Enzyme Activity In Leukocytes Research Articles

Lysosomal enzyme activity in pulmonary alveolar macrophages, peritoneal macrophages, and blood mononuclear leukocytes in the hypothyroid rat

Chronic hypothyroidism can result in an increased susceptibility to severe pulmonary or genitourinary tract infections. We postulate that decreased lysosomal activity in the pulmonary alveolar macrophage (PAM), peritoneal macrophage, and mononuclear leukocyte may be a factor which results in the impaired response to infections in hypothyrosis. In this study rats underwent total thyroidectomy and had their PAMs, peritoneal macrophages, and mononuclear leukocytes analyzed after 4, 8, and 12 weeks. The total lysosomal activity (postnuclear homogenate + lysosomal-rich cell fraction) of PAMs was 50% of age-matched controls. Peritoneal macrophage lysosomal enzyme activity was 25–30% of control activity. In separate experiments, rats were administered physiologic replacement of triiodothyronine (T 3) 8 weeks after thyroidectomy. This resulted in PAM and peritoneal macrophage lysosomal enzyme activity similar to control activity. Mononuclear leukocyte lysosomal enzyme activity from hypothyroid rats showed decreases in only one of the three enzymes monitored (β-glucuronidase). Hypothyroidism did result in a decrease in total number of mononuclear leukocytes harvested per animal. Administration of T 3 resulted in mononuclear leukocyte β-glucuronidase activity similar to euthyroid rats (44 ± 8 U/mg protein vs 42 ± 3 U/mg protein). In summary, hypothyroidism in the rat results in decreases in subcellular lysosomal enzyme activity in PAMs, peritoneal macrophages, and mononuclear leukocytes.

Lysosomal enzyme activities in polymorphonuclear leukocytes, macrophages, serum, and spleen of conventional, germ-free, and antigen-free Minnesota miniature swine.

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of gnotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.

Tay-Sachs disease heterozygote detection in Brazil: comparison between tears and leukocytes as beta-hexosaminidase A source.

Hexosaminidase A activities were determined in tears and peripheral leukocytes of carriers and noncarriers for Tay-Sachs Disease (TSD) as a percentage of total hexosaminidase activity. Correlation between enzyme activities in tears and leukocytes was highly significant (r = 0.75, p less than 0.01). Compared to leukocytes, screening in tears revealed 100% sensitivity, 100% negative predictive value, 86% specificity and 80% positive predictive value. These results indicate that tears are a satisfactory material for mass-screening of TSD-carrier state, but positive results must be confirmed in peripheral leukocytes. Seven heterozygotes were detected among 298 young Ashkenazi Jewish volunteers screened, giving an adjusted frequency of the TSD gene of 0.024.

Human glycerol kinase deficiency: enzyme kinetics and fibroblast hybridization.

Glycerol kinase deficiency has been associated with neuromuscular, skeletal and adrenal abnormalities and has also been seen in individuals without these clinical findings. Examination of residual enzyme activity in patients' liver, kidney, leukocytes and fibroblasts showed a generalized, heritable defect: the apparent Km for glycerol was increased 5-200-fold over control values, whereas the apparent Km for ATP was not significantly altered. This kinetic defect was similar in fibroblasts from clinically different individuals with this inborn error of metabolism. Hybridization of fibroblasts from these individuals showed no evidence of complementation for glycerol kinase activity.

Detection of fabry hemizygotes and heterozygotes by the assay of specific α-galactosldase-a in single hair roots

Fabry’s disease is an X-linked recessive disorder caused by a deficiency of lysosomal enzyme α-galactosidasc-A that results in accumulation of excess trihexosylceramide in blood vessels throughout the body which leads to corneal dystrophy, skin lesions, diffuse cardiovascular disease and renal failure. Genetic counselling requires the unequivocal identification of heterozygotes, but this is not practicable with certainty from measurement of the enzyme activity in leucocytes nor cultured skin fibroblasts. However, it has been possible to detect heterozygotes in several X-linked genetic disorders by using hair roots in which the many thousands of cells are derived from possibly single or just a few primordial cells. Therefore, and according to the Lyon hypothesis, it may be expected that a hair root would show either full normal enzyme activity, or virtually absent-mutant-activity. We have adapted our ultra-micro assay system already in use in this laboratory for detection of Hunter Syndrome heterozygotes, to the identification of Fabry carriers. The technique involves the fluorogenic assay of u -galactosidase-A (α-galactosidasc-B activity representing about 40% of the total α-galactosidase activity was inhibited by incubation in 75 nmol/l GalNAc) and the simultaneous assay (radiolabelled substrate GM 1 ganglioside) of a control enzyme activity on each of 30 50 individual hair roots. Three related hemizygotes and an unrelated fourth adult (but symptomless) hemizygote had hair root enzyme α-galactosidase-A to control enzyme ratios of virtually zero. Five female relatives of the group of three hemizygotes were studied; about 50%, of hair roots from 2 of these females showed reduced or zero activity compared to normal control subjects and their other 3 female relatives.

Biochemical Events Related to Phagocytosing Cells

Publisher Summary This chapter discusses the biochemical events related to phagocytosing cells. Phagocytosing cells include the polymorphonuclear leukocytes or neutrophils, the mononuclear leukocytes that can give rise to macrophages in tissues, and the eosinophils. Neutrophils are the most abundant phagocytes that ensure the early tissue response to infection. A decrease or increase in a number of leukocyte enzyme activities can be demonstrated in pathological situations, but the specificity of such changes and the way in which enzymes are brought about are not elucidated. The metabolic pathways are studied as multienzyme systems or by characterizing their individual enzymes. The enzymatic equipment of the polymorphonuclear cell is complete and the main metabolic pathways are present in white cells. Biochemical disorders in leukocytes may present a picture of metabolic impairment of the white cells themselves or that of general pathological manifestations in the human organism (Fl). Therefore, leukocytes have become a valuable tool for the study or even the diagnosis of inborn errors and acquired deficiencies and disorders of ubiquitous enzymes and metabolic systems because it can be isolated and purified with relative ease.

Functional and metabolic studies of polymorphonuclear leukocytes in the congenital Pelger-Huet anomaly

Polymorphonuclear leukocytes (PMNL) from two individuals with congenital Pelger-Huet anomaly (PHA) were examined to determine whether functional or metabolic defects accompanied the known morphological abnormality. No abnormalities of the PHA cells, as compared to normal control cells, were found when tested for quantitative leukocyte enzyme activities, nitroblue tetrazolium reduction, hexose monophosphate shunt activity, superoxide production, generation of chemiluminescence, or iodination. The PHA cells, as compared to normal PMNL, demonstrated normal chemotaxis and random migration, as well as bactericidal activity.

Functional and Metabolic Studies of Polymorphonuclear Leukocytes in the Congenital Pelger-Huet Anomaly

Polymorphonuclear leukocytes (PMNL) from two individuals with congenital Pelger-Huet anomaly (PHA) were examined to determine whether functional or metabolic defects accompanied the known morphological abnormality. No abnormalities of the PHA cells, as compared to normal control cells, were found when tested for quantitative leukocyte enzyme activities, nitroblue tetrazolium reduction, hexose monophosphate shunt activity, superoxide production, generation of chemiluminescence, or iodination. The PHA cells, as compared to normal PMNL, demonstrated normal chemotaxis and random migration, as well as bactericidal activity.

A case of primary hyperparathyroidism with low serum alkaline phosphatase levels.

A 23-year-old man with primary hyperparathyroidism which was typical except for reduced alkaline phosphatase activity is reported. Histological examination of surgical specimens revealed chief cell hyperplasia of the parathyroid glands. Systemic abnormalities of alkaline phosphatase were demonstrated, i.e., marked reduction of all isoenzymes and undetectable osseous enzyme in the serum, abnormal distribution of the enzyme in hepatocytes and diminished enzyme activities in leukocytes. In addition, diminished bone remodeling activity was revealed in a biopsy specimen of the rib. The association of hypophosphatasia is highly unlikely, because of normal urinary excretion of phosphoethanolamine, lack of osteomalacia, and no indication of an hereditary factor. The causal relationship between low remodeling activity and abnormalities in alkaline phosphatase was suggested.

Concanavalin A Increases Glyoxalase Enzyme Activities in Polymorphonuclear Leukocytes and Lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.

Galactocerebrosidase activity in canine globoid leukodystrophy.

In an investigation of canine globoid leukodystrophy, cerebroside beta-galactosidase activities were assayed in 24 brains from neonatal and older dogs and in 90 canine leukocyte pellets from nine samplings. The neonatal brains had significantly less enzyme activity than the brains of older dogs, which indicates a potential complication in making neonatal and fetal enzymatic diagnoses. For leukocytes, heterozygous activities averaged 51 percent and globoid leukodystrophy activities 18 percent of mean enzyme activity of normal leukocytes. Variability of leukocyte enzyme activities among the nine samplings was large, but within each sample, variability per genotypic category was moderate. A statistical model was developed to facilitate enzyme diagnosis in the dog and, by implication, in human globoid leukodystrophy and other sphingolipidoses.

A micro-radiochemical assay forα-1,4-glucosidase

An assay system for α-l,4-glucosidase (acid maltase) which is deficient in glycogen storage disease Type 2 (Pompe's disease) has been developed. The assay, which represents an improvement in sensitivity of at least 100-fold over the more conventional colorimetric methods is used to measure the enzyme activity in peripheral blood leucocytes, cultured amniotic cells and skin fibroblasts, has a total volume of 8 μl and uses (U- 14 C) maltose as the substrate, The product (U- 14 C) glucose is separated from the substrate by cellulose thin layer chromatography. The procedure allows the assay of acid maltase in cell extracts equivalent to about 2000–5000 cultured cells or 0.5-1.0 × 10 5 leucocytes isolated from 400 μl of whole blood. The assay has been used to measure the enzyme activity in fibroblasts and leucocytes derived from normal subjects, an affected patient and to assess the genotype of two foetuses suspected of being affected by Pompe's disease.

A new disorder of leucine metabolism

Investigations are described on a child admitted to hospital with acute acidosis and hypoglycaemia. Initial TLC and GLC showed presence of unidentifiable organic acids in the urine. Further investigations of these organic acids by GLC-MS demonstrated abnormally high concentration of 3-hydroxy-isovaleric, 3-methylglutaric, 3 methylglutaconic and 3-hydroxy-3-methylglutaric acids. This profile differed from that seen in the three known defects of leucine catabolism and suggested a deficiency in the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetate and acetyl CoA. Studies on cultured skin fibroblasts and peripheral leucocytes have shown a markedly reduced activity of the enzyme 3-hydroxy-3-methyl-glutaryl CoA lyase. Estimation of this enzyme activity in the parents' peripheral leucocytes suggest a genetic basis for the condition.

Hypophosphatasia: A Developmental Anomaly of Alkaline Phosphatase?

This report deals with quantitative and qualitative investigations of alkaline phosphatase in two unrelated infants with the severe infantile form of hypophosphatasia. Both affected infants had no detectable leukocyte alkaline phosphatase activities and both sets of parents and one sibling tended to have low but variable leukocyte enzyme activities. Normal duodenal juice alkaline phosphatase activity was present in the one patient in whom it was measured and a wide range of variation in enzymic activity was observed in the stools. There was no significant difference in the stool enzyme activity between both patients with hypophosphatasia (42.01 +/- 9.77 U) and control infants (40.55 +/- 6.29 U). However, the heterozygous parents had values significantly lower than the control adults (2.10 +/- 0.47 as compared with 19.10 +/- 4.44 U). Intestinal bacteria did not contribute significantly to the stool alkaline phosphatase activity. Enzyme activity was present in the bile of one of the patients and nearly absent in that of the other. Three "inducers" of alkaline phosphatase were given to both patients (phenobarbital, vitamin A, and corticosteroid). No clinical improvement or rise in serum alkaline phosphatase activity was observed during the trial of therapy with these agents. However, a significant increase in the activity of serum acid phosphatase was demonstrated during the course of vitamin A administration, suggesting an in vivo action of vitamin A on the lysosomes through decreasing the stability of the membrane and releasing acid phosphatase to the serum. Quantitative determination of tissue alkaline phosphatases from autopsy tissues was highly variable: no activity was found in bone, lungs, or spleen of either infant; there was a discrepancy in liver and kidney alkaline phosphatase values (zero in one patient and present in the other) and activity was present in the intestinal mucosa of both. Qualitative analysis of kidney, liver, and intestinal alkaline phosphatase revealed some differences between the patients and control subjects in heat inactivation and phenylalanine inhibition (Table 3). Starch gel electrophoresis of the liver preparation of one patient disclosed a single band which had greater mobility than that of six control subjects matched for age. Liver extracts from a premature and from full term newborns showed two bands. The single band of the patient's liver enzyme corresponded to the newborn's fast moving component. In addition, the intestinal enzyme prepared from the same patient had an extra band when compared with age-matched control subjects.

Ethnic Variation in Red Cell Glutathione Peroxidase Activity

Glutathione peroxidase activity was measured in blood and cultured fibroblasts from healthy persons of several different population groups. Individuals of Jewish ancestry and others of Mediterranean origin were found to manifest a decrease of red cell but not of leukocyte or fibroblast enzyme activity. Oriental populations differed in that the scatter in red cell enzyme activity was significantly lower than in Occidental populations. The erythrocyte enzyme of individuals with low activity was found to be less stable to heating than was the enzyme from persons with high activity. As a possible explanation for these data, a provisional genetic model is presented: a low GSH Px allele with a frequency of 0.556 in the Jewish population and of only 0.181 in the United States-Northern European population. Our results suggest that an association between GSH Px deficiency and hemolytic anemia need not represent a cause-and-effect relationship.

Ethnic variation in red cell glutathione peroxidase activity

Glutathione peroxidase activity was measured in blood and cultured fibroblasts from healthy persons of several different population groups. Individuals of Jewish ancestry and others of Mediterranean origin were found to manifest a decrease of red cell but not of leukocyte or fibroblast enzyme activity. Oriental populations differed in that the scatter in red cell enzyme activity was significantly lower than in Occidental populations. The erythrocyte enzyme of individuals with low activity was found to be less stable to heating than was the enzyme from persons with high activity. As a possible explanation for these data, a provisional genetic model is presented: a low GSH Px allele with a frequency of 0.556 in the Jewish population and of only 0.181 in the United States-Northern European population. Our results suggest that an association between GSH Px deficiency and hemolytic anemia need not represent a cause-and-effect relationship.

Disparate enzyme activity in erythocytes and leukocytes. A variant of hypoxanthine phosphoribosyl-transferase deficiency with an unstable enzyme.

A family is reported in which each of two sisters has a son with no detectable hypoxanthine phosphoribosyltransferase (HPRT) (EC 2. 4. 2. 8) in his erythrocytes, a finding considered pathognomonic of Lesch-Nyhan disease. However, neither has the stigmata of the disease. One boy is neurologically normal, and the other is moderately retarded. There was only a slight increase in urinary uric acid, but the amounts of hypoxanthine and xanthine, and their ratios, were similar to those found in Lesch-Nyhan disease, strongly indicating that excesses of these last two oxypurines are not responsible for the symptomatology in that disease. In contrast to the nondetectable HPRT activity in the red blood cells, leukocyte lysates from the two boys have 10-15% of normal activity, possibly reflecting continuing synthesis of an unstable enzyme. This hypothesis is supported by the demonstration that at 4 degrees C HPRT activity was rapidly lost in the propositus while the activity increased in control subjects. The mother's cells were intermediate between the two. The intact and disrupted leukocytes of the hemizygote, in the absence of added phosphoribosyl converted as much hypoxanthine to inosinate as the normal cell, and appropriate tests indicated that under these circumstances enzyme concentration is not rate limiting whereas the concentration of the cosubstrate, phosphoribosyl pyrophosphate, is. The capacity for normal function in the intact mutant cell is more representative of in vivo conditions than the lysate, which may explain the important modification of clinical symptomatology, the relatively mild hyperuricosuria, and the presence of mosaicism in the circulating blood cells of the heterozygotes. A similar explanation may apply to other genetic diseases in which incomplete but severe enzyme deficiencies are found in clinically normal individuals. An associated deficiency in glucose-6-phosphate dehydrogenase in this family permitted confirmation of previous observations on linkage with hypoxanthine phosphoribosyltransferase.

Histochemical disclosure of endogenous and H 2O 2-dependent reducing enzyme activities in leukocytes and other cytological entities from the human oral cavity

In the presence of added optimal concentrations of H 2O 2 (0.024 M), many polymorphonuclear neutrophils (PMN) in the sediment of human oral lavages exhibited intense nitro-blue tetrazolium (Nitro-BT) reducing enzyme activity. This H 2O 2-dependent reaction in PMN was inhibited markedly by cyanide, azide, hydroxylamine and catalase, and totally by 0.15 M H 2O 2, indicating the participation of a peroxidase (probably myeloperoxidase). In contrast, intense endogenous Nitro-BT reducing enzyme activity in granular masses (GM) was inhibited by all concentrations of H 2O 2 employed until 100 per cent inhibition was obtained at 0.15 M. Small numbers of PMN also yielded intense endogenous activity, which was correlated with the phagocytosis of microorganisms and the formation of H 2O 2 in the PMN. As the endogenous reactions in the PMN and GM were not inhibited by cyanide, azide, hydroxylamine or catalase, it is postulated that critical concentrations of endogenous bound H 2O 2 and peroxidase are present in these GM and PMN and are required for the endogenous activities. The exogenous H 2O 2-dependent reaction in the PMN and endogenous reactions in the GM and PMN were inhibited completely after 10 min exposure to 60 °C and by low concentrations of iodoacetate and cupric ion. The latter indicates that the system involved in both PMN and GM consists of a complex containing one or more heat-labile and -SH group-dependent components in addition to peroxidase.

IMP: and AMP:pyrophosphate phosphoribosyltransferase in leukemic and normal human leukocytes.

SummaryAn in vitro assay measuring AMP: and IMP: pyrophosphate phosphoribosyltransferase (EC 2.4.2.7 and EC 2.4.2.8) levels in intact leukocytes has been described. Intact erythrocytes incorporate insignificant amounts of precursor compared with leukocytes. The mean level of IMP pyrophosphate phosphoribosyltransferase in leukocytes from nine patients with acute myelogenous leukemia in relapse was significantly higher (0.01 0.001)) than in normal leukocytes. The levels of leukocyte AMP: pyrophosphate phosphoribosyltransferase were higher in leukemics but not significantly so compared with normal values. No significant reduction of IMP: pyrophosphate phosphoribosyltransferase activity was observed in leukocytes from leukemics clinically resistant to purine analogs. No correlation could be found between leukocyte enzyme activity and relapse, remission or therapy of leukemia. Enzyme levels in leukocytes isolated from patients with other malignancies were in the normal range. However, in a number of leukemic...

Enzymatic abnormality of the carrier state in metachromatic leukodystrophy

Arylsulfatase A activity in leukocytes from patients and from their close relatives was investigated in three families with metachromatic leukodystrophy. The activity of arylsulfatase A in leukocytes from three patients with metachromatic leukodystrophy was approximately one tenth of the mean control value. The enzyme activity in leukocytes from the parents of affected patients and from some of their relatives was approximately half that found in control subjects, demonstrating a gene-dose effect. There appeared to be no overlap between the enzyme activities in leukocytes from the parents of patients and from normal individuals. These findings suggest that the heterozygous carrier state in metachromatic leukodystrophy could be predicted by the assay of arylsulfatase A in leukocytes and autosomal recessive mode of inheritance may be assumed.