Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

1. 1. The specific activity of the lysosomal hydrolases in cultured skin fibroblasts varies according to the phase of growth in culture.

2. 2. Diagnosis of heterozygous genotypes for lysosomal enzyme deficiency diseases is unreliable with cultured fibroblasts, at least partly because of the growth curve-associated variations in specific activity.

3. 3. Fluctuations in specific activity during the beginning of the growth curve in vitro can be avoided by initiating cultures with cells which are in the early log phase of growth.

4. 4. Primary amniotic fluid cell cultures show no relationship between length of time in culture and lysosomal enzyme specific activity.

5. 5. Secondary amniotic fluid cell cultures exhibit growth curve-related variations in lysosomal enzyme specific activity as they assume fibroblast-like growth kinetics.

6. 6. Prenatal and postnatal diagnosis on cultured amniotic fluid cells and fibroblasts requires the use of appropriate controls which are matched for stage of growth and length of time after the last trypsinization.

     

Genotyping and prenatal assessment of collagen lysyl hydroxylase deficiency in a family with Ehlers-Danlos syndrome type VI.

Collagen lysyl and prolyl hydroxylase activities were measured in cultured fibroblasts from a child with clinical features of Ehlers-Danlos syndrome. Lysyl-to-prolyl hydroxylase activity ratios in cells from the proband, mother, father, and control were .24, .86, .52, and 1.00, respectively, providing a biochemical diagnosis of Ehlers-Danlos syndrome type VI and indicating an autosomal recessive mode of inheritance in this family. Prenatal assessment of lysyl hydroxylase deficiency was requested and accomplished for the first time during a subsequent pregnancy in the family. A series of control cultures established lysyl hydroxylase activity to be similar in cultured amniotic fluid cells (AF and F cells) and in cultured dermal fibroblasts. Cultured F and AF cells from the monitored pregnancy had enzyme activity similar to controls, indicating that the fetus should not be affected by lysyl hydroxylase deficiency. This finding was confirmed by demonstration of normal lysyl hydroxylase activity in fibroblasts cultured from the newborn baby. These studies show that cells cultured from second trimester amniotic fluid have collagen lysyl hydroxylase activity similar to that in dermal fibroblasts, making prenatal diagnosis of lysyl hydroxylase deficiency possible.     

Utilization of electron microscopy in the prenatal diagnosis of genetic disease.

The use of electron microscopy as a further method of diagnosis of disease in cultured skin fibroblasts and cultured amniotic fluid fibroblasts is presented. It was demonstrated that Tay-Sachs disease, Fabry's disease, and metachromatic leukodystrophy had distinctive abnormalities in both cultured skin fibroblasts and cultured amniotic fluid fibroblasts. It was shown that control of culturing conditions made it possible to distinguish normal cell lines from certain cell lines carrying known genetic diseases.     

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.     

Citrullinemia: prenatal diagnosis of an affected fetus.

We monitored a pregnancy in a family at risk for citrullinemia due to argininosuccinic acid (ASA) synthetase deficiency. ASA synthetase activity in cultured epithelioid amniotic fluid cells from the fetus at risk was less than 2% of control epithelioid amniotic fluid cell activity. An increased concentration of citrulline was found in the at-risk amniotic fluid (0.14 mumol/ml) as compared with fluid from six controls and one at-risk but unaffected pregnancy (trace). The pregnancy was terminated, and the in utero diagnosis was confirmed by assay of ASA synthetase activity in cultured fetal skin fibroblasts (4.4% of control activity). In addition, all five fetal tissues studied had significant accumulation of citrulline, whereas control fetal tissues had none. These data provide evidence that, if precise control is maintained over tissue culture variables, citrullinemia can be diagnosed successfully in utero by microassay of ASA synthetase activity in cultured amniotic fluid cells. They also suggest that amniotic fluid citrulline concentrations provide strong adjunctive evidence for this prenatal diagnosis.     

Branching enzyme activity of cultured amniocytes and chorionic villi: prenatal testing for type IV glycogen storage disease.

Although type IV glycogen storage disease (Andersen disease; McKusick 23250) is considered to be a rare, autosomally recessive disorder, of the more than 600 patients with glycogenosis identified in our laboratory by enzymatic assays, 6% have been shown to be deficient in the glycogen branching enzyme. Most of the 38 patients with type IV glycogen storage disease who are known to us have succumbed at a very early age, with the exception of one male teenager, an apparently healthy 7-year-old male, and several 5-year-old patients. Fourteen pregnancies at risk for branching enzyme deficiency have been monitored using cultured amniotic fluid cells, and four additional pregnancies have been screened using cultured chorionic villi. Essentially no branching enzyme activity was detectable in eight samples (amniocytes); activities within the control range were found in five samples (three amniocyte and two chorionic villi samples); and five samples appeared to have been derived from carriers. In two of the cases lacking branching enzyme activity, in which the pregnancies were terminated and fibroblasts were successfully cultured from the aborted fetuses, no branching enzyme activity was found. Another fetus, which was predicted by antenatal assay to be affected, was carried to term. Skin fibroblasts from this baby were deficient in branching enzyme. Pregnancies at risk for glycogen storage disease due to the deficiency of branching enzyme can be successfully monitored using either cultured chorionic villi or amniocytes.     

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.     

Rapid prenatal testing for human beta-glucuronidase deficiency (MPS VII)

Prenatal diagnosis for the lysosomal storage disorders is typically achieved by enzymatic analysis of the relevant lysosomal enzyme in cultured amniocytes or chorionic villi. While prenatal diagnosis of some genetic diseases can be done by analysis of pertinent metabolites in amniotic fluid, there are few data regarding prenatal diagnosis of lysosomal disorders by enzyme analysis of amniotic fluid. Prenatal diagnosis by enzyme analysis of amniotic fluid has the potential advantage of providing a more rapid prenatal test result. In this study we describe an assay for the prenatal diagnosis of the mucopolysaccharidosis beta-glucuronidase deficiency (MPS VII; MIM #253220) using amniotic fluid and we confirm its reliability in detecting an affected fetus in an at-risk pregnancy by enzyme analysis of cultured amniocytes and fetal fibroblasts. Because MPS VII is rare and few instances of prenatal diagnosis for this and nearly all other lysosomal disorders have been accomplished by enzyme analysis of amniotic fluid, confirmation of results obtained from enzyme analysis of amniotic fluid should be carried out by enzyme or mutation analysis using cultured amniocytes or chorionic villus specimens.     

Analysis of normal and mutant forms of human adenosine deaminase — A review

Summary A deficiency of the enzyme adenosine deaminase is associated with an autosomal recessive form of severe combined immunodeficiency disease in man. The molecular forms of the normal human enzyme have now been well characterized in an effort to better understand the nature of the enzyme defect in affected patients.In some human tissues adenosine deaminase exists predominantly as a small molecular form while in other tissues a large form composed of adenosine deaminase (small form) and an adenosine deaminase-binding protein predominates. The small form of the enzyme purified to homogeneity by antibody affinity chromatography is a monomer of native molecular weight of 37,600. The adenosine deaminase-binding protein, purified by adenosine deaminase affinity chromatography, appears to be a dimer of native molecular weight 213,000 and contains carbohydrate. Based on direct binding measurements, chemical cross-linking studies and sedimentation equilibrium analyses, small form adenosine deaminase has been shown to combine with purified binding protein in a molar ratio of 2:1 respectively to produce the large form adenosine deaminase.Reduced, but widely ranging levels of adenosine deaminating activity, have been reported in various tissues of adenosine deaminase deficient patients. Further, the characteristics of this residual enzyme activity have been analyzed immunochemically to substantiate genetic heterogeneity in this disorder.While many types of immunodeficiency are currently recognized in man, in most cases the molecular defect is unknown. The discovery of a deficiency of the enzyme, adenosine deaminase, ADA, (EC 3.5.4.4), in some patients with severe combined immunodeficiency disease represented an early clue to the pathogenesis of immune dysfunction at the molecular level^1-4. Affected patients with markedly reduced levels of ADA exhibit a defect of both cellular and humoral immunity characterized clinically by severe recurrent infections with a fatal outcome if untreated. Attempts to elucidate the nature of the genetic mutation(s) leading to the reduction of ADA activity in these immunodeficient patients have been complicated in part by an incomplete understanding of the nature of ADA in normal tissues. In this review we will consider the structural characteristics of the normal and mutant forms of ADA as they are currently understood.     

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.     

Aromatase activity in human skin fibroblasts: characterization by an enzymatic method

Human skin fibroblasts were incubated for 24 h with 10(-6) M androstenedione and the estrone + estradiol released in the culture medium were measured by an enzymatic assay. Aromatase activity was expressed as pmol (estrone + estradiol) formed in the medium per mg cell protein per day. Using this method we were able to investigate the kinetic properties of aromatase in different cell strains and its stimulation by dexamethasone. Values of 92 nM and 9.1 pmol/mg protein/day were obtained respectively for Km and Vmax in cultured fibroblasts derived from genital skin of normal prepubertal subjects. In patients with complete androgen insensitivity syndrome CAIS, the Km was 156 nM and the Vmax 42 pmol/mg protein/day. Aromatase activity varied from 7.9 +/- 1.2 pmol/mg protein/day (mean +/- SD; n = 19) in normal prepubertal boys to 24.5 +/- 4.7 pmol/mg protein/day (mean +/- SD; n = 11) in those from normal postpubertal boys. The values were even higher in fibroblasts cultured from genital skin of prepubertal patients with CAIS. Cell concentrations did not modify the pattern of estrogen formation and aromatase activity did not vary with serial subcultures. The stimulatory effect of dexamethasone on aromatase activity in cultured fibroblasts was measured after preincubation of the cells for 48 h with dexamethasone, by determining estrogen formation after 24 h incubation of the cells with androstenedione 10(-6) M using this enzymatic method. This data suggest that aromatase activity measured in cultured fibroblasts could be a useful tool for studying extraglandular estrogen formation in physiological and pathological conditions.     

Prenatal diagnosis of hypophosphatasia; genetic, biochemical, and clinical studies.

This report has considered three approaches to the prenatal diagnosis of the severe, early onset form of hypophosphatasia. Two of these approaches, ultrasonography and the determination of the bone/liver isozymes of alkaline phosphatase (ALP) in cultured amniotic fluid cells, have proven useful diagnostically. The third method, assay of the bone/liver isozyme activity or total activity in supernatant amniotic fluid, was not informative for the affected fetus we studies. Failure to visualize a well-defined fetal skull after 16 weeks of pregnancy when the level of alpha-fetoprotein in the amniotic fluid is normal should arouse the suspicion of hypophosphatasia. Because the disease is known to manifest clinical variabiltiy, studies to detect both the biochemical defect as well as the structural manifestations should be considered. The combined use of ultrasonography, analysis of amniotic fluid alpha-fetoprotein, and the measurement of the bone/liver ALP in cultured amniotic fluid cells would appear to be the best approach to the prenatal diagnosis.     

Effect of temperature on extracellular accumulation of beta-D-N-acetylglucosaminidase in I-cell disease fibroblast cultures.

The activities of most lysosomal enzymes are elevated in the culture fluid of skin fibroblasts derived from patients with I-cell disease with a corresponding reduction in the intracellular activities when the cells are cultured at 37°C. When I-cell fibroblast cultures are incubated at 27°C for 8–24 hr, the β-D-N-acetylglucosaminidase (EC 3.2.1.30) activity accumulated in the culture fluid is reduced to approximately 25% of the activity in 37°C control cultures without a corresponding change in intracellular activity. No significant effect of temperature is observed on the intra- and extracellular distribution of β-D-N-acetylglucosaminidase in non-I-cell fibroblast cultures. These findings suggest the existence of two lysosomal enzyme pools in I-cell fibroblasts, one of which is temperature-dependent and destined for excretion while the other remains intracellular and appears to be unaffected by temperature.     

Different functional subsets of cultured murine T cells express characteristic levels of adenosine deaminase activity

The level of adenosine deaminase (ADA) activity in mouse T-lymphocyte cultures was studied under different growth-supporting conditions and in mixed lymphocyte culture-derived long-term T-cell lines and clones. Early after the initiation of in vitro culture, the levels of ADA (2000 U/mg) were similar in bulk cultures either depleted or not depleted in Lyt-2+ T cells. Enrichment for cytolytic T lymphocytes (CTL) obtained by addition of exogenous interleukin 2 (IL-2), was accompanied by a net decrease of ADA activity (110 +/- 15 U/mg). All the tested CTL-A lines derived from such cultures were also characterized by a low or undetectable level of this enzyme (at best 160 +/- 70 U/mg) as previously observed. In contrast, "Lyt-2-" T-cell bulk cultures grown, without addition of exogenous IL-2, in the presence of gamma-irradiated H-2d stimulators maintained a constant level of ADA activity (1770 +/- 340 U/mg) for at least 3 months. Functionally distinct types of Lyt-2- T-cell lines were also analyzed: T-cell lines competent to activate B lymphocytes to growth and terminal maturation as well as others devoid of detectable functions showed a stable ADA level comparable to that expressed by the original bulk culture 1685 +/- 620 U/mg). The present results demonstrate that, like tumor cell lines, most normal T lymphocytes express a high level of ADA activity in culture, which strongly suggests that the low level of ADA activity exhibited by CTL is a characteristic of this functional subset.     

The use of enzyme therapy to regulate the metabolic and phenotypic consequences of adenosine deaminase deficiency in mice. Differential impact on pulmonary and immunologic abnormalities

Adenosine deaminase (ADA) deficiency results in a combined immunodeficiency brought about by the immunotoxic properties of elevated ADA substrates. Additional non-lymphoid abnormalities are associated with ADA deficiency, however, little is known about how these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as severe pulmonary insufficiency. ADA enzyme therapy was used to examine the relative impact of ADA substrate elevations on these phenotypes. A "low-dose" enzyme therapy protocol prevented the pulmonary phenotype seen in ADA-deficient mice, but did little to improve their immune status. This treatment protocol reduced metabolic disturbances in the circulation and lung, but not in the thymus and spleen. A "high-dose" enzyme therapy protocol resulted in decreased metabolic disturbances in the thymus and spleen and was associated with improvement in immune status. These findings suggest that the pulmonary and immune phenotypes are separable and are related to the severity of metabolic disturbances in these tissues. This model will be useful in examining the efficacy of ADA enzyme therapy and studying the mechanisms underlying the immunodeficiency and pulmonary phenotypes associated with ADA deficiency.     

Prenatal diagnosis of Sanfilippo disease type B

Summary The prenatal diagnosis of a fetus affected with Sanfilippo disease type B is described. The deficiency of -N-acetylglucosaminidase in the cultured amniotic fluid cells was shown by a microassay enabling early prenatal diagnosis. In addition an increased level of heparan sulphate was demonstrated in the amniotic fluid by two-dimensional electrophoresis of glycosaminoglycans. The latter result confirmed the value of this test as an adjunctive method in the prenatal diagnosis. The pregnancy was terminated and the prenatal diagnosis was confirmed by enzyme analysis of cultured fetal fibroblasts and fetal liver.     

1-Thioglycerol: inhibitor of glycerol kinase activity in vitro and in situ

The infantile form of glycerol kinase (GK) deficiency (McKusick No. 30703) (1) is characterized by adrenal cortical insufficiency, adrenal hypoplasia and developmental delay. The underlying biochemical mechanism(s) responsible for the observed clinical presentations are undetermined. Pursuant to our examination of the molecular pathogenesis of this enzyme deficiency, we have endeavored to develop a model for this disorder. 1-thioglycerol (1-TG) was investigated as a potential GK inhibitor in adrenal gland, an organ consistently affected, and in cultured fibroblasts, available from affected individuals. In 105,000 g bovine adrenal supernatant the Ki for 1-TG was 1.9 mM. In human fibroblast 105,000 g supernatant, the Ki for 1-TG was 3.4 mM. In both tissues the inhibition was purely competitive with respect to glycerol. Using incorporation of [14C(U)]-glycerol into protein as an index of GK activity in situ in human skin fibroblasts, GK deficient fibroblasts incorporate less than 10% of that observed in normal fibroblasts. Addition of 1-TG to normal fibroblasts resulted in inhibited incorporation rates. The specificity of these effects in situ was examined. Our findings indicate that 1-TG may be a suitable inhibitor of GK activity for the development of a model for glycerol kinase deficiency.     

Cell disease: desialylation of beta-hexosaminidase and its effect on uptake by fibroblasts

The pinocytosis by fibroblasts of beta-hexosaminidase (EC 3.2.1.30) excreted by cultured skin fibroblasts from a patient with I-cell disease was not enhanced by neuraminidase treatment of the enzyme. The uptake of sialic acid-rich normal plasma beta-hexosaminidase was minimal and neuraminidase treatment did not appreciably enhance uptake. In contrast, sialic acid-rich normal seminal fluid beta-hexosaminidase was readily pinocytosed regardless of neuraminidase treatment. Thus the presence of sialic acid on beta-hexosaminidase does not influence uptake and a neuraminidase deficiency in I-cell disease may not be directly responsible for excessive extracellular enzyme.     

Heme content of normal and porphyric cultured skin fibroblasts

Partial deficiencies in enzyme activities of the heme biosynthetic pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients with protoporphyria (PP) and acute intermittent porphyria (AIP). We have quantitatively and qualitatively assessed the heme and free porphyrin content in cultured PP, AIP, VP (variegate porphyria, in which an enzymatic deficiency has not been identified), and normal skin fibroblasts during routine culture conditions in order to assess the overall metabolism of heme in these cells. The total heme concentration was not significantly different between control and porphyric lines; 189 +/- 15 pmoles/mg protein (mean +/- SEM) in controls, 154 +/- 17 in PP, 175 +/- 20 in AIP, and 181 +/- 81 in VP. The hemoprotein difference spectra were similar in all lines. Free porphyrins were not detected in any of the disorders. Despite partial deficiencies in enzyme activities of the heme pathway, porphyric fibroblasts thus maintain normal heme content during routine culture conditions without detectable porphyrin accumulation.