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From www.bloodjournal.org by guest on August 9, 2017. For personal use only. Lymphocyte By and L. HENRY Granulocyte with Down’s NADLER, UMEROUS in the Down’s syndrome.’ activity is leukocyte in blood cells sults have the technic rophosphate mass, upon syndrome with hand, higher trisomic locyte In an responsible utilized Engel2 published age enzyme Toimu demonstrating and levels. among suggested Two patients that that result IN0UYI: HsIA enzymes have in Patients a the presence recent studies increase trisomic level shortened in the an with of enzyme lifespan of of younger with the white conflicting re- to ) answer this question. Gaibraith and Valberg,3 using leukocytes in vitro with radioactive diisopropyl fiuoto study the granubocyte half-life, the blood granulocyte turnover and et al.,4 using syndrome, rate four were patients comparable were unable with technics able to to Down’s demonstrate involving demonstrate any syndrome. On DFP32 an increase the labeling in granu- rate. attempt for granulocytes cell might normals Down’s turnover been cell granulocyte Raab have Activity MONTELEONE, YI-YUNG blood and Down’s between other have Hook dependent the difference in of white attempted of labeling ( DFP32 and DAVID REPORTS activity L. PATRICIA AND N Enzyme Syndrome the in to demonstrate the elevation method Garvin’s in which white blood enzyme activities of Rabinowitz5 glass bead in the blood ( C), of lymphocytes and AND was who METHODS obtained from 10 patients with trisomic Down’s are residents at the Dixon State School, Dixon, and sex and had an age range of 6 to 10 years. 30 minutes with 6 per cent dextran in normal saline (Abbott) mixed in a ratio of 1 ml. of dextran per 5 ml. of blood. The supernatant was removed to within 0.2 cm. of the red blood cells and centrifuged at 100 g for 10 minutes. The white cell pellet was then resuspended in 4 ml. of serum. The remainder of the supernatant was centrifuged at 1500 g for 10 minutes and this cell-free serum used for both the preparation procedure and the elution of the column. The general procedure of preparation of the column and cells and the actual separation The blood was sedimented for separation be we columns.6 MATERIALS Twenty-five ml. of heparinized syndrome ( M ) and 10 controls Ill. The two groups were matched cell component might in Down’s syndrome, age at 37 C. for From the Genetic Clinic of the Children’s Memorial Hospital and the Department of Pediatrics, Northwestern University Medical School, Chicago, Ill. These studies were aided by grants from the Otlao S. A. Sprague Memorial Fund, the Illinois Mental Fund, and the U. S. Public Health Service (1-SOL-FR 5070) (1-SOLFR 5475) (T1-AM-5186). submitted March 2, 1967; accepted for publication April 13, 1967. HENRY L. NADLER, M.D.: Associate in Pediatrics, Northwestern University, Children’s Memorial Hospital, Chicago, ill. PATRIcIA L. MONTELEONE, M.D.: Instructor in Pediatrics, Northwestern University, Children’s Memorial Hospital, Chicago, Ill. Tomiu IN0uYE, PH.D.: Assistant Professor of Biochemistry, University of Illinois, Chicago, Ill. DAVID YI-YUNC H5IA, M.D.: Professor of Pediatrics, Northwestern University, Children’s Memorial Hospital, Chicago, Ill. First 669 BLOOD, VOL. 30, No. 5 (NOVEMBER), 1967 From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 670 NADLER Irji +I+I+I U INr\Ie) 1+1+1+11 I IQtI I I. I’? +1 +1 +1 IN.t- t.o Irt I.. U 1+1+1+1 U I v II - V INtr)c I+1+1+, 2 IO\t H- ‘) 00 = -0 U I+i+i+i Isr’i - I - I z <L I H ET AL. From www.bloodjournal.org by guest on August 9, 2017. For personal use only. LYMPHOCYTE AND GRANULOCYTE ENZYME 671 ACFIVITY of cells were those of Rabinowitz.5 Mir’imal essential media modified for suspension culture (G. I. B. Co. ) was substituted for Hanks BSS. In addition, the column was incubated at 37 C. for 20 minutes instead of 30 minutes. Plastic tubes, pipettes, and siliconized glassware were used throughout. A sample of the sedimented white blood cells ( W) was placed on the column, the lymphocyte fraction ( L ) was obtained by elution with 100 per cent cell-free serum, and the polymorphonuclear leukocyte fraction ( P) was obtained by elution with EDTA solution ( G. I. B. Co. ). Aliquots of W, L and P were used for microscopic analysis, cell count ( using a Coulter counter ) , and enzyme studies. Acid phosphatase ( AcP ) was assayed at pH 4.90 and alkaline phosphatase#{176} (AkP) at pH 9.3, using p-nitrophenol phosphate as substrate.7 Glucose-6-phosphate dehydrogenase ( C-6-PD) was determined spectroscopically by the reduction of NADP at 340 m, using C-6-P as substrate.’ Separation of white cells was always performed in pairs with one C and one M being studied simultaneously. The column was considered satisfactory if 50 per cent of P and 50 per cent of L were recovered from W in both columns. Approximately 70 per cent of the columns filled these criteria and were Included in the results. The L fraction contamed 97 per cent lymphocytes, 3 per cent polymorphonuclear leukocytes, and was contaminated by 2 to 3 red blood cells per lymphocyte. The P fraction contained 95 per cent polymorphonuclear leukocytes and 5 per cent lymphocytes and monocytes. The cell fractions were ith the treated with results of hypotonic the saline enzyme to remove any red cells which might interfere assay. RESULTS As test shown in Table 1, significant could be found between C and differences D for W, measured by the Student L, and P in all instances. “T” DIscussIoN Several eral have explanations blood of suggested “structural” have patients that genes for ed that the extra for certain specific “C” localizing a specific with The most demonstrate and activity Nadler white changes in the authors12”3 to periph- of investigators911 might contain contain evidence enzyme that blood cell, been convincingly proved. labels the neutrophilic al.19 of fibroblasts of AcP, an suggesting of the and et Other might date, no for the A number chromosome enzymes. locus lifespan Valberg3 in were patients with Down’s syndrome. A number of different enzymes red blood cells8’’13’15’1#{176} of patients tivity to explain chromosome As of this hypothesis a shortened Galbraith specific genetic age leukocyte, has not DFP32 specifically while offered have the suggest- the “regulator” genes has been presented the extra chromosome mongolism. recent to the certain group enzymes. associated is related been with Down’s syndrome. the extra “G” group have derived AkP, and failed from C-fl-PD 4 of unable have with to the in both of enzyme the Cartwright series. Raab 6 patients activity polymorphonuclear et al.14 has shown that et al.4 were able to with Down’s to demonstrate any syndrome, difference been reported to be increased Down’s syndrome. DeMars,17 demonstrate trisomic alteration specifically any mongols. the W difference The and the #{176}0.1 ml. of 0.3M magnesium chloride is required for the P fraction, threefold increase of AkP activity. Neither magnesium chloride nor effect upon AkP activity in the W or L fractions. in 4 in the Cox,18 in enzyme finding L and of ac- increased P fractions in since this results in a zinc chloride has any From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 672 NADLER this study is not demonstrates localized related to cells some which The that the to any particular specific, though may be distribution alteration in fraction. white blood It would appear generalized, characteristic of of enzyme cell Down’s activities of predominantly terest to The that requirement in the with in note An various The Mg+ specific blood genetic, be be related increase circulatory is blood cell beads is of in- chloride. loss or to the different is in It magnesium to an intracellular is that fractions that the activity has been shown to technics.21’22 P require glass possibility the procedure valuable metabolic, the to the activity this of Mg+ elution isoenzymes + ion procedure are present components. of a histochemical separation, + may alternative cell numerous of adhering application to cell process EDTA. prove P by for active the in after AL. syndrome. in the accord with previous studies. Rabinowitz2#{176} has demonstrated of G-6-PD is four times greater in P than L. AkP activity reside enzyme that phenomenon ET adjunct and of to clinical white the blood standard cell separation methods used should in studying problems. SUMMARY Patients with increases of acid dehydrogenase arated from in enzyme trisomic in white activities chromosome Down’s syndrome phosphatase, causing both blood alkaline lymphocytes cells by appears Esseva trovate to be que patientes causa de phosphatase directly related le syndrome de have significant glucose-6-phosphate leukocytes sepThe alteration to genes con IN located on the le INTERLINGUA tnisomic syndrome acide, de de phosphatase alcalin, cosa-6-phosphato in le lymphocytos e etiam in le leucocytos ab le altere leucocytos per le procedimento de Rabinowitz. activitate enzymatic non es directemente relationate con genes (jtIC nivellos and syndrome. SUMMARIO elevate to found and polymorphonuclear procedure of Rabinowitz. the not Down’s were phosphatase, Down ha significativemente e de dehydrogenase de glupolymorphonucleari separate Ii pare que Ic alteration del locate super Ic chromosomas Down. ACKNOWLEDGMENTS The authors wish to express their appreciation to Drs. James Carvin for sharing with us their experience with for permitting us to study the patients, and to Elvira Pribila, and Bonnie Williams for technical assistance. Yale Rabinowitz, Fritz Bach, and the columns, to Dr. Paul Tillman Kavaliunas, Janet Pi, Rosemary REFERENCES 1. Hsia, 2. Shih, L. Y.: Biochemical changes in chromosomal abnormalities. Ann. N. Y. Acad. Sci. (in press, 1967). Hook, E. B., and Engel, R. R.: Leuco- D. Y. Y., Nadler, H. cyte life-span, leucocyte phosphatase, and the 21st some. Lancet 1:112, 1964. 3. L., and 4. alkaline chromo- Caibraith, P. R., and Valberg, L. S.: Granulopoiesis in Down’s syndrome. Pediatrics 37:108, 1966. 5. S. 0., Mellman, N. J., Oski, F. A., and Baker, D.: Abnormal leukocyte kinetics-an explanation for the enzyme abnormalities observed in trisomy-21 (Down’s syndrome). Abst. 36th Annual Meeting Soc. Ped. Res., Atlantic City, May 1966, p. 9. Rabinowitz, Y.: Separation of lymphocytes, polymorphonuclear leukocytes and monocytes on glass columns, in- Raab, From www.bloodjournal.org by guest on August 9, 2017. For personal use only. LYMPHOCYTE 6. 7. 8. AND 10. 11. 12. 13. ENZYME eluding tissue culture observations. Blood 23:811, 1964. Garvin, J. E.: Factors effecting the adhesiveness of human leucocytes and platelets in vitro. J. Exp. Med. 114: 51, 1961. Bessey, 0. A., Lowry, 0. H., and Brock, M. J.: A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164:321, 1946. Shih, L. Y., Wong, M., and Makler, 9. (;RANULOCYTE P., Inouyc, and Mahanand, D.: Biochemical differentiation of trisomic Down’s syndrome (mongolism) from that due to translocation. New Eng. J. Mcd. 273:1356, 1965a. 14. 15. 16. T., Hsia, D. Y. Y.: Enzymes in Down’s syndrome. Lancet 2:746, 1965. Alter, A. A., Lee, S. L., Pourfar, M., and Dobkin, C.: Studies of leukocyte alkaline phosphatase in mongolism: a possible chromosome marker. Blood 22:165, 1963. Brandt, N. J., Froland, A., Mikkclsen, M., Nielsen, A., and Toistrup, N.: Galactosaemia locus and the Down’s syndrome chromosome. Lancet 2: 700, 1963. Baikie, A. C., Loder, P. B., deCruchy, G. C., and Pitt, D. B.: Phosphohexokinase activity of erythrocyte in mongolism. Another possible marker for chromosome 21. Lancet 1:412, 1965. Mellman, W. J., Oski, F. A., Tcdesco, T. A., Coelho, A. M., and Harris, H.: Leucocyte enzymes in Down’s syndrome. Lancet 2:674, 1964. Rosner, F., Ong, B. A., Paine, R. S., 673 ACTiVITY 17. 18. 19. Cartwright, C. E., Athens, J. W., and Wintrobe, M. N. : The kinetics of granulopoiests in normal man. Blood 24:780, 1964. Donnell, C. N., Ng, W. C., Bergren, W. R., Melnyk, J., and Koch, R.: Enhancement of crythrocyte-galactokinase activity in Langdon-Down Trisomy. Lancet 1:553, 1965. Hsia, D. Y. Y., Inouye, T., Wong, P., and South, A.: Studies on galactose oxidation in Down’s syndrome. New Eng. J. Med. 270:1085, 1964. DeMars, R.: Some studies of enzymes in cultivated human cells. Nat. Cancer Inst. Monogr. 13:181, 1964. Cox, R. P.: Regulation of alkaline phosphatase in skin fibroblast bultures from patients with mongolism. Exp. Cell Res. 37:690, 1965. Nadler, H. L., Inouye, T., and usia, D. Y. Y.: Enzymes in cultivated human fibroblasts derived from patients without Down’s syndrome (mongolism). 20. 21. 22. Rabinowitz, Y.: DNA polymcrase and carbohydrate metabolizing enzyme content of normal and leukemic glass column separated leukocytes. Blood 27:470, 1966. Kaplow, L. S.: A histochemical procedure for localizing and evaluating leucocyte alkaline phosphatase activity in smears of blood and marrow. Blood 10:1023, 1955. Brodell, H., and Swisher, S. N.: Studies of leucocyte alkaline phosphatase determined by a clinically applicable histochemical method. Clin. Res. Proc. 2:58, 1954. From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1967 30: 669-673 Lymphocyte and Granulocyte Enzyme Activity in Patients with Down's Syndrome HENRY L. NADLER, PATRICIA L. MONTELEONE, TOHRU INOUYE and DAVID YI-YUNG HSIA Updated information and services can be found at: http://www.bloodjournal.org/content/30/5/669.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.