Polyamine compartmentalization in various human disease states

1

Clinica Chimica Acta, 82 (1978) 1-7 0 Elsevier/North-Holland Biomedical Press

CGA 8950

POLYAMINE COMPARTMENTALIZATION DISEASE STATES

IN VARIOUS

HUMAN

KEVIN D. COOPER **, JAYESH B. SHUKLA *** and OWEN M. RENNERT * Division of Genetics, Endocrinology and Metabolism, Departments of Pediatrics, Biochemistry and Neuroscience, Uniuersity of Florida College of Medicine, Gainesville, Fla. 32610 (U.S.A.) (Received May 27th, 1977)

summary Whole blood was separated into preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets and plasma. Each preparation was analyzed for the concentration of the polyamlines putrescine, spermidine and spermine. This was done in 17 controls, 14 patients with psoriasis, four patients with hereditary elliptocytosis, two patients with chronic lymphocytic leukemia and one patient each with lung cancer, non-Hodgkin’s lymphoma, sickle cell anemia with mild psoriasis, and progeria. In patients with elevated blood polyamine levels, absorption onto erythrocytes was relatively common, and the spermine/spermidine ratio was useful in localizing abnormalities and characterizing the nature of the polyamine alteration. Proliferative states were associated with elevated spermine/spermidine ratios relative to controls while this relationship was reversed in erythropathies such as hereditary elliptocytosis and sickle cell anemia.

Introduction Polyamines are universally distributed in all living cells and in all probability have essential roles in cellular metabolism related to cell growth [l], RNA and protein synthesis [ 11, cyclic nucleotide metabolism [ 21 and membrane bound enzymes [3,4]. In human disease states, whole blood polyamines are elevated * Address all correspondence and reprint requests to: O.M. Rennert. M.D., University of Oklahoma Health Science Center. Department of Pediatrics, Oklahoma City, P.O. Box 26901, Okla. 73190. U.S.A. ** Resent address: Department of Dermatology, University of Oregon Health Science Center. Portland, Oreg.. U.S.A. *** Present address: Department of Biochemistry, University of Oklahoma Health Science Center. Oklahoma City, Okla.. U.S.A.

2

in patients with cancer [ 51 and leukemias [ 61, polycythemia vera [ 71, psoriasis [ 81, sickle cell anemia [ 91, and are altered in patients with cystic fibrosis [lo]. To define a framework for understanding these abnormalities we studied the polyamine localization in cellular components of human blood and found that although nucleated cells (lymphocytes and granulocytes) contain lo3 times more polyamine than anucleated cells (RBC’s and platelets), erythrocytes contribute the most significant proportion of whole blood polyamine, whereas minute amounts circulate in plasma [ll]. Polyamine concentrations are markedly increased in young erythrocytes (reticulocyte rich) over red cells of successively increasing ages [ 111. With further elucidation of alterations of blood polyamines in mind, we have undertaken exploration of cellular polyamine distribution in disease states characterized by proliferation of specific blood cellular elements, non-hematologic cellular proliferation, and erythrocyte abnormalities, in particular an intrinsic red cell membrane defect as proposed for hereditary elliptocytosis [ 12-181. Distribution data is presented here on chronic lymphocytic leukemia [ 21, lymphoma [ 11, psoriasis [ 141, large cell anaplastic carcinoma of lung [ 11, progeria [ 13, sickle cell anemia coexisting with mild psoriasis [ 11, and four family members with hereditary elliptocytosis. Methods, A. General

The patients presented were not receiving chemotherapy, radiation therapy, or blood transfusions for their disease except for the patient with non-Hodgkin’s lymphoma who had received a single course of Cytoxan-Hydroxydaunorubicin (adriarnycin)-Oncovin (vincristine)-Prednisone (CHOP) chemotherapy six weeks prior to the study. Heparinized venous blood was collected 2 h p.c. and all component cellular separations were immediately performed. B. Erythrocyte

separation

Whole blood was centrifuged at 700 Xg for 20 min prior to removal of serum and careful aspiration of buffy coat leukocytes. The red blood cells were subsequently washed five times in Balanced Salt Solution and centrifuged as above with buffy coat aspiration each time. l-2 ml resuspended red blood cell suspension was prepared for polyamine analysis. C. Whole blood, preparations

mononuclear

cell, polymorphonuclear

cell, platelet

and plasma

Whole blood, mononuclear cell (MNC), polymorphonuclear cell (PMNC), platelet and plasma separation were performed as described previously [ 111. Leukocytes were separated by Ficoll-Hypaque discontinuous density gradients and platelets and plasma were separated by differential sedimentation. Acid precipitation of each sample followed by lyophilization was performed prior to analysis on a Durrum D500 (Durrum Instrument Corp.) high pressure amino acid analyzer [ 111.

3 TABLE I SPERMINE CONCENTRATIONS

IN CELLULAR

COMPARTMENTS

OF BLOOD

Whole blood and plasma expressed in nm/ml. otherwise as nm/lOg cells. +S.E.M. where applicable. WB. whole blood; RBC, red blood cells; MNC. mononuclear cells; PMNC. polymorphonuclear cells: Plts, platelets; Pla, plasma: Tr, unquantifiable trace amount of substance detectable.

Controls (N = 17) CLLWM (N = 1) CLLBS (N = 1) Lymphoma (N = 1) Lung cancer (N = 1) Psoriasis (N = 14) Progeria (N = 1) HgbSS+(iV=l) mild psoriasis Hereditary eIIiptocytosis (N = 4)

WB

RBC

MNC

PMNC

3.92 ?: 0.36 23.05 12.1 14.05 13.8 6.69 k 0.99 6.14

0.48 + 0.04 0.48 1.60 1.31 1.88 0.89 + 0.14 0.55

440 + 61 943 246 718 549 480 ? 67 321

493 f 56 287 160 389 525 256? 43 145

< 0.043 Tr Tr Tr Tr Tr -

12.8

1.69

-

-

-

Tr

Tr

Tr

4.15

0.36

301

Pla

Pits

100

0.030 0.15 0.15 Tr Tr 0.031 T?

+ 0.01

f 0.01

Results Values obtained for the various diseases in each cellular compartment for spermine, spermidine and putrescine are presented in Tables I, II, III and IV respectively and the spermine/spermidine (Spm/Spd) ratios depicted graphically in Fig. 1. Perturbations were mainly limited to spermine and spermidine with putrescine remaining remarkably stable, which may be due to tight control of circulating putrescine levels since putrescine exerts important modulating effects on ornithine decarboxylase and spermine and spermidine synthetase [ 11. In fact, putrescine may be tightly bound to blood peptides which would not be detectable by our measurement of the acid soluble free polyamines (Seale, T., personal communication). Polymorphonuclear cells demonstrated marked fluctuations in Spm/Spd ratio and this may be due to an artifact of the manipulations involved in cell separation, since granulocytes are quite sensitive to environmental changes.

TABLE II SPERMIDINE CONCENTRATIONS

Controls (N = 17) CLLWM (N = 1) CLLBS (N = 1) Lymphoma (N = 1) Lung cancer (N = 1) Psoriasis (N = 14) Progeria (N = 1) HgB SS + mild psoriask (N = 1) Hereditary eIliptocytosis (N = 4)

IN CELLULAR

COMPARTMENTS

OF BLOOD

WB

RBC

MNC

PMNC

Pits

Pla

6.56 ? 0.39 12.3 18.1 12.85 6.50 7.25 + 0.53 9.6

0.815 2 0.07 0.767 1.84 1.16 1.07 1.03 2 0.10 1.13

226 f 28 126 117 333 275 216 + 66 139

207 f 45 13 47 55 12 93 + 18 11

0.44 + 0.05 0.29 -

0.08 f 0.02 0.10 -

0.61 0.53 0.42 t 0.06 -

0.15 0.10 0.14 f 0.03 0.72

27.5

3.74

14.8

1.62

191

80.3

0.60

0.45 0.03

4 TABLE

III

PUTRESCINE

CONCENTRATIONS

IN CELLULAR

WB

RBC

Controls (N = 17) CLLWM (N = 1) CLLBS (N = 1) Lymphoma (N = 1) Lung cancer (N = 1) Psoriasis (N = 14) Progeria (N = 1)

0.214 0.65 0.66 0.40 0.28 0.217 0.19

HgB SS + mild psoriasis (N = 1)

0.40

0.06

Hereditary elhptocytosis

0.29

0.08

TABLE

(N = 4)

t 0.02

0.065 0.05 0.19 0.07 0.13 0.069 0.03

t 0.02

t 0.02

OF BLOOD

MNC

PMNC

Pits

220 t 50 17 4 179 137 120 t 30 84

410? 80 23 77 167 50 130 ? 50 290

0.21 0.14 0.69 0.31 0.18 0.22

263

80.2

0.42

COMPARTMENTS

OF BLOOD

Pla + 0.03

? 0.06 -

0.08 0.10 0.08 0.10 0.10 0.12 0.10

-

0.10

+ 0.01

i 0.02

0.035

IV

SPERMINE/SPERMIDINE

RATIOS

IN CELLULAR

WB Controls (N = 17) CLLWM (N = 1) CLLBS (N = 1) Lymphoma (N = 1) Lung cancer (N = 1) Psoriasis (N = 14) Progeria (N = 1) HgB SS + mild psoriasis (N = 1) Hereditary elliptocytosis (N = 4) HgB SS (N = 3)

b

? 0.01

COMPARTMENTS

Whole Blood

0.60 1.87 0.67 1.09 2.12 0.97 0.64

RBC + 0.05

f 0.14

0.70 0.63 0.87 1.13 1.76 0.93 0.49

0.46

0.45

0.28 0.19

0.22 0.21

PMNC

MNC + 0.39

+ 0.68

0.184 7.48 2.10 2.16 2.00 1.97 2.31

f 0.27

f 0.31

3.36 20.8 3.4 7.1 43.7 4.16 13.2

f 0.80

f 1.6

1.58

-

1.24

-

Erythrocytc

0

l l

Fig. 1. Spm/Spd ratios in whole blood and erythrocytes. Data is presented graphically to demonstrate elevation in Spm/Spd ratios in proliferate diseases and depression in erythropathies (P&S. SS, HE). Co, controls; Ps. psoriasis; CL, chronic Iymphocytic leukemia (WM); Oat, lung cancer; Lym. Iymphoma; Prg, progeria; CLb, chronic lymphocytic leukemia (BS); P&S. sickle cell anemia with mild psoriasis: SS. sickle ceII anemia; HE, hereditary elliptocytosis.

5 TABLE V HEREDITARY

ELLIPTOCYTOSIS

Data on four family members with marked elliptocytosis

in peripheral blood.

Iict. hematocrit:

Retie.

reticulocyte count: WBC. white blood ceII count; WB. whole blood: RBC. red blood cells. Patient

Controls (N = 17) PP EP GP GR j;

Hct

Retie

(a)

(%)

42+ 31 33 42 33

5

O-l.5 2.5 1.2 2.8 1.6

RBC @m/lo9

WB @m/ml)

cells)

WBC (x106/

Put

Spd

Spm

Put

Spd

Spm

ml)

0.26 0.43 0.19 0.30 0.24 0.29

6.23 18.7 9.1 16.1 15.3 14.8

3.96 3.9 2.7 5.8 4.2 4.15

0.078 0.076 0.056 0.045 0.137 0.078

0.856 2.37 1.21 1.05 1.84 1.62

0.515 0.379 0.310 0.386 0.377 0.363

7.8 -I 3 6.1 4.0 5.4 7.2 5.7

Table V presents data on four members of a family with hereditary elliptocytosis. The hematocrit, reticulocyte count and white blood cell count are shown with the corresponding whole blood and erythrocyte polyamine levels. A substantial increase in whole blood and red cell spermidine only is observed, with distinctive depression of the Spm/Spd ratio in both whole blood and erythrocytes (Fig. 1). This is not related to white cell count or hematocrit (red cell count) but may be proportional to the degree of reticulocytosis. Discussion The two patients with chronic lymphocytic leukemia (CLL) studied had markedly elevated white cell counts (CL&, = 99 000 X 106/ml, 97% of which were small lymphocytes and smudge cells; CLLss = 92 000 X 106/ml, 64% lymphocytes, 10% immature lymphocytes, and 20% granulocytes). Based on erythrocyte count8 only 10% of blood spermine and 32% of blood spermidine is contributed by erythrocytes in CLLwM and in CLLss 36% of the blood spermine and 27% of the blood spermidine is contributed by red cells, as contrasted to these controls in which 2/3 of both whole blood spermine and spermidine is contributed by erythrocytes. Thus the polyamine elevations in these two patients is probably due to an increased white cell count. This is also demonstrated by an elevation in the Spm/Spd ratio in CLbM lymphocyte8 (MNC) which is reflected in the elevated whole blood Spm/Spd ratio, whereas in CLLas, where the lymphocyte Spm/Spd ratio is not remarkable, there is no elevation in whole blood Spm/Spd ratio (Fig. 1). In the patient with non-Hodgkin’s lymphoma, poorly differentiated lymphoma, Stage IVB, there was localized lymphocytic proliferation but no circulating abnormal lymphocyte8 on peripheral smear. Both spermine and spermidine are increased in the erythrocytes and whole blood and an elevated Spm/Spd ratio is seen in the blood, red cells and lymphocytes. The patient with lung cancer had a large cell anaplastic carcinoma metastatic to brain and liver. It is reasonable to postulate that the increased polyamine production associated with the rapid tumor growth in these patients result8 in

6

blood equilibration of polyamines between tumor, plasma, erythrocytes and leukocytes. Psoriasis is characterized by a rapidly proliferating, relatively undifferentiated epidermis with a dramatically shortened epidermal turnover time from 28 days to 3 or 4 days [ 191. Again we see elevated whole blood and erythrocyte spermine and spermidine levels with an increased Spm/Spd ratio in red cells and blood, but this time without leukocyte involvement. Of interest is that psoriatic epidermis may have deficient epidermal adenylate cyclase activity [20,21]. This finding is in question [22]. A tempting postulate would be that elevated polyamines suppress epidermal adenylate cyclase [2] resulting in lowered CAMP levels and worsening of psoriasis. The elevated polyamine levels probably reflect the accelerated formation and degradation of keratinocytes and correlates with the extent of cutaneous involvement. Progeria is a childhood disease of unknown etiology but which is often considered a disease of premature aging. There are no marked polyamine abnormalities except for slightly decreased erythrocyte putrescine and some leukocyte Spm/Spd elevations. We separated the blood of a patient with sickle cell anemia (SS HgB) and mild psoriasis. Polyamine alterations are that of sickle cell disease [23] : markedly increased spermidine concentrations and appreciably elevated spermine levels with consequent distinctive lowering of the Spm/Spd ratio in whole blood and erythrocytes. This depression of the Spm/Spd ratio was also seen in the whole blood and erythrocytes of three patients with sickle cell anemia (Fig. 1, Table IV). Hereditary elliptocytosis is an autosomal dominant erythropathy characterized by the presence of greater than 25% (up to 90%) of oval and elliptical erythrocytes in the peripheral blood [ 181. Evidence suggests there is an intrinsic membrane defect with abnormal membrane cholesterol [14], ATP and 2, 3DPG instability [17], increased sodium efflux [15], ATPase and aldolase deficiencies [ 121, and an abnormal membrane protein electrophoretic pattern r131. In these patients we found an increase in blood and red cell spermidine and a suggestive decrease in red cell spermine, and consequently a lowering of the Spm/Spd ratio as was seen in sickle cell anemia. Patient EP was the mother of this family and had a concurrent iron deficiency anemia secondary to multiparity which has blunted her reticulocyte count. She also has the lowest spermidine levels of the family members studied. This is consistent with the postulate that the spermidine elevation is due to a young, reticulocyte rich erythrocyte population [ 121, as is also seen in sickle cell anemia. Our data on red cell aging revealed that old erythrocytes had a Spm/Spd ratio of 0.69 while young erythrocytes had a Spm/Spd ratio of 0.42, also consistent with this idea. Alternatively, the unique polyamine profile in these patients may be due to altered polyamine binding on erythrocytes with an intrinsic membrane defect. Thus the abnormal HE erythrocyte has altered polyamine binding sites which results in a selective binding of spermidine only. There is evidence for multiple independent binding sites for polyamines on normal and sickling red blood cells [23] and that polyamines, when bound to these sites, cause relocalization of the surface charge density of the red cell and a perturbation of the membrane

7

in terms of increased thickness and interfacial viscosity [ 231. Thus it is conceivable that an abnormal polyamine binding pattern secondary to the intrinsic membrane defect in hereditary elliptocytosis may play a role in the morphology of the disease. Conclusion In human diseases with elevated blood polyamine levels, absorption onto erythrocyte polyamine sites is relatively common, but the Spm/Spd ratio is useful in localizing abnormalities and characterizing the nature of the polyamine alteration. Proliferative states are associated with elevated Spm/Spd ratios while in erythropathies such as hereditary elliptocytosis and sickle cell anemia this relationship is reversed. White blood cell count elevations may elevate whole blood polyamine levels. Acknowledgements This work was funded by a training grant from the National Institutes of Health No. AM07164, and research grants from the Cystic Fibrosis Foundation, the Boehringer Mannheim Corporation and the Hayward Foundation. Special thanks go to Dr. Alan Keitt for the use of the Coulter counter, and to Dr. Craig Kitchens and Elwood Headley, Department of Medicine, for providing the patients for this study. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Raina, A. and Janne, J. (1975) Med. Biol. 53.121-147 Wright. R., BeuhIer, B.. Schott, S. and Rennert, 0. (1977) Pediatr. Res.. in press Kossorotow, A., Wolf, H.U. and SeiIer, N. (1974) Biochem. J. 144.21-27 Peter, H.W., Wolf, H.U. and SeiIer, N. (1973) Hoppe Seyler’s Z. Physiol. Chem. 354. 1146-1148 Marton, L.J.. Vaughn, J.G.. Hawk, LA.. Levy. C.C. and Russell. D.H. (1973) in Polyamines in Normal and Neoplastic Growth (RusseU. D.H.. ed.), p. 636, Raven Press, New York Rennert. 0.. Miale. T., ShukIa, J., Lawson, D. and Frias, J. (1976) Blood 47.695-701 Desser. H.. Hacker, P.. Weiser, M. and Bohnel. J. (1975) CIin. Chim. Acta 63, 243-247 Proctor, M.S., Fletcher. H.V.. ShukIa. J.B. and Rennert. 0. (1975) J. Invest. Dermatol. 65.409411 Chun, P.W., Rennert. O.M., ShukIa. J.B.. Saffen. E.E. and Taylor, W.J. (1977) Biophys. Chem. 6. 321-326 Rennert, 0. and Mangos, J. (1976) G.A.P. Conference on Polyamines. N.I.H., Bethesda, Md.. Cystic Fibrosis Foundation, Janusry 28-30.1976 Cooper, K.D., ShukIa. J.B. and Rennert, O.M. (1976) CIin. Chim. Acta 73.71-88 Torlontano, G., Fontana. L.. De Laurezi. A., Papa, G. and Proietti. M. (1972) Acta Haematol. 48, l-11 Gomperts. E.D., Cayannis, F., Metz, J. and ZaiI. S.S. (1973) Br. J. Haematol. 25.415-420 Murphy, J.R. (1965) J. Lab. CIin. Med. 65, 756 Schartum-Hansen, H. (1935) Acta Med. Stand. 86.348 Rebuck, J.W. and Van Slyck. E.J. (1968) Am. J. Clin. Pathol. 49.19 De Gruchy, G.C. (1962) Br. J. Haematol. 8,168 Wintrobe. M.W. (1974) Clinical Hematology, Lea and Febiger. PhiIadelphia Weinstein, G.D. and Van Scott. E.J. (1965) J. Invest. Dermatol. 45. 257 Voorhees, J.J.. Stawiski. M.. DuelI, E.A.. Haddox, M.K. and Goldberg, N.D. (1973) Life Sci. 3.639653 Voorhees. J.J., Marcels, C.L. and Due& E.A. (1975) J. Invest. Dermatol. 65. 179-190 K.. Levine, V.. Mui, M.M. and Hsia, S.L. (1975) J. Invest. HaIprin. K.M.. Adachi. K., Yoshikawa, Dermatol65.179-178 Chun, P.W., Rennert. O.M., Saffen, E.E., DiTore. R.J., ShukIa. J.B.. Taylor, WJ. and Shah. D.O. (1976) CoUoids Interface Sci. 5, 23-51