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Technical Briefs |
1 Division of Medical Genetics, E.K. Shriver Center, Waltham, MA2 Foundation for Blood Research, Scarborough, ME; 3 Departments of Neurology, Pathology and Pediatrics, Massachusetts General Hospital and Department of Pathology, Harvard Medical School, Boston, MA;
aaddress correspondence to this author at: Departments of Neurology and Pathology, Cleveland Clinic Foundation S-71, 9500 Euclid Ave., Cleveland, OH 44195; fax 216-445-9139, e-mail natowim{at}ccf.org)
Lysosomal enzymes are crucial for the degradation of numerous macromolecular substrates. Deficiencies of many of the known lysosomal enzyme activities have been associated with different clinical disorders, collectively termed the lysosomal storage diseases (1)(2)(3)(4). Accurate measurement of lysosomal enzyme activities, therefore, is important in establishing diagnoses of lysosomal storage diseases. In addition, accurate measurement of these lysosomal enzymes is critical in identifying carriers or heterozygotes for these conditions and in monitoring patients with these diseases who have undergone bone marrow transplantation and other enzyme replacement therapies.
Clinical laboratories that measure lysosomal enzyme activities typically use sonicates or detergent extracts of mixed leukocyte pellets for enzymologic studies of lysosomal disorders. The use of peripheral blood leukocytes for this purpose has the merit of using a readily accessible tissue that, in addition, has considerable concentrations of most lysosomal enzymes.
There are few reported analyses of the comparative activities of human lysosomal enzymes in different types of leukocytes. Limited and conflicting data have been published regarding lysosomal enzyme activities in the different populations of human peripheral leukocytes. Differences in the activities of lysosomal enzymes among populations of leukocytes, if real and of substantial magnitude, could be important clinically. In this study we comparatively analyzed six commonly measured lysosomal enzymes of clinical importance. Our data indicate significant differences in the activities of these enzymes in the different types of leukocytes.
The lysosomal enzymes studied here were assayed by standard methods (below). All 4-methylumbelliferyl glycoside and nitrocatechol substrates, 4-methylumbelliferone, and buffer reagents were obtained from Sigma Chemical Co. Tissue culture reagents and fetal calf serum were purchased from Gibco BRL Products-Life Technologies. Ficoll-Paque was purchased from Pharmacia Biotech AB.
Blood was obtained from healthy adult donors with informed consent. Leukocytes were fractionated with Ficoll-Paque as follows: Blood (10 mL) was drawn into evacuated tubes (Vacutainer; Becton Dickinson) containing sodium heparin, transferred to a 40-mL plastic centrifuge tube, diluted with 20 mL of Hanks Balanced Salt Solution (HBSS), and gently mixed. The diluted blood was gently layered on 15 mL of Ficoll-Paque in a 20 x 150 mm centrifuge tube and centrifuged at 360g for 50 min at room temperature; the supernatant was carefully aspirated and discarded. The mononuclear cells at the interface with the plasma were pipetted into a plastic centrifuge tube, washed with HBSS, and centrifuged twice at 170g for 10 min. The mononuclear pellets were then rinsed with saline solution (9 g/L NaCl) to remove residual HBSS and used for the experiments (hereafter referred to as the mononuclear fraction). The mononuclear fraction contained 90–93% lymphocytes and 3–5% monocytes when evaluated by Wright staining. The granulocyte/erythrocyte fraction that was present at the bottom of the initial Ficoll-Paque separation was washed twice with isotonic saline, and the erythrocytes were subsequently removed by hypotonic lysis, giving a granulocyte fraction (hereafter referred to as granulocytes) consisting of 94–98% granulocytes. Cell pellets were stored at –20 °C, and all enzyme assays were carried out within 1–5 days after isolation of the cells.
-Iduronidase activity (EC 3.2.1.76) was determined by the method of Rome et al. (5). Fluorescence was measured for this and all other assays with 4-methylumbelliferone-based substrates with an excitation wavelength of 365 nm and an emission wavelength of 450 nm; the results were compared with a calibration curve prepared with 4-methylumbelliferone. Cell protein concentrations for this and all other assays were measured by the Lowry method (6). ß-Hexosaminidase A and B (Hex A and B; EC 3.2.1.52) and ß-glucosidase (EC 3.2.1.45) activities were assayed as described previously (7)(8). ß-Mannosidase activity (EC 3.2.1.25) was measured by the method of Panday et al. (9). Arylsulfatase A activity (EC 3.1.6.8) was determined by the method of Lee-Vaupel and Conzelmann (10); absorbance was read at 515 nm with a spectrophotometer and compared with a calibration curve for p-nitrocatechol. Results are reported as the mean ratio (with 95% confidence intervals) of enzyme activity in matched samples.
All of the leukocyte lysosomal hydrolases investigated in this study showed highly significant differences in activity per mg of cell protein between mononuclear cells and granulocytes (Table 1
). Activity was higher in mononuclear leukocytes for -iduronidase, arylsulfatase A, ß-glucosidase, and total Hex A and B activities. By contrast, activity was lower in mononuclear leukocytes for Hex A and ß-mannosidase. The largest proportional changes occurred for -iduronidase and for ß-mannosidase (Table 1
).
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Hex A and B are arguably the most commonly assayed and thoroughly studied of the lysosomal enzymes. A deficiency of the Hex A isoenzyme resulting from mutations in the -subunit gene of that enzyme causes Tay–Sachs disease; mutations in the ß-subunit of the Hex B gene lead to a deficiency of both Hex A and Hex B and cause the clinically similar condition Sandhoff disease (11). These isoenzymes are frequently assayed, partly because of the existence of international heterozygote screening programs for Tay–Sachs disease and because of their challenging and complex clinical chemistry (7)(11)(12)(13). Two studies reported similar concentrations of total ß-hexosaminidase activity in mononuclear cells or lymphocytes vs granulocytes (14)(15), whereas Tanaka (16) noted markedly lower total ß-hexosaminidase activity in granulocytes and Casal et al. (17) reported minimally increased ß-hexosaminidase activity in granulocytes. In this study, we noted significant differences in total ß-hexosaminidase activity between mononuclear cells and granulocytes, with the former having 
1.5-fold greater total ß-hexosaminidase activity (Table 1
). Beutler et al. (14) did not study the distribution of Hex A and B isoenzymes in different cell types, whereas Nakagawa et al. (15), Casal et al.(17), and Ellis et al. (18) observed a lower percentage of Hex A activity in mononuclear cells or lymphocytes compared with granulocytes. Nakagawa et al. (15) quantified the difference and reported that mononuclear cells have 52% Hex A whereas granulocytes have 88% Hex A, and Casal et al. (17) reported 54% Hex A in mononuclear cells and 72% Hex A in granulocytes. We also noted a marked difference in the percentage of Hex A between the mononuclear cells and granulocytes; the former have 38% Hex A and the latter have 75% (Table 1
).
A clinical scenario in which these findings would likely be relevant might be the determination of heterozygosity for Tay–Sachs disease for an individual who has a pathologic deviation of the normal leukocyte differential, perhaps as a result of a severe inflammatory condition or a hematopoietic cell dyscrasia. An excess of lymphocytes in the peripheral blood might lead to a false-positive diagnosis of heterozygosity when standard enzyme-based (differential isoenzyme thermolability) determinations of heterozygosity are used; an excess of granulocytes could lead to a false-negative diagnosis of heterozygosity.
ß-Glucosidase deficiency is associated with Gaucher disease, the most common lysosomal lipid storage disorder (19). Here, too, heterozygosity testing is often done by measuring leukocyte ß-glucosidase activity (8), although carrier testing by molecular genetic analyses has important advantages in some clinical contexts (19). Studies of ß-glucosidase activity in different populations of leukocytes indicate minimally to markedly increased activity of this enzyme in lymphocytes compared with granulocytes, depending on assay conditions (14)(20). We noted a 1.4-fold increase of activity of this enzyme per gram of protein in mononuclear cells compared with granulocytes with our assay conditions (Table 1
). As is the case for the ß-hexosaminidases, the likeliest clinical situation in which differences in ß-glucosidase activity among different types of leukocytes could have clinical significance may be for enzyme-based heterozygosity testing: a skewed representation of one population of the leukocytes may lead to incorrect assignment of genotype.
A deficiency of arylsulfatase A activity leads to the relatively common lysosomal storage disorder metachromatic leukodystrophy (21). Accurate determination of arylsulfatase A activity is important because partial deficiencies of this enzyme that are associated with substantial residual enzyme activity, so-called "leaky" mutants, have been described that have neurologic manifestations (21). In addition, this is one of the lysosomal storage disorders for which bone marrow transplantation therapy is sometimes done (21)(22); careful monitoring of post-bone marrow transplantation arylsulfatase A activities can be helpful in monitoring the status of the graft.
We are aware of only two published reports regarding the comparative activities of arylsulfatase A in different populations of leukocytes. Georgopoulos and Manowitz (23) did not find a significant difference in the arylsulfatase A activity of lymphocytes and neutrophils, whereas Shah et al. (24) reported that lymphocytes had 1.5-fold greater arylsulfatase A activity than a mixed leukocyte preparation. Our study indicated that the leukocyte mononuclear cell fraction has 1.5-fold greater enrichment of arylsulfatase A enzyme activity compared with granulocytes (Table 1
). In addition to possible relevance in heterozygosity testing, these findings are likely to be important in the biochemical monitoring of patients with metachromatic leukodystrophy who have had bone marrow transplantation therapy. We have noted, for example, one patient who underwent a bone marrow transplant and who later developed a significant decrease in leukocyte arylsulfatase A activity; that patient previously had stable, approximately heterozygote values of enzyme activity after bone marrow transplantation. There was no evidence of loss of engraftment of the transplant or of any usual manifestations of disease, and the best explanation for the transient change in leukocyte arylsulfatase A activity was the change in the proportions of leukocyte cell types at the time that the blood specimen was obtained (M. Natowicz, personal observation).
We also compared the activities of -iduronidase and ß-mannosidase in different populations of leukocytes. We are aware of no similar studies regarding the relative activity of either of the enzymes in different populations of leukocytes. Deficiencies of -iduronidase activities are associated with a variety of clinical phenotypes, including Hurler syndrome, Scheie syndrome, and Hurler–Scheie phenotype (25); although rare, deficiencies of ß-mannosidase are also associated with a spectrum of clinical presentations and natural histories (26). The activity of -iduronidase per gram of protein is approximately twofold greater in mononuclear cells or lymphocytes than in granulocytes (Table 1
). In contrast, ß-mannosidase activity in granulocytes is 2.4-fold greater than that in mononuclear cells (Table 1
). The clinical significance of these results will also likely be in the realm of heterozygosity testing. In addition, some individuals with -iduronidase deficiency have undergone bone marrow or cord blood transplantation therapy (22)(27), and here, too, biochemical monitoring of enzyme activities in mixed leukocyte preparations could at times provide misleading results, suggesting the possibility of loss of engraftment when, instead, the distribution of types of leukocytes in the peripheral blood has changed.
In summary, this study provides data regarding six lysosomal enzymes that are frequently measured and shows that all six enzymes have significantly different activities per gram of cell protein in mononuclear cells compared with granulocytes. These data have clinical implications in the areas of biochemical diagnosis of patients having partial deficiencies of lysosomal enzyme activities, in heterozygosity testing, and in the monitoring of patients who have undergone bone marrow or cord blood transplantation.
Footnotes
1 current address: Taksim Teaching and Research Hospital, Biochemistry Department Beyoglu, Istanbul, Turkey; 
References
-L-iduronidase, to receptors on cultured fibroblasts. Proc Natl Acad Sci U S A 1979;76:2331-2334.-mannosidosis, ß-mannosidosis, fucosidosis, and sialidosis. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited disease 2001:3507-3533 McGraw-Hill New York. .
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