Polysorbate 80 and E-Ferol Toxicity

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Polysorbate

80 and E-Ferol

Toxicity

Solomon

L. Alade,

PhD,

R. E. Brown,

MD, and Andrew

Paquet,

Jr, PhD

From the Department of Pathology, Cook-Fort Worth Children’s Medical Center, and Department of Biology, Texas Christian University, Fort Worth

ABSTRACT. The relatively recent introduction and use of an intravenous form of a vitamin E preparation (E-Ferol) has been associated with the development of an unusual syndrome and fatalities among low birth weight (<1,500 g), premature infants in neonatal intensive care units. We have observed an inhibitory effect by this

vitamin E preparation on the in vitro response of human

lymphocytes to phytohemagglutinin (PHA). E-Ferol sup-pressed the expected response to low doses of PHA. However, this suppression was not due to the a-to-copherol acetate (vitamin E) component, because a-to-copherol acetate by itself was not inhibitory; in fact, it often enhanced the PHA response. Because a mixture of polysorbate 80 and polysorbate 20 is used as a carrier in E-Ferol, these components were also tested and were found to be responsible for the suppression, especially the polysorbate 80. Concurrent with this suppression of

PHA-induced mitogenesis was a decrease in the

percent-age of Til lymphocytes. Pediatrics 1986;77:593-597;

vi-tamin E, a-tocopherol, polysorbate 80,

phytohemaggluti-fin, mitogenesis.

In December 1983, E-Ferol was marketed as an intravenous vitamin E preparation to provide nu-tritional supplementation for premature neonates and, as implied, for the possible prevention of

reti-nopathy of prematurity (E-Ferol package insert, St

Louis, O’Neal, Jones and Feldman

Pharmaceuti-cals, December 1983). Unfortunately, the use of

E-Ferol in these infants has been associated with the development of a syndrome characterized by

pul-monary deterioration, hepatomegaly, cholestatic

jaundice, ascites, splenomegaly, renal failure, azo-temia, and thrombocytopenia.’3 In April 1984, E-Ferol was recalled.

Received for publication Sept 5, 1985; accepted Nov 18, 1985. Presented at the 85th annual meeting of the American Society for Microbiology, March 7, 1985, Las Vegas.

Reprint requests to (S.L.A.) Department of Pathology,

Cook-Fort Worth Children’s Medical Center, 1400 Cooper, Fort Worth, TX 76104.

Prior to the recall, we had been studying

phyto-hemagglutinin (PHA)-induced lymphocyte trans-formation as an assay of serum inhibition of lym-phocyte function in patients with childhood

leuke-mia.4 Because vitamin E was also being considered

as an adjuvant in the chemotherapy of these

pa-tients, we began measuring the effects of E-Ferol on PHA responses and noticed a seemingly para-doxical inhibitory effect.5’6

The purpose of this paper is threefold: (1) to document that the aforesaid inhibitory effect on PHA-induced lymphocyte transformation can be linked to the polysorbate 80 emulsifier in E-Ferol

solutions; (2) to strengthen the thesis that polysor-bate 80 may also be responsible for the E-Ferol syndrome in vivo by: (a) discussing relevant, poten-tially toxic effects of its component chemicals and (b) citing toxic effects of polysorbate 80 that have

been observed in hepatic and renal tissues of ex-perimental animals; and (3) to raise concerns about

similar aqueous preparations that may be delivering the same potentially toxic polysorbate to other groups of pediatric patients.

MATERIALS AND METHODS

E-Ferol Aqueous Solution was obtained from O’Neal, Jones and Feldman Pharmaceutical of St Louis, MO. It contained 25 United States

Phar-macopeia units/mL of vitamin E as dl-a-tocopherol acetate mixed with 9% polysorbate 80 and 1% poly-sorbate 20 in water for injection. Polysorbate 80

which, by definition, is a mixture of oleate esters of

sorbitol and sorbitol anhydrides, consisting pre-dominantly of the monoester, condensed with

ap-proximately 20 mol of ethylene oxide and polysor-bate 20 which, by definition, is identical with

polysorbate 80 except that laurate is substituted for

oleate8 were obtained from ICI, Americas

Incorpo-rated, Wilmington, DE, as purified (National

For-mulary grade) Tween 80 and 20. They were diluted

(2)

and 1%, respectively. Purified a-tocopherol acetate

(Eli Lilly, Indianapolis) was dissolved in 95% ethyl alcohol and used for comparison. RPMI 1640 (Bio-fluids, Incorporated, Rockville, MD) containing glutamine, 20 mmol/L

N-2-hydroxyethylpipera-zine-N’-2-ethane sulfonic acid, and 50 g/mL of gentamicin and supplemented with 5% fetal calf serum was used as the culture medium. PHA

(PHA-M) was obtained from Difco Laboratory, Detroit.

The monoclonal antibodies Til and B4 (Coulter Immunology, Hialeah, FL) were used for cell

enu-meration studies.

Heparinized blood was obtained from normal

vol-unteers who had given informed consent. The mononuclear cells were isolated from these speci-mens by centrifugation on Ficoll-Hypaque gradient (Pharmacia, Piscataway, NJ) and washed three

times in culture medium. For cell enumeration, 1 x

106 cells were incubated with 200 tL of monoclonal antibody according to the Coulter indirect immu-nofluorescent procedures and examined microscop-ically. For PHA-induced mitogenesis, cells were resuspended to 4 x 106 cells per mL and dispensed in 0.1-mL volumes into the wells of flat-bottom 96-well microtest II (Falcon, Cockeysville, MD) culture

plates. A 0.1-rn! volume of PHA diluted in culture medium to give a concentration of either 10 or 100

tg/rnL was added to quadruplicate wells containing

lymphocytes. This resulted in a final PHA concen-tration of 5 and 50 tg/rnL, respectively. A 0.1-rn! volume of culture medium alone was added to

con-tro! wells. E-Ferol aqueous solution or the individ-ual polysorbates (9% polysorbate 80 and 1%

poly-sorbate 20) were diluted 1:10 to culture medium,

and then 5 L of this dilution was added to the

wells containing the lymphocyte-PHA mixtures. This series of dilutions was designed to result in final concentrations for vitamin E (a-tocopherol acetate), polysorbate 80 and polysorbate 20 of 6, 22, and 2.4 mg/dL, respectively. A 1:10 mixture of the

combined polysorbates (80 + 20) was also tested in

this manner as well as a-tocopherol acetate

dis-solved in 95% ethyl alcohol and 95% ethyl alcohol

by itself. Control cultures received 5 L of culture medium.

The cells were cultured for 72 hours at 37#{176}Cin a

humidified atmosphere of 5% CO2 in air.

Repre-sentative cultures were checked for cell viability using the trypan blue technique. Eight hours before

harvesting, 1 zCi of tritiated thymidine (50 zL volume) was added to each well. Cultures were collected on fiberglass filters using a semiautomatic sample harvester and the amount of incorporated radioactivity measured by liquid scintillation spec-trometry. The mean count of each variable was

calculated from the quadruplicate samples and used

to determine percentage inhibition relative to the

measured response in the PHA-stimulated control alone.4 Statistical comparisons were made using the Student’s t test.

RESULTS

Preliminary trials in our laboratory had

estab-lished that concentrations of PHA at 5 and 50 jg/

mL represent suboptimal and optimal mitogenic

doses, respectively. The 5 zg/mL dose would allow

significant levels of stimulation (approximately tenfold) over unstimulated control cells and yet at

the same time should allow any inhibition of said

stimulation to be measured. PHA at a concentra-tion of 50 tg/mL routinely produces 25- to 30-fold increases in stimulation over control cells.

After initial studies had revealed that E-Ferol inhibited the PHA response, we decided to analyze

the individual components.

The results from 25 individuals are shown in the figure. The complete E-Ferol solution caused a 37% inhibition (P .01) of the mean response to 5 g/ mL of PHA. However, the polysorbate 80 by itself also produced significant inhibition (44%, P .01). In contrast, when a-tocopherol acetate or the 95%

ethyl alcohol solvent was tested, a stimulation of

the PHA response occurred. When the PHA con-centration was increased to the optimal level of 50 g/mL, E-Ferol still appeared inhibitory, but the difference was not statistically significant.

In an effort to understand the mechanism of this inhibition, we enumerated the percentage of B cells and Til cells in cultures containing E-Ferol or its components before and after 72 hours of incuba-tion. B cells were counted, but there was no signif-icant difference between any of the groups; all remained unchanged from the normal value of ap-proximately 5%. There was also no significant change in the percentage of Til cells before and after 72 hours of incubation in the control cultures and in cells cultured with 5 zg/mL of PHA. How-ever, significant reduction in the percentage of Til

cells were seen in cultures containing E-Ferol or

one or more of its polysorbate components (see Table). a-Tocopherol acetate or its ethanol solvent, on the other hand, caused no significant reduction in the percentages of Til cells. The viability of cells treated with E-Ferol or its components was

similar to control cultures.

DISCUSSION

The ability of E-Ferol to suppress PHA-induced

lymphocyte transformation as demonstrated in this study appears to be related primarily to its

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Lii

PHA

5 ug/mI

. po.o1

PHA 5Oug/ml

E-F 80 20

MEDIA ADDITIONS

aTA #{149}toti E-F

0

C

0

I

0.

0

Figure. Mean response of normal human lymphocytes to phytohemagglutinin (PHA) (5

and 50 zg/mL; open and hatched bars, respectively) was established as the expected

(100%) response. Additions to the media of PHA-stimulated human cells as listed on the abscissa include E-F (E-Ferol), 80 (polysorbate 80), 20 (polysorbate 20), 80 & 20

(polysor-bate 80 + 20), aTA (a-tocopherol acetate), and etoh (95% ethyl alcohol). Suppression or

stimulation by the additions is represented as mean percentage of PHA control and

depicted by bars below and above dotted (100%) line. Vertical line above bars indicates standard deviation from mean. Black circle above vertical lines indicates statistically

significant difference (P .01) compared with PHA alone.

TABLE. Effect of E-Ferol on T Cell Enumeration*

Activator % Til Cells

Before culture 81 ± 7.2

After 72 h of culture

Control culture 79 ± 9.1

Phytohemagglutinin

5 tg/mL 77 ± 9.6

+E-Ferol 50 ± 6.3t

+Polysorbate 80 45 ± 9.4t

+Polysorbate 20 48 ± 8.8t

+Polysorbates 80 + 20 43 ± 1.8t

+a-Tocopherol acetate 76 ± 3.4

+Ethyl alcohol (95%) 72 ± 2.7

* Values are means ± SD from N = 25.

t

Treatment means with E-Ferol and components are

significantly different, P < .05, compared with

phytohe-magglutinin (5 zg/mL) alone.

findings accord with in vivo, experimental data that show an immunosuppressive effect of Tween 80

(polysorbate 80), possibly at the level of the T helper cell.#{176}There are numerous sites at which such

suppression could be effected. Several of these

in-volve the plasma membrane and include (1) an alteration of lectin-receptor agglutinability or a re-duction in the fluidity of the lymphocyte

mem-brane1#{176}and 2) inhibition of membrane-associated

acyltransferase activation or competition between

one or more polysorbate 80 constituents and ara-chidonic acid for this enzyme that preferentially

incorporates arachidonate into the phospholipids

of activated lymphocytes.’#{176} Both mechanisms are likely in this instance. The former is supported by the concurrent finding of a decrease in the percent-age of Til cells without an appreciable effect on cell viability in the polysorbate 80-exposed groups. This could reflect an alteration of the plasma

mem-brane to the extent that antigenic markers have been either masked or destroyed. The fact that the

chemical composition of polysorbate 80 consists of

a mixture of oleate esters of sorbitol and sorbitol

anhydrides, consisting predominantly of the mon-oester, condensed with approximately 20 mol of ethylene oxide provides a basis for the latter. Be-cause some inhibition of PHA-induced transfor-mation of human lymphocytes can be produced

with the methyl ester of oleic acid and also has been noted in circumstances in which either the arachidonic to oleic acid ratio has been relatively reduced and/or the oleic acid concentration

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polysorbate 80 is likewise contributing to the inhi-bition.

More importantly, the aforesaid chemical

corn-position of polysorbate 80, when taken together

with what is currently known about the catabolism and excretion of polysorbates in vivo, could provide a theoretical basis to explain several of the

char-acteristic findings of the E-Ferol syndrome. That is to say, because fatty acid-’4C-labeled polysorbate has been recovered largely in respired air and

be-cause polyoxyethylene-’4C-labeled polysorbate has been recovered primarily in urine following intra-venous administration to rats,boa we believe that it

is important to consider further the possibility that the oleic acid and polyoxyethylene moieties released

during in vivo hydrolysisboa of polysorbate 80 are contributing to the pulmonary deterioration and

renal failure, respectively, in the E-Ferol syndrome. Moreover, the fact that intravenous administration

of oleic acid to dogs can produce dose-related

pul-monary changes ranging from the leakage of fluid and occasional RBCs into alveoli to extensive

a!-veolar-capillary necrosis” lends support to this pos-sibility. Similarly, the other constituent, ethylene

oxide, because of its potential conversion to

ethyl-ene glycol,’2 a known nephrotoxic agent,’3 could account for E-Ferol-associated renal failure and

azotemia.

Finally, this contention is supported by data

gained from experiments with polysorbate 80 in animal systems. Specifically, polysorbate 80 has been associated with impaired uptake and secretion

of bile acids and loss of microvilli from the

canali-cular surface of isolated hepatocytes from newborn rabbits’5 and, following chronic administration, with degenerative changes in the kidney and liver of rats.’6 The latter include marked congestion and

dilation of central veins and sinusoids, changes that appear to parallel histopathologic features of the E-Ferol-associated hepatopathy in low birth weight

infants.’7

IMPLICATIONS

a-Tocopherol (vitamin E) and its acetate deny-ative have been held suspect in the E-Ferol syn-drome.3”8 However, based on the findings in this

study and a comparison of the reported adverse effects in humans of vitamin E’9 with the

charac-teristic findings in the E-Ferol syndrome, we believe

that vitamin E is an unlikely participant in its etiopathogenesis. Perhaps, as a result of our

find-ings and discussion, attention will be focused on polysorbate 80, not only for future studies on the E-Ferol syndrome but also in reconsidering the

mechanism of the toxicity of other vitamin E prep-arations. For example, it may indeed be the

poly-sorbate 80 in Aquasol E (USV Canada, Mississauga,

Ontario) and not the hyperosmolality that is

re-sponsible for the increased incidence of necrotizing

enterocolitis in infants with birth weights <1,250 g who received this oral vitamin E preparation.2#{176}

Additionally, we hope that our pediatric and pharmaceutical colleagues will be sensitized to the potential toxicity of polysorbate 80 and will famil-ianize themselves with other preparations

contain-ing this agent so that these may be used with

caution and with an eye to their potentially

dele-tenious effects. Our concern extends to the long-term use of oral preparations containing polysor-bate 80 given the fact that the oral administration of Tween-80 at relatively low concentrations of 1%,

2%, and 4% for 90 days resulted in the

aforemen-tioned degenerative change in the liver and kidney of experimental rats.’6 Therefore, to facilitate this, we have elected to list the pharmaceutical products in common usage in our hospital pharmacy that contain polysorbate 80. It includes (1) Aquasol E (Armour Pharmaceutical Company, Tarrytown, NY), (2) Poly-Vi-Sol (Mead Johnson Nutritional Division, Evansville, IN), and (3) Tri-Vi-Sol (Mead Johnson Nutritional Division, Evansville, IN).

SUMMARY

E-Ferol and its polysorbate 80 constituent are capable of both decreasing the response of normal human lymphocytes to PHA and, concurrently, de-creasing the percentage of Til lymphocytes. The establishment, by this in vitro study, of a direct adverse effect of polysorbate 80 on human cells and the toxic effects of polysorbate 80 on hepatic and renal tissues in experimental systems serve to im-plicate polysorbate 80 as a principal agent in the

pathogenesis of the E-Ferol syndrome.

ACKNOWLEDGMENT

This work was supported by funds from Cook-Fort

Worth Children’s Medical Center and Texas Christian

University Research Foundation.

We thank Lynette Hill, RPh, for researching the

phar-maceuticals in common usage in Cook-Forth Worth

Chil-dren’s Medical Center for the presence or absence of

polysorbate 80 and Karen Hurd for assistance in

prepar-ing the manuscript.

REFERENCES

1. Unusual syndrome with fatalities among premature infants: Association with a new intravenous vitamin E product.

MMWR 1984;33:198-199

2. Bodenstein CJ: Intravenous vitamin E and deaths in the intensive care unit, letter. Pediatrics 1984;73:733

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intravenous vitamin E product. Pediatrics 1985;75:598-602 4. Brown RE, Steele RW, Marmer DJ, et al: Fatty acids and

the inhibition of mitogen-induced lymphocyte transforma-tion by leukemic serum. J Immunol 1983;131:1011-1016 5. Watson RR, Petro TM: Cellular immune responses,

corti-costeroid levels, and resistance to Listeria monocytogenes

and murine leukemia in mice fed a high vitamin E diet, in Lubin B, Machlin U (eds): Vitamin E: Biochemical

Hema-tological, and Clinical Aspects. New York, New York

Acad-emy of Science, 1982, pp 205-208

6. Corwin LM, Gordon RK: Vitamin E and immune regulation, in Lubin B, Machlin U (eds): Vitamin E: Biochemical,

Hematological and Clinical Aspects. New York, New York

Academy of Science, 1982, pp 437-451

7. beleted in proof

8. Estrin NF, Crosley PA, Haynes CR (eds): CTFA Cosmetic Ingredient Dictionary, ed 3. Washington, DC, The Cosmetic, Toiletry and Fragrance Association, mc, 1982, pp 246-247 9. Barnett JB, Bryant RL: Adjuvant and immunosuppressive

effects of retinol and Tween 80 on IgG production in mice.

mt

Arch Allergy AppI Immunol 1980;63:145-152

10. Meade CJ, Mertin J: Fatty acids and immunity. Adv Lipid Res 1978;16:127-165

lOa.Nelson MF, Poulos TA, Gongwer LE, et al: Preparations of

carbon-14-labeled polyoxyethylene (20) sorbitan

monolaur-ate and their metabolic fate in rats. J Food Sci 1966;31:253-258

11. Sugerman HJ, Hirsch JI, Tatum JL, et al: Comparative scintigraphy in oleic acid pulmonary microvascular injury.

Crit Care Med 1982;10:31-33

12. Ethylene oxide, ethylene chlorohydrin, and ethylene glycol.

Federal Register 1978;43(Jan 27):3800-3815

13. Darmady EM, Maclver AG (eds): Renal Pathology. Boston, Butterworth Inc, 1980, chap 9, pp 198-223

14. Deleted in proof

15. Bhat R, Jiang J-X, Walsh JM, et al: Effect of vitamin E and polysorbate on bile acid transport in newborn rabbit hepatocytes, abstracted. Pediatr Res 1985;19:213A

16. Nityanand S, Kapoor NK: Effect of chronic oral adminis-tration of Tween-80 in Charles Foster rats. Indian J Med Res 1979;69:664-670

17. Balistreri WF, Bove KE, Kosmetatos N, et al: Distinctive hepatopathy in low birthweight infants in association with E-Ferol infusion, abstracted. Pediatr Res 1985;19:212A

18. Phelps DL: E-Ferol: What happened and what now? Pedi-atrics 1984;74:1114-1116

19. Roberts HJ: Perspective on vitamin E as therapy. JAMA 1981;246:129-131

20. Finer NN, Peters KL, Hayek Z, et al: Vitamin E and necrotizing enterocolitis. Pediatrics 1984;73:387-393

POWER

OF CLINICAL

OBSERVATIONS

Clinical examination is far more powerful than laboratory evaluation in

establ!shing diagnoses, prognoses, and therapeutic plans for most patients in

most places. In a general practice, for example, Crombie documented that 88% of diagnoses were established by the end of a brief history and physical exam subroutine. In a general medical clinic, Sandler showed that 56% of cases had been assigned correct diagnoses by the end of the history and this figure rose to 75% by the end of the physical examination. Even when patients are referred to specialty centers after exhaustive workups elsewhere, attention is appropri-ately refocused on the patient’s “story” and the physical examination. Indeed, when a colleague in gastroenterology was asked to consult on a patient who already had undergone a large battery of endoscopic, radiographic, and biochem-ical studies elsewhere, he proclaimed, “All that there is left for us to do is a history and physical!”

Submitted by Student

From Sackett DL, Haynes RB, Tugwell P: Clinical Epidemiology, A Basic Science for Clinical

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1986;77;593

Pediatrics

Solomon L. Alade, R. E. Brown and Andrew Paquet, Jr