpha-mannosidase were examined in the skeletal muscle of patients with neuromuscular diseases, and the ratios of these enzyme activities in leukocytes to those in myocytes (L/M ratio) were determined. The activities of enzymes with a high L/M ratio were markedly increased in the muscles of patients with Duchenne muscular dystrophy (DMD), myotonic dystrophy (MyD), or polymyositis (PM). In contrast, those which showed a low L/M ratio were increased in the muscles of amyotrophic lateral sclerosis (ALS) and disuse muscle atrophy (DUA). The isozyme pattern of alpha-mannosidase in DMD muscle resembled that in leukocytes, while those in ALS and DUA muscle resembled that in normal muscle. These results may suggest that the increased activity of lysosomal enzymes in the muscles of patients with DMD, MyD, or PM is due primarily to infiltrating leukocytes, while that in patients with ALS or DUA is due to intramyofiber lysosomes.

Diagnosis of fructose-1,6-bisphosphatase deficiency using leukocytes: normal leukocyte enzyme activity in three female patients

In the past 5 years we have discovered 8 boys and 3 girls who suffer from fructose-1,6-bisphosphatase deficiency. Although they all showed the typical symptoms of the deficiency such as frequent vomiting, hypoglycemia, lactic acidosis, and hepatomegaly, the age at diagnosis varied from 2 months to 4 years. All the boys revealed the deficient enzyme activity in leukocytes but none of the girls. The liver biopsy was investigated in six patients to confirm the diagnosis. These results suggest the existence of tissue-specific isoenzymes for fructose-1,6-bisphosphatase possibly with a different gene origin.

Orthotopic Liver Transplantation in Two Adults with Niemann-Pick and Gaucher's Diseases: Implications for the Treatment of Inherited Metabolic Disease

Two adults were seen with cirrhosis caused by different lipid storage diseases. A 42-yr-old woman with Niemann-Pick disease type B had marked hepatomegaly, ascites and recent variceal bleeding. Her evaluation showed chronic bilateral pulmonary infiltrates, multiple stigmata of chronic liver disease including the recent cessation of menses, diuretic-resistant sterile ascites, hepatic encephalopathy and variceal bleeding. Five percent of normal sphingomyelinase activity was measured in peripheral leukocytes. A 42-yr-old man with Gaucher's disease and a history of bilateral hip replacements presented with hepatomegaly, jaundice, refractory ascites and renal insufficiency. His evaluation showed 20% to 23% of normal glucocerebrosidase activity in peripheral leukocytes. Both patients underwent orthotopic liver transplantation with resolution of all aspects of decompensated liver function. Assessment of the underlying metabolic defect before and 6 to 14 mo after transplantation showed that after transplantation the patient with Niemann-Pick disease had above normal hepatic sphingomyelinase activity, a less-marked increase in peripheral leukocyte enzyme activity and lower than normal hepatic sphingomyelin and cholesterol content. In contrast, the patient with Gaucher's disease had only a 61% increase in hepatic glucocerebrosidase activity but had an elevated hepatic glucocerebroside content that was only 15% of the pretransplant level and decreased peripheral leukocyte enzyme levels. These findings suggest that variable relationships may exist between posttransplant hepatic and peripheral leukocyte enzyme activities in the different lipidoses, which may have implications for recurrence of glycolipid-induced liver damage.

Amelioration of clinical disease following bone marrow transplantation in fucosidase-deficient dogs.

Canine fucosidosis was studied as an animal model for the treatment of neurovisceral lysosomal storage disease. Following successful bone marrow engraftment, dogs with fucosidosis had increased levels of alpha-L-fucosidase enzyme activity in leukocytes, plasma, and neural and visceral tissues. This widespread increase in enzyme activity was accompanied by a rapid improvement in the peripheral nerve and visceral lesions of fucosidosis and a more gradual improvement in the central nervous system pathology. Long-term engraftment from an early age reduced the severity and slowed the progression of clinical neurological disease. Transplantation after the onset of clinical signs was not effective. These findings suggest that the neurological damage caused by some inherited metabolic disorders, such as fucosidosis, may be preventable but emphasise the need for early diagnosis and treatment.

Hereditary branching enzyme dysfunction in adult polyglucosan body disease: A possible metabolic cause in two patients

We describe 2 unrelated patients with adult polyglucosan body disease (APBD) diagnosed by sural nerve biopsy. Both patients were offspring of consanguineous marriages. They presented clinically with late onset pyramidal tetraparesis, micturition difficulties, peripheral neuropathy, and mild cognitive impairment. Magnetic resonance imaging of the brain revealed extensive white matter abnormalities in both. In search of a possible metabolic defect, we evaluated glycogen metabolism in these patients and their clinically unaffected children. Branching enzyme activity in the patients' polymorphonuclear leukocytes was about 15% of control values, whereas their children displayed values of 50 to 60%, suggesting a possible autosomal recessive mode of transmission. This is the first report of an inherited metabolic defect in patients with adult polyglucosan body disease. We suggest that branching enzyme dysfunction may be implicated in the pathogenesis of some patients with adult polyglucosan body disease.

In vivo regulation of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase: increased enzyme protein concentration and catalytic efficiency in human leukemia and lymphoma.

The activity of microsomal HMG-CoA reductase in freshly isolated leukocytes from patients with a variety of hematologic malignancies was significantly increased (up to 20-fold) when compared to enzyme activity in leukocytes from normal subjects (average 10.3 +/- 0.8 pmol/min per mg). Increased enzyme activity was not due to nonspecific leukocyte stimulation or to the presence of a malignancy, since normal enzyme activity was observed in subjects with either viral illnesses or solid tumors. Increased HMG-CoA reductase activity accompanying hematologic malignancy could also not be attributed to alterations in enzyme-substrate kinetic parameters (Km), or to alterations in the phosphorylation state or thiol-disulfide status of the enzyme, nor was it correlated with differences in serum lipid or lipoprotein concentrations. The increase (3.6-fold) in HMG-CoA reductase activity in leukocytes from patients with preleukemia was due entirely to a rise in enzyme catalytic efficiency (specific activity), whereas the increase (4.3-fold) observed in leukocytes from patients with overt leukemia or non-Hodgkin's lymphoma was due to a concomitant increase in both enzyme catalytic efficiency (2.5-fold) and enzyme protein concentration (1.6-fold). Similar increases in HMG-CoA reductase activity and catalytic efficiency were also noted for both transformed, nonmalignant, and malignant cultured leukocytes, suggesting that increased enzyme catalytic efficiency is not a nonspecific consequence of physiological changes occurring in response to the malignancy but may be an integral aspect of the malignant phenotype. HMG-CoA reductase protein concentrations, however, were not elevated in either transformed, nonmalignant, or malignant cultured leukocytes, suggesting that increases in enzyme protein levels may be secondary to other physiological changes that occur during the development of overt leukemia. Taken together, these observations suggest that an increase in the activity of HMG-CoA reductase, the rate-controlling enzyme in cholesterol synthesis, is a common occurrence in human hematologic malignancies and that a biphasic elevation of enzyme activity may exist in malignant leukocytes, such that changes in catalytic activity may occur early in tumorigenesis and may be followed by secondary changes in enzyme levels.

Hepatic phosphorylase b kinase deficiency with normal enzyme activity in leukocytes and erythrocytes

Hepatic phosphorylase b kinase deficiency with normal enzyme activity in leukocytes and erythrocytes

Leukocyte and plasma N‐laurylsphingosine deacylase (ceramidase) in Farber disease

Severe deficiency of acid ceramidase activity (4-5% of normal) was demonstrated in cultured skin fibroblasts, leukocytes and plasma from a 1-year-old boy who was diagnosed as being affected with Farber disease. Determination of ceramidase activity in plasma was achieved by a highly sensitive assay employing a ceramide substrate containing radiolabeled C12 N-acyl moiety (N-lauryl). The enzyme activity in the parents' leukocytes and plasma was found to be reduced to 18-47% of the respective normal values, and that determined in a plasma specimen from a patient with I-cell disease was about 4 times elevated above the normal level.

Long-term follow-up of biochemical and cognitive functioning in patients with mannosidosis.

Longitudinal assessments of three brothers with alpha-mannosidosis were performed biochemically by determining levels of leukocyte enzyme activity, and neurodevelopmentally by testing of general intelligence, language, visual spatial skills, and overall adaptive abilities. During the follow-up examination, enzyme activity was assessed in fibroblasts to evaluate the uniformity of biochemical deficits. The biochemical findings demonstrated profound deficits of leukocyte alpha-mannosidase that remained remarkably stable over time and were very similar to levels of the same enzyme activity in fibroblasts. The cognitive findings showed that the patients manifested mild cognitive deficits. Cognitive deficits were generally uniform with no signs of progressive deterioration, except receptive language abilities. Suggestions are made for careful follow-up of auditory abilities in patients with mannosidosis.

The effect of infection on leukocyte and serum lysosomal enzyme activities in protein-calorie malnutrition

Representative leukocyte and serum lysosomal enzymes, namely B-glucuronidase, acid phosphatase and hexosaminidase, were studied in 53 protein-calorie malnourished children and 26 controls. The leukocyte lysosomal enzyme activities were elevated in non-infected malnourished children while significant decreases in these enzymes were observed in the leukocytes of the infected malnourished group whose serum lysosomal hydrolase levels were also significantly elevated. These observed alterations in serum and leukocyte hydrolase activities may partially explain the impaired leukocyte microbicidal activity and the increased propensity for the development of endotoxic shock in children with protein-calorie-malnutrition.

Hepatic phosphorylase b kinase deficiency with normal enzyme activity in leukocytes

Glycogen phosphorylase b kinase (EC 2.7.1.38) is responsible for the activation, by phosphorylation, of phosphorylase b to phosphorylase a. Deficiency o f this enzyme complex in man (McKusick 30600) was first documented by Hug et a12 Three girls and two boys were found to be deficient in PBK activity in liver homogenate. These and subsequent patients having an autosomal recessive mode of inheritance of PBK deficiency, were found clinically tO have moderate hepatomegaly, variable muscle weakness, normal growth and development, and normal response to glucagon. 2 Another group of patients, all boys, were found to have varying hepatomegaly, fasting hypoglycemia, a reduced response to glucagon, and retarded growth and development with an X-linked pattern of inheritance2 Patients with the autosomal recessive form of PBK deficiency ~'iJave 20% to 30% of normal control PBK activity in erythrocytes, leukocytes, and liver. 2 Male patients with the X-linked form of the deficiency have almost no PBK activity in liver, approximately 20% in leukocytes, and normal activity in muscle. 2 We report a patient with reduced liver homogenate and erythrocyte lysate PBK activity with normal leukocyte and muscle homogenate enzyme activity.

The significance of cytochemical studies of leukocytes in the diagnosis and evaluation of the effectiveness of treatment of acute purulent destructive pneumonia in pediatric practice

To assess diagnostic significance of enzyme activity in peripheral blood leukocytes when determining individual stages of acute purulent destructive pneumonia in children we performed cytochemical studies in 52 patients aged from one month to 10 years. In 14 children the disease was infiltrative, in 38 - destructive. The control group consisted of 15 healthy children of the same age.

Changes in host cell membrane activities in response to adhesion of Neisseria gonorrhoeae

Physiological changes in host cell model membranes (intact human erythrocytes and ghosts) as a consequence of bacterial adhesion were studied with special reference to Neisseria gonorrhoeae. Membrane activities examined were transport of K+, Cl- ions, pyruvate kinase, Na-K-dependent ATPase, and cAMP. We found that K+ and Cl- transport were affected, more so in membranes with attached pilated (P+) organisms than in those with apilated (P-) isogenic strains. In N. gonorrhoeae and in several other species of gram-negative bacteria studied, hemagglutination titres were directly correlated with effects on anion transport, suggesting that perturbations in anion transport are an immediate result of adhesion. Of three P+ gonococcus strains tested, two depressed Na-K-ATPase activity in the membrane, indicating a possible effect on the Na-K pump. Pyruvate kinase activity associated with the membrane appeared to be stimulated by attached gonococci, again by P+ strains to higher levels than P- organisms. Clearly, some enzyme properties of host membranes are intrinsically affected by bacterial adhesion. Human polymorphonuclear neutrophils were also investigated, and with some exceptions, changes observed in leukocyte enzyme activities tended to parallel those in erythrocytes. Since hypochlorous acid production is considered to be an important microbicidal mechanism in neutrophils, interference with Cl- transport could jeopardize their role in host defense.