Thymulin (Zn-Facteur Thymique Serique) activity in anorexia nervosa patients13

Thymulin (Zn-Facteur in anorexia nervosa Sa/imata

Wade,

Philippe

Chapuis,

Fanny Daniel

Thymique patients13

Bleiberg,

Arlette

Moss#{233},Jean

Roche,

Daniel

Lemonnier

Serique) Lubetzki,

and

Hubert

Mireille

activity Flavigny,

Dardenne

KEY WORDS T-lymphocyte

Anorexia subpopulations

nervosa,

Introduction

thymulin

(FTS-Zn)

delayed 1 1),

Protein-energy malnutrition, children, is known to be thymic

atrophy,

depression

at least in associated with in T-cell

numbers

and functions. This apparently powerful and specific effect of malnutrition on the thymus has been extensively studied in malnourished individuals

from

the

developing

countries.

However, in these populations, infections are commonly associated with malnutrition and can interfere with the immune responses

(1-4).

Anorexia nervosa is a syndrome of profound weight loss in young persons in which the refusal to eat is directly related to psychic disturbances. Despite their severe body-wasting, anorexia nervosa patients are described as exceptionally free from infections (5). Extensive literature involving the clinical and some biochemical changes in this illness has been widely reported in the past (6, 7), but the effect of anorexia nervosa on the immune function remains conflicting and unclear (811): thus, patients with anorexia nervosa display a relatively modest impairment of The American Journal of Clinical Nutrition 42: AUGUST © 1985 American Society for Clinical Nutrition

activity,

zinc, cortisol,

hypersensitivity but

when

their

skin weight

thyroid

hormones,

responses loss

is

far

(8, ad-

vanced, a significant decrease in cell-mediated immunity can occur (8). Recently, several investigators have mdicated the possibility that the epithelial function of the thymus may be altered by nutritional deficiencies (12-15). In order to further investigate the effect of anorexia thymic function, a circulating

nervosa thymic

on hor-

From the U I INSERM, H#{244}pitalBichat, 170 Bd 75018 Paris; U25 INSERM, H#{244}pitalNecker, 161 rue de S#{232}vres, 75015 Paris; Service de Psychiatric, H#{212}pital International de l’Universit#{233}de Paris, 42 Bd Jourdan, 75014 Paris; Service de M#{233}decineInterne et d’Endocrinologie-Nutrition, Laboratoire de Biochimie, Laboratoire de Radio-Immunologic, H#{244}pitalLariboisi#{232}re,2 rue Ambroise-Par#{233},75010 Paris. 2Supported by grants from the Conseil Scientifique de l’UER Xavier-Bichat and from the PRC INSERM N#{176} 128020. 3Address reprint request to: Salimata Wade, U 1 INSERM, Unite de Recherches sur Ia Nutrition et l’Alimentation, H#{244}pitalBichat, 170 Bd Ney, 75018 Paris-France. Received May 10, 1984. Accepted for publication January 29, 1985. ‘

Ney,

1985,

pp 275-280.

Printed

in USA

275

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ABSTRACT Thymulin (or FTS-Zn) a well-defined thymic hormone was studied in fifteen female patients hospitalized for anorexia nervosa. The circulating hormone was measured together with the plasma levels of thyroid hormones, cortisol and zinc. Thymulin activity determined by the rosette assay was significantly reduced in the anorexia nervosa patients compared to sex- and age-matched healthy control subjects. The patients were characterized by very depressed plasma levels of tniodothyronine (T3) but exhibited normal concentrations of thyroxine (T4), thyroxinebinding globulin (TBG), cortisol and zinc. The distribution of their peripheral lymphocyte cells into several subsets was not affected. The observed decrease of thymulin activity in this illness might be the consequence of thymic atrophy secondary to malnutrition and/or hormonal disturbances. Our results suggested that the fall in thymulin level might explain the variability of cellular immune responses in anorexia nervosa patients and occurrence of anergy when their weight loss is far advanced. Am J Clin Nuir l985;42:275-280.

276

WADE

mone, thymulin (a zinc-facteur thymique serique complex previously called FTS) was quantified in anorexia nervosa patients and in

healthy

control

subjects.

Thymulin

is a

nonapeptide exclusively produced by the thymic epithelial cells but its activity is detectable in peripheral blood. Thymulin level is thus

a good

marker

of thymic

epithelial

Patients

and methods

Patients The study, conducted in accordance with the INSERM ethical committee for human experimentation, involved fifteen female patients (14 to 35 years of age) suffering from anorexia nervosa. They were admitted for treatment either at the psychiatric unit ofthe H#{244}pital International de l’UniversitC de Paris or the Medicine and Endocrine Unit of the H#{244}pitalLariboisi#{232}re, Paris. The patients satisfied the diagnostic criteria of Feighner et al (20); they were characterized by a distorted attitude towards food, eating and weight, a greater than 30% loss in body

TABLE Clinical

1 features

Patients

Set

F F F F F F F F F F F F F F F

RB LePA SC NF PMC GN KJ BJ VBt BF MM Alt CL Gc AV Mean a)

of patients

±

SEM

20.6

with anorexia

-

Immune

studies

Lymphocytes were isolated from peripheral blood by Ficoll-Hypaque gradient density centrifugation. Monoclonal anti-T cell antibodies were used to evaluate the proportion of various T-lymphocyte cell subsets by indirect immunofluorescence: the percentage of mature Tcells, helper and cytotoxic-suppressor cells were determined using OKT3, OKT4 and OKT8 respectively (22). slg-bearing lymphocytes (B-lymphocytes) were quan-

nervosa Body weight

body’ weight (IBW)

Age

Duration of anorexia nervosa

Height

(yrs)

(yrs)

(cm)

(kg)

(kg)

15 20 18 15 18 16 35 29 18 20 26 30 19 14 16.5

1 5 4 0.5 0.3 1 23 12 4 0.7 9 2 0.5 1.5 0.75

151 151 149 155 162 163 160 161 147 167 161 175 167 138 158

34 28 31.5 38 34 34.8 38.6 31.3 27 45 38 45 39.5 20.6 40

50.6 50.6 49.4 53 57.2 57.8 56 56.6 48.2 60.2 56.6 65 60.2 42.8 54.8

±

1.64

Ideal body weight (IBW) was calculated edemic on admission.

t Patients

weight, a persistance in an idealized slim self-image, amenorrhea and an absence of other medical and psychiatric illness. Their clinical features are reported in Table I. Calculations for ideal body weight (IBW) were based on Lorentz’ formula (21): height - 100 [(height 150)/2.5]. The control group (thirteen females) comprised students and nurses and were age-matched. None ofthe subjects, including the controls had clinical evidence of infection. Consent for venipuncture was obtained from the patients and the medical staff after the purpose and nature of the study had been explained. Our anorexia nervosa patients were tested within 1 to 5 days of admission. After overnight fasting, blood was drawn and collected in cooled (4#{176}C) heparinized tubes. Because of the cortisol assay, samples were obtained between 8 and 8:30 AM. Plasma was immediately separated and kept frozen at -20#{176}Cuntil analyzed. Plasma zinc was assayed by atomic absorption spectrophotometry. Cortisol was quantified by radioimmunoassay (RIA) (Kit Cortctk 125-Oris, CEA, Gif-sur-Yvette, France). Thyroxine (T4), triiodothyronine (T3) and thyroxinebinding globulin (TBG) were determined in plasma samples by RIA (Kit CEA, Gif-sur-Yvette, France).

4.4

±

1.6

acccording

158

±

at

Ideal

hospitalization

2.41

to Lorentz’

35.0 formula:

±

1.72

IBW = height

54.6 -

±

100

% weight loss

33 45 37 29 46 40 31 45 44 25 33 31 35 52 27 36.9

1.45

- height

-

2.5

150

Downloaded from ajcn.nutrition.org by guest on February 23, 2015

function. Thymulin activity has been found in the serum of various species including man. Its level is relatively constant in humans during ontogeny but declines with advancing age (16-18). Thymulin possesses several biological activities (16, 18). It has also been shown that it acts on the T-cells involved in delayed type hypersensitivity (18, 19).

ET AL

THYMIC

FUNCTION

IN

-I-

2

20

15

A

801

E 0 0

a

S.

. .

S. S.

60

S

S. S

a

S.

S

S.

-J

40

10

0 (I)

S

a)

e.

U

z

S

S. S

20

N

S. S

5

0 U

S. S.

S S

S.

0

0

I

in anorexia controls l.

tified by direct immunofluorescence with a fluorescein conjugated Ig-antisera. Thymulin activity was measured by the method described by Dardenne and Bach (23); the assay analyzes the conversion of relatively azathioprine(Az) resistant spleen cells of adult thymectomized mice to 0-positive rosette-forming cells that are more sensitive to Az. Briefly, plasma samples were filtered by centrifugation at 4#{176}C on CF Amicon membranes (molecular weight cut off at 50.000 to remove an FTS inhibitor). The ultrafiltrates were incubated for 90 mm at 37#{176}C with spleen cells from mice thymectomized 10 to 15 days before the test, and Az (10 zl/ml) was added simultaneously. At the end of the incubation time 12 X l0 sheep red blood cells were added to the cells. This cellular preparation was centrifuged (at 4#{176}C 150 X g, 5 mm) and resuspended by low-speed rotation (10 rpm) on a roller. Rosette-forming cells were counted in a hemocytometer. In the presence of thymulin, Az inhibited rosette formation at a concentration of 10 eg/ml. The highest dilution of the plasma sample which induced the rosette inhibition by Az was considered as the active dilution. All the determinations were carried out in duplicate and the results were expressed as 1og2 reciprocal titer. analysis

Differences determined

of data

in mean using

TABLE 2 Concentration

values between the groups were

the Student’s

of thyroxine

I

test.

(T4), triiodothyronine

At the time of the study the mean weight and height of the patients were 35.0 ± 1.7 kg and 158 ± 2.4 cm, respectively. The duration of the syndrome varied between 0.3 to 23 yr suggesting that recurrent episodes of the illness had occurred in some patients. However only two of them were edemic (clinical observation). Body weight loss was greater than 35% (Table 1). The healthy control group was 2 1.2 ± 1.6 (range 15 to 35) yr old; the mean weight and height were 54 ± 1.6 kg (range 46 to 60) and 163 ± 1.6 cm (range 152 to 173), respectively. Their weight/height index was more than 90% of the standard (2 1 , 24). The plasma zinc concentration (Fig 1) was unchanged in comparison with healthy subjects. The mean plasma cortisol was slightly and not significantly higher in the patients than in the control group (Fig 1). T4 concentration and free T4 index were comparable in the anorexia nervosa patients and in the controls, while T3 was significantly reduced. No statistical difference was observed in TBG, although mean TBG levels were slightly higher in the control than in the patient groups (Table 2). This slight elevation was probably due to oral contraception. Thymulin activity, expressed as log2 reciprocal titers is shown in Figure 2. Because of the age-related decline of thymulin level, results are shown according to age. Plasma thymulin level was significantly (p < 0.01) reduced whatever the duration of anorexia nervosa. Yet, the distribution of T-lymphocytes in the peripheral blood into mature, helper, cytotoxic-suppressor cells remained unchanged as well as the ratio helper/cytotoxic-suppressor cells. The total B-lymphocyte count was also normal (Table 3).

(T3) and thyroxine T,

(ng/liXt

Anorexia nervosa Controls *

Ratio

Means

277

X ±

SEM;

patients

86.5 129.8

versus

(g/IOO

±

6.4t

±

7.5

9.5 ± 0.8 9.2 ± 0.4

the control

group.

globulin

(TBG)

TBO

T4 ml)

10. t p 5

....

t 4

#{149}.

C....

consequence of protein-energy without superimposed infections

-U.;-.

4

atrophy and/or other hormonal disturbances. The immune system, although regulated to a large extent by intrinsic cellular and humoral events, is sensitive to hormonal

...

z3

-

..

>. I

.-0

.‘-

changes.

-y__--

15-21

Patients

25-35

years

Controls

Patients

years ______ Controls

Plasma thymulin activity was significantly reduced in anorexia nervosa. We have previously reported that thymulin level was normal in moderately as well as in severely malnourished children with acute infections (25). However nutritional deficiency has complex effects on the immune system depending upon the age at which the nutritional insult occurs, variations in nutritional requirements and the presence or absence of infections. Decreased thymulin activity has been shown in malnourished children without infections (12) and in experimental animals fed nutrient-deficient (zinc, pyridoxine, vitamins) diets (15, 26, 27). The presence of zinc is essential for the biological activity of the circulating thymic hormone (17). In our study, the plasma zinc concentrations of the patients were normal and there was no correlation between its level and that of thymulin whatever the degree and duration of patient’s malnutrition. This

lymphocyte

cells into several

years,

several

investigators

subsets5 Cytotoxic-

slg-beanng B.cells

Anorexia Controls

nervosa

Results expressed Means ± SEM.

patients

13

± 1.2

11±1.4

as

%.

Mature

Helper

T-cells

(a)

64 ± 2.9

39 ± 2.3 40±2.7

59±4.5

SU1C55O1

(b)

23 ± 1.8 22±1.6

Ratio:

b

1.8 ± 0.20 1.9±0.27

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Discussion

of peripheral

In recent

have demonstrated that hormones such as glucocorticoids, growth hormone, thyroxine and sexual hormones may exert their action on the lymphoid system (28). It has been shown that high glucocorticoid levels may decrease thymulin concentration (29). Our patients exhibited a normal cortisol concentration suggesting that this factor was not involved in the depression oftheir circulating thymic hormone. In humans, alterations of thyroid turnover (low T3 syndrome) cause reduction of thymulin activity (28). Experimental thyroxinedeprived animals show a significant decrease in thymulin activity, which is restored by daily injection with exogenous triiodothyronine (30). Low serum T3 and increased serum reverse T3 levels have been previously reported by others (31-33) in anorexia nervosa. The patients we studied exhibited normal T4 and TBG levels but a significant drop in plasma T3 concentrations compared to the healthy controls. The value recorded for T3 in the presence of unchanged T4 and TBG concentrations suggests a decrease in fractional conversion of T4 to T3. Virtually all the metabolic action of T3 is derived from T4 generated from it. In normal subjects a minimum of about 40% ofT4 is metabolized via monodeiodination (34). These data confirm previous findings (33) indicating that patients with anorexia nervosa were euthy-

FIG 2. Plasma thymulin activity in anorexia nervosa patients and healthy controls grouped according to age. Individual values were reported. p