Salicylate effects on the response of human lymphocytes to phytohemagglutinin isolectins

GLINtCAL IMMUNOLOGY

AND

I?v~%ILJNOPATHOLOGY IO, I- 10 (1978)

alicylate Effects on the Response mphocytes to Phytohemagglutinin MERRILL

J. EGORIN,

ROKALD

L. FELSTED,

of Human Bsolectins

AND NICHOLAS

R. BACXUR

Although aspirin (ASA) inhibits the response of cultured lymphocytes to phytohemagglutinin (PHA). it is controversial whether lymphocytes from ASA-treated humans respond normally to PHA. We treated 12 volunteers with ASA at levels of 600 or 900 mg four times a day. Their lymphocytes were isolated before and during therapy and 1 week after the cessation of therapy, cultured in RPM1 1640 medium or minimal essential medium (MEM) with purified PHA isolectins. and assessed for L3H]thymidine incorporation and glucose utilization. Lymphocytes from normal donors were also purified and cultured with ASA in RPM1 1640 medium, MEM, or autologous plasma and assessed under PHA isolectin stimulation. The lymphocytes from ASA-treated volunteers responded normally to L, and E, PHA isolectin stimulation whether obtained during ASA treatment or not and regardless of the culture medium used. However, addition of up to 40 mg/lOO ml of ASA to L,-stimulated cultures produced a dose-dependent inhibition of blastogenesis and glucose utilization. This inhibition was substantially more pronounced in MEM than in RPM1 1640 or plasma. Supplementation of MEM with amino acid and vitamin components of RPM1 1640 failed to reduce the degree of ASA inhibition. Aging of RPMI 1640 reduced its protective effect against ASA inhibition, indicating that the responsible factor was labile. Salicylate did not affect the binding of L, to lymphocytes in either medium. INTRODUCTION

Acetylsalicylic acid, aspirin, is one of the most commonly used drugs, due to its efficacy as an analgesic, antipyretic, and antiinflammatory agent and its lsw cost and relative safety. Investigations have been directed at the effects of aspirin on numerous aspects of cellular physiology. In the case of phytohemaggluti~i~ (PI-IA)-induced lymphocyte transformation, controversy exists over the effects of salicylate treatment. Zn ~,if~o studies have shown that aspirin added to the cultures of normal human lymphocytes inhibits the blastogenic response of these ceils to crude preparations of PHA (1, 2). In contrast, lymphocytes from patients treated with therapeutic doses of aspirin gave varying responses when stimulated by crude PHA (3, 4). Since those earlier studies, we have resolved and purified PHA into five tetrameric isolectins: L,, L3E1, L,L,, L,E3, and E, (5). The mitogenic and eryrlsroagglutinating activities of this family of purified plant proteins have been characterized and shown to be proportional to the number of L and E subunits per tetrameric protein molecule (6). Because of the controversy regarding the effects of aspirin on lymphocyte response to PHA and the availability of purified PHA isolectins that allow a more precise investigation of the process, we have studied the PHA-induced transfor-

0090-122917810101 -OOO:$Ol.OO/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

2

EGORIN,

FELSTED,

AND

BACHCR

mation of lymphocytes from aspirin-treated volunteers, the effect of aspirin added to cultures of PHA-stimulated lymphocytes from untreated donors, and the influence of tissue culture media on these processes. MATERIALS

AND METHODS

Experimentcli Desigrl Twelve Caucasian males, 17-22 years of age, were studied in groups of three and treated with 600 or 900 mg of aspirin orally (PO) four times a day (qid) for 1 week. No volunteer had any significant past medical history, allergies, asthma, history of bleeding disorders, or history of adverse reactions to aspirin. No other medications were used by any study subject. Lymphocytes from these volunteers were obtained and studied prior to and after 1 week of aspirin administration and 1 week after cessation of aspirin treatment. After the conclusion of these in vitro studies, lymphocytes from the previously treated and other comparable untreated volunteers were isolated and cultured with purified PHA isolectins and different concentrations of aspirin in plasma or chemically defined media. The results of the in \,itro studies were compared to the previous in \jilBo experiments. PHA Purification PHA isolectins L, and E, were isolated as previously described (5) from extracts of red kidney beans (Phaseollrs l,ulgaris) by affinity chromatography on Sepharose-bound porcine thyroglobulin and subsequent ion-exchange chromatography on SP-Sephadex (Pharmacia, Upsala, Sweden). Isolectin purity was confirmed by polyacrylamide disc gel electrophoresis according to the method of Reisfeld (7). Protein concentrations were determined by the method of Lowry er al. (8) with bovine serum albumin (Sigma Chemical Co., St. Louis, Missouri) as a standard. Lymplwcyte Isolatiorl Lymphocytes were purified from heparinized peripheral blood by sedimentation in a Ficoll (Sigma)-Hypaque (Winthrop Laboratories, New York, New York) gradient followed by three washes with 0.154 A4 saline (9). The sedimented lymphocytes were resuspended to a final cell concentration of 3 I 0.2 x 106/ml in either RPM1 1640 medium (NIH Media Unit, Bethesda, Maryland) or minimal essential medium containing 50 units of penicillin, 50 pg, of streptomycin, 2 pmol of L-glutamineiml, and 22.5% fetal bovine serum (Flow Laboratories, Rockville, Maryland). Cell counts were obtained on a Model ZB, Coulter counter. The cellular composition of the final lymphocyte preparations was evaluated with Wrightstained smears. Lymphocytes composed 98% or more of total leukocytes. Erythrocyte contamination was negligible, and there were two to five platelets per lymphocyte in the final suspension. Incubntions \tYtll lsolectins In vivo aspirin treatment. The incubations were done in quadruplicate. One milliliter of cell suspension was placed into a sterile 16 x 150-mm glass culture tube and fitted with a stainless steel cap (Bellco Glass, Vineland. New Jersey).

SALICYLATE

EFFECTS

ON

HUMAN

LYMPHOCYTES

3

Solutions of L, or E, in either RPM1 1640 medium or MEM without fetal calf serum were sterilizedby filtration through 0.22-pm filters (Millipore Corp.? Cambridge, Massachusetts), and 0.5-ml aliquots containing appropritate amounts of isolectin were added to each of the quadruplicate cultures. The cultures were incubated at 37°C in an atmosphere of 95% air, 5% CC&, and 95% humidity. 1r1 vitro aspirin treatment. The incubations were done as in the in vivo aspirintreated cultures with two notable differences. First, lymphocytes from each donor were studied simultaneously in RPM1 1640 and MEM and at times also in human plasma or MEM supplemented with various amino acids and vitamins. Second. the only concentrations of L, and E, used were 10 and 30 pgiculture, respectively (i.e., those that gave maximal stimulation in the in ~i\~o treatment experiments). Lectins were added in 0.25-ml amounts to the appropriate medium. Medium. 0.25 ml, containing the appropriate amounts of acetylsalicylic acid (Malinkrodt Chemicals, St. Louis, Missouri) constituted the remainder of the 1.5-ml final culture volume. Assay of [3H]thyrnidine ilzcovporatiorz. Four hours prior to the termination of incubation, 0.5 ml of medium containing 2 &i of methyl [3H]thymidine (New England Nuclear, Boston, Massachusetts) was added to three of every four sets of cultures. At the end of the incubation [3H]thymidine incorporation into trichloroacetic acid-precipitable material was assayed by the previously described method (5). Assay of glucose. Aliquots of the fourth tube of each quartet group were assayed for glucose content by the previously described o-toluidine calorimetric method (10). Viability arid contamination. Aliquots, 0.5 ml, of the fourth tube of each set of cultures were examined using light microscopy and trypan blue. All cultures were greater than 95% viable with no bacterial or fungal contamination. Tlzin-layer Chromatography of in Vitro Salicylate Additilaes Acetylsalicylic acid was converted to its sodium salt with 2 N sodium hydroxide and dissolved in tissue culture medium. Ascending thin-layer chromatography of these solutions was performed on 250-pm silica gel G plates (E. Merck, Darmstadt, Germany) in system I (methanol:acetic acid:diethyl ether:benzene, 1: 18:60: 120) or system II (butyl acetate:chloroform:formic acid, 60:40:20). Salicylates were detected by examination under ultraviolet light and compared against simultaneously chromatographed aspirin and salicyclic acid standards. Saiicyiate Concentrations Plasma salicylate concentrations of each volunteer were determined by the method of Trinder (11) on blood samples obtained when lymphocytes were obtained. In the in vitro studies, a similar methodology was used to determine salicylate concentrations in aliquots of culture supernatants. L4 Birldirlg Radioiodination of L, isolectin was accomplished by the chloramine-T method with lrsI (New England Nuclear, protein iodination grade in 0.1 M NaGH) as described by Kornfeld (12). This procedure did not alter the erythroagglutinating

4

ECORIN,

FELSTED,

4ND

BACHCR

titer or the electrophoretic characteristics of the iodinated protein as compared to the unlabeled L, used in the reaction. Binding studies were performed in 12 x 75-mm polypropylene tubes (Falcon, Oxnard, California) that had been precoated with a solution of 0.5 g/l00 ml of bovine serum albumin in 0.154 M NaCl. Isolated lymphocytes were suspended to a cell concentration of 107/ml in either RPM1 1640 or MEM, each containing 10 mgiml of bovine serum albumin. Cell suspension, 0.2 ml, was added to 0.2 ml of the appropriate medium containing 10 pg of 12s1-labeled L, and concentrations of salicylate comparable to those used in the in vitro culture experiments. Reactions were performed in duplicate and carried out with gentle shaking at 25°C in a water-saturated 5% CO, atmosphere. After 2 hr of incubation 3 ml of ice-cold 0.154 M NaCl was added to each tube and the contents of the tube were deposited onto a OS-km Celotate filter (Millipore Corp., Bedford, Massachusetts) with a multiple filtering manifold (NIH Biomedical Engineering Instrumentation Branch, Bethesda, Maryland). The reaction tubes were rinsed once, and the filters were subsequently washed twice with 0.154 M NaCl before the individual filters were placed into gamma counting tubes and assayed on a Baird Atomic Model 530 gamma spectrometer. RESULTS

In Viva Salicylate Treatment Salicylate concentrations nnd adl,erse effects ,tlith therapy. Prior to or 1 week after the cessation of salicylate therapy, plasma blood salicylate concentrations of volunteers were less than 1 mgi100 ml in all cases except one. That sample concentration was 2 mg/lOO ml. Salicylate concentrations in the three individuals treated with 600 mg qid were 5.4, 8.0, and 10.7 mg/lOO ml, whereas salicylate concentrations in subjects treated with 900 mg qid were 8.0, 10.0, 12.0, 12.0, 14.0, and 15.0 mgi100 ml. The major complaint attributable to salicylate therapy was mild gastrointestinal upset. Two subjects in the higher dosage group complained of transient tinnitus during treatment. There were no episodes of melena or decreases in hematocrit or hemoglobin greater than expected for the volume of blood drawn for the study. FHlThymidine incorporation. Aspirin therapy did not affect any aspect of the DNA synthesis elicited by L, and Eq isolectin stimulation of lymphocytes cultured in either RPM1 1640 or minimal essential medium. More specifically, aspirin therapy failed to reduce peak DNA synthesis or to alter the time course of DNA synthesis in response to PHA (Fig. 1). Lymphocytes isolated from subjects before and during aspirin treatment showed no alteration in the dose-response relationship to either PHA isolectin (Fig. 2). Aspirin therapy did not affect the relative mitogenic activity of the L, and E, isolectins, with L, remaining approximately 30 times more potent. Finally, [JH]thymidine incorporation in lymphocytes obtained 1 week after cessation of salicylate treatment was similar to that in pretreatment and treatment cultures. Glucose utilization. Aspirin therapy did not affect the rate of glucose utilization of resting or La-stimulated lymphocytes (Table 1). L4, 1.1 ,ug, increased glucose metabolism over unstimulated cultures, and L, continued to provoke the greatest increase in glucose utilization. Although the E, doses used in this study were

SALICYLATE

EFFECTS

OS

HUMAN

LYMPHOCYTES

5

FIG. i. Time course of [3H]thymidine incorporation by lymphocytes from treated volunteers. (A) &e-therapy, (B) during therapy, and (C) post-therapy. L,, L, isolectin (10 ~giculture); E,, E, isolectin (30 ~giculture). Each point represents the mean i: SEM of six vo!unteers. Each experiment was done in triplicate.

insufficient to produce the maximal glucose utilization possible with this isoiectin, there were no differences in the rates of utilization produced in untreated and aspirin-treated lymphocytes. As previously described (lo), concentrationdependent isolectin stimulation of glucose utilization paralleled the stimulation of [3H]thymidine incorporation (Fig. 3), and salicylate therapy did not alter this relationship.

L4 A-----4

A

FIG. 2. Dose response to isolectin stimulation of [‘Hlthymidine incorporation by lymphocytes from aspirin-treated volunteers. (A) Pre-therapy, (B) during therapy, and(C) post-therapy. Each point represents the mean t SEM of six volunteers. Each experiment was done in triplicate.

Isolectin dose Control L,, 10 ,kg/culture L,, 3.3 ~giculture L,, 1.1 pgiculture E,. 30 ~giculture E,. 10 ~giculture E,. 3.3 pgiculture

Pre-therapy 1.0 26.1 22.3 10.6 5.5 2.4 1.6

i- 0.4” i 1.6 t 1.8 t 1.7 +- 1.4 -c 0.5 +- 0.5

During therapy 2.1 27.9 26.1 12.8 9.4 4.3 3.4

it 2 t ir i i

0.7 1.0 1.5 0.8 2.5 1.1 1.2

” The rates are expressed as micrograms of glucose per hour per 3 the mean f SEM of mean rates of six volunteers.

x

Post-therapy 2.9 26.4 21.7 11.4 7.1 4.1 4.3

i r 2 t ik 2

0.7 1.6 2.0 2.1 1.8 0.9 0.7

10Fcells. The values represent

In Vitro Scrlicylate TI-entment rH]Thymidirze incorpoxrtiorz. Chemically determined salicylate concentrations of the experimental cultures agreed closely with the expected theoretical concentrations. In paired experiments with lymphocytes from the same donor, the addition of increasing concentrations of salicylate to lymphocyte cultures in either RPM1 1640 or human plasma under optimally stimulatory concentrations of L, produced only moderate inhibition of LA-induced blastogenesis (Fig. 4). However, this inhibitory effect of salicylate was much greater in the lymphocytes cultured in MEM. This medium-dependent difference was noted in all in \itvo experiments, although RPM1 1640 and MEM were equally effective in supporting LA-stimulated thymidine incorporation by control cultures (Table 2). Mitigating medium component in RPMI 1640. To determine why RPM1 1640 suppressed the ability of aspirin to inhibit the PHA-induced lymphocyte transformation, several modifications and alterations of the two media were carried out. The exposure of RPM1 1640 medium at room temperature to fluorescent lights

FIG. 3. Dose response to isolectin stimulation of glucose utilization by lymphocytes from aspirintreated volunteers. (A) Pre-therapy. (B) during therapy, and (C) post-therapy. Each point represents the mean t SEM of the mean rate of six volunteers.

SALICYLATE

FIG.

ohocytes. Methods. described

EFFECTS

ON

HUMAN

LYMPHOCYTES

4. III l~itro salicylate-mediated inhibiton of L,-induced Lymphocytes were grown for 2.5 days and assayed Aspirin was neutralized and added, and salicylate under Materials and Methods. Each point represents

[)H]thymidine incorporation in iymfor [3H]thymidine as described under concentrations were determined as the mean f SEM of three samples.

destroyed the ability of this medium to reduce the salicylate effect. On the other hand, the salicylate effect in MEM supplemented with the components unique to RPM1 1640 was no less than that observed in unsupplemented MEM. The salicylate effect observed in a “hybrid” medium composed of equal parts of RPM1 1440 and MEM was greater than that observed in RPM1 1640 but less than that in MEM. Glrrcose utilization. Salicylate did not affect glucose utilization by control cuilured lymphocytes (Table 3). However, these lymphocytes utilized glucose at a greater rate from MEM than from RPM1 1640. Glucose utilization by L,stimulated lymphocytes was the same in either RPM1 1640 or MEM. Added salicylate produced a dose-dependent decrease in glucose utilization by L,-stimulated cells cultured in MEM (Table 3), whereas this effect was much less pronounced in TABLE EFFECX

OF MEDIA

ON MAXIMUM

IS L,-STIMCLATED

2 [BHITHYWDINE

Day Medium RPM1 MEM

of incubation 2.5

1.5 1640

162.289 167,540

i 31,302” i- 45,778

‘I Tne values represent the mean disintegrations Each determination was done in triplicate.

ISCORPORATIOK

LYMPHOCYTES

266,095 249,009 per minute

4.5

2 76.540 i 33.100 per culture

133,228 147.164 t

SEM

t 38,078 i 12,756

of nine patients.

ASA (mg/lOO ml) 0 10 20 30 40

Control lymphocytes RPM1 1640 0.5" 0.1 0.3 1.3 0.1

L,-stimulated

MEM

RPM1 1640

3.8 3.9 4.1 4.1 4.4

19.4 18.2 27.4 16.0 17.0

lymphocytes MEM 19.0 17.4 16.1 15.2 13.3

o The values are expressed as micrograms of glucose per hour and represent the mean of three experiments.

cells grown in RPM1 1640. Therefore, the addition of increasing concentrations of salicylate to cultures of lymphocytes stimulated with 10 pgiculture of L, produced alterations in glucose utilization, paralleling the effects of salicylate on j3H]thymidine incorporation by these cells. PHA Billding The binding of L, to lymphocytes was the same in RPM1 1640 and MEM. Salicylate in concentrations up to 40 mgi100 ml had no effect on L, binding to lymphocytes in either medium. Thin-layer Chr-omatography of Salicylate Additilpes Thin-layer chromatography of neutralized stock salicylate solutions in both solvent systems I and II showed that all salicylate in the culture additives was in the form of unesterified salicylate. In system I, the Rf values of acetylsalicylate, salicylate, and the salicylate added to the culture were 0.78, 0.82, and 0.82, respectively. In system II, the Rf values of acetylsalicylate, salicylate, and the salicylate added to cultures were 0.68, 0.82, and 0.82, respectively. DISCUSSION

Our studies reveal that oral salicylate treatment did not affect the ability of the recipient’s lymphocytes to respond to PHA isolectin stimulation. These results agree with those of Smith et al. (14) and, as such, fail to confirm the findings of Crout and colleagues (3). Our studies are comparable to both of these previous investigations with respect to aspirin dosage, the length of salicylate therapy, and lymphocyte isolation and culture techniques. The absence of an ASA effect might be expected in view of the in vitro studies of Opelz et al. (2), who showed that lymphocytes that had been washed free of salicylate reacted normally to PHA. In our studies, procedures used to isolate lymphocytes also removed all plasma. Therefore, cultures of these lymphocytes were established with cells washed free of salicylate, By utilizing purified La and E, isolectins, we not only confirm the findings of Smith er al. (4) but carry this observation further and show no alteration by salicylate therapy of several aspects of the mitogenic response of lymphocytes to

SALICYLATE

EFFECTS

Oh’

HUMAN

LYMPHOCYTES

9

ese purified plant proteins. In addition, we show that aspirin therapy did nol alter the glucose utilization by resting or isolectin-stimulated cells. In contrast to our in I+Q studies and in agreement with two previous studies i 1, 2), salicylate addition to L,-stimulated lymphocytes produced a dose-dependent inhibition of [3H]thymidine incorporation. Unlike previous reports dealing with salicylate effects on lymphocyte transformation, we maintained an internal control. We studied lymphocytes from the same individuals for their ability to respond to L, after oral aspirin therapy and to L, when cultured in the presence of salicylate. Since these lymphocytes are inhibited by in r)ifuo salicylate, this argues against a refractory nature of the lymphocytes. Rather. this observation is strong evidence that oral aspirin therapy does not induce any irreversible change in the2 capability of lymphocytes to respond to isolectin stimulation. The action of salicylate in l,itro as an inhibitor of lymphocyte transformation does not result from an interference with isolectin binding to lymphocyte membranes. Our binding studies with 12sI-labeled L, show no change in the quantity of isolectin bound to lymphocytes in inhibitory salicylate concentrations. Despite the fact that the neutralized “aspirin” added to our lymphocyte cultures had been deacetylated to free salicylate. we feel that these experiments may legitimately be contrasted to those in which aspirin was ingested by volunteers. Pachman et al. (1) showed identical activity of aspirin and salicylic acid when used to inhibit blastogenesis in PHA-stimulated lymphocyte cultures. In addition, ingested acetylsalicylic acid is rapidly deacetylated, resulting in free salicylate as rhe significant plasma form of the drug (13-15). The impressive disparity noted between the inhibitory effect of salicylate in plasma or RPM1 1640 and MEM was surprising. The inhibition observed in MEM cultures is comparable to that reported by Pachman et al. (1) in Eagle’s MEM Spinner modification and that reported by Opelz et nl. (2) in medium 199. Our studies. however, indicate that RPM1 1640 is a more suitable medium for these studies, since the response of cells in RPM1 1640 more closely resembles their response in human plasma. The basis for RPM1 1640’s mitigation of salicylate’s effect is unknown, although our studies argue against altered binding as the reason. RPM1 1640 contains numerous amino acids and cofactors not found in MEM. The addition of these missing components to MEM did not reduce salicylate inhibition. We are continuing our investigation of this phenomenon. Our measurements of salicylate stimulation of glucose utilization by resting lymphocytes is less than the 161% increase reported by Pachman et al. (1). There was no effect of salicylate on resting cultures in RPM1 1640. The higher rates of glucose consumption by resting cultures in MEM with respect to those in RPMI 1640 probably reflects the absence of various amino acids in MEM and the reliance on carbohydrate and intermediary metabolism to generate these materials. The ability of salicylate to reduce glucose utilization by L,-stimulated cultures might be expected in view of the previously reported parallelism in [3H]thymidine incorporation and glucose utilization by lymphocytes stimulated with this mitogen (10). Moreover, the greater reduction in glucose utilization by lymphocytes grown

10

EGORIN,

FELSTED,

AND

BACHUR

in MEM with respect to that by cells grown in RPM1 1640 could be anticipated because of the greater inhibition of [3Hjthym,idine incorporation seen in MEM. In summary, our studies show no effect on oral salicylate therapy on lymphocyte response to purified PHA isolectins. Our itz rlitvo results confirm the previously described aspirin inhibition of PHA-induced lymphocyte transformation and provide evidence that those earlier workers, by their choice of culture medium, overestimated the acutal inhibitory effect of salicylate. Our data show that the behavior of lymphocytes cultured with aspirin and L, in RPM1 1640 more accurately reflects the behavior of these cells cultured with these compounds in 100% autologous plasma and that the concentrations of aspirin required for significant inhibition of PHA isolectin-induced transformation are far above those routinely encountered in clinical practice. ACKNOWLEDGMENTS We appreciate the technical assistance of Jeffrey Cohen, Forrest Litvin, and Norman Schlossberger and the efforts of Barbara Dressel in the preparation of this manuscript.

REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9.

10. Il. 12. 13. 14. 15.

Pachman. L. M., Esterly, N. B.. and Peterson, R. D. A., J. C/i/t. I~r,est. 50, 226, 1971. Opelz, G., Terasaki, P. I., and Hirata. A. A.. La,tcrf 2, 478, 1973. Crout. J. E., Hepburn, B., and Ritts, R. E.. N. EI?~I. J. Med. 292, 221. 1975. Smith, J. M., Hoth. M., and Davis. K.. Ann. Inrer-n. Med. 83, 509, 1975. Felsted, R. L., Leavitt, R. D., and Bachur. N. R., Biocltim. Biopltys. Acr~1 405, 72, 1975. Felsted. R. L., Egorin. M. J., Leavitt. R. D., and Bachur, N. R.. J. Biol. C/tent., 1977. in press. Reisfeld, R. A., Lewis, V. J.. and Williams. D. E., Nature (London) 195, 28. 1962. Lowry, 0. H.. Rosebrough, N. J., Fart-, A. L.. and Randall. R. J.. J. Bib/. Chern. 193, 265, 1951, Boyum. A., Stand. J. C/i/7. Inl,est. S~ippl. 21, 97. 1968. Egorin. M. J.. Felsted. R. L., and Bachur. N. R.. L$e Sci. 20. 341, 1977. Trinder, P.. Biochent. J. 57, 301. 1954. Kornfeld. R., Gregory, W. T.. and Kornfeld. S. A., 117“Methods in Enzymology” (V. Ginsburg. Ed.). pp. 344-349, Academic Press, New York, 1972. Mandel, H. G., Gambosos, N. M.. and Smith, P. K., J. Pharmacol. Exp. Ther. 112, 495. 1954. Rowland, M.. and Riegelman. S.. J. Pharm. Sci. 57, 1313, 1968. Rowland, M., Riegelman, S. Harris, P. A., Sholkoff. S. D.. and Eyring. E. J.. NNtlrr-e (Landon) 21.5. 413. 1967.