Original Paper Ann Nutr Metab 1992;36:197-202
Pilar Varda
Ascensión Marcos María Pilar Navarro
Zinc Status in Anorexia nervosa
Instituto de Nutrición y Bromatología (CSIC), Facultad de Farmacia, Ciudad Universitaria, Madrid, España
Abstract Some aspects of zinc nutritive status in patients suffering from anorexia nervosa were evaluated. Basic anthropometric mea surements, Zn levels in serum, urine and hair as well as serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH; Zn-dependent enzymes) and delayed dermal hypersensitivity were tested both in patients and in control subjects. The patients showed higher Zn levels in serum (123%), hair (85%) and urine (215%) than controls, whereas ALP and LDH val ues were 38 and 21 %, respectively, lower than those from the control group. Half of the patients showed hypoergy, and less than 50% of them were able to show a normal immune response. These results suggest that Zn-dependent functions may be impaired in anorexia nervosa as a consequence of Zn unavailability.
Introduction Anorexia nervosa is a psychophysiological disorder predominantly occurring in adoles cent girls that is characterized by behavior directed toward weight loss, resulting in ema ciation, hypermotility, fear of becoming obese and distortions in the body image [1], The restricted dietary intake of these patients in creased the risk of developing marginal or severe deficient nutritive status [2], Disturbed zinc metabolism has been sug gested to be a potential factor in the etiology
Received: May 21, 1991 Accepted: April 2. 1992
of anorexia nervosa and to explain some of the symptoms of this syndrome, such as skin lesions, alopecia, nausea, hypogeusia, depres sion, irritability and anorexia [3,4], Oral sup plementation with zinc sulfate has been re ported to be successful in the treatment of anorexia nervosa [5, 6], Zinc is a cofactor in over 100 metalloen zymes in different species. It is essential for the normal functioning of cells and the spe cialized enzymatic function of cells in many organs [7],
Pilar Varela Instituto de Nutrición y Bromatología (CSIC) Facultad de Farmacia Ciudad Universitaria E-28040 Madrid (Spain)
© 1992 S. Kargcr AG. Basel 0250-6807/92/ 0364—0 197S2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 11:14:36 PM
Key Words Zinc status Anorexia nervosa I mmunocompetence Metalloenzymes
Subjects and Methods Subjects The study involved 10 female patients (15-21 years of age) suffering from anorexia nervosa. They were admitted for treatment at the Children Psychiat ric Service of the Red Cross Hospital in Madrid. The patients satisfied the diagnostic criteria of Feighner et al. [12]: they were characterized by a distorted attitude towards food, eating and weight, a greater than 25% loss in body weight, a persistence in an idealized slim self-image, amenorrhea and an absence of other medi cal and psychiatric illness. The control group (10 females) comprised students and was matched by age and sociocultural level. Consent for venipuncture, urine collection and hair samples was obtained from the patients and the medi cal staff after the purpose and nature of the study had been explained. The control population studied was drawn from a public secondary school in Madrid on the official school list and of intermediate socioeconomic status. The socioeconomic status of the families w'as as sessed using the father’s working position or, when there w'as no father in the family, information concern ing the mother’s occupation.
198
Methods Anthropometric Measurements. Height, weight, percentage of ideal body weight (I BW) according to the Metropolitan Life Insurance tables [ 13] and body mass index [BMI: weight (kg)/height- (m2)] w'ere taken in both groups (control and anorectic) by the same trained investigator using standard techniques. The procedures followed were in accord with the Helsinki Declaration as updated in Tokyo, Japan, in 1975. Biochemical Measurements. After an overnight fast all subjects were seen in the outpatient clinic for the sampling of blood. Urine collections were begun at 10 h a.m. for the next 24 and 48 h. Urine was voided directly into plastic containers in acid medium, kept under refrigeration (4 °C), the volumes were recorded and the samples aliquoted were frozen until analysis. Collection syringes, needles, tubes and urine con tainers were determined to be essentially free of zinc and thus did not contaminate the serum and urine. All chemicals used for serum, urine and hair sam ples were of high purity, and distilled-deionized water was used throughout. Duplicate samples of serum and urine, diluted as necessary', were analyzed directly for Zn by flame atomic absorption spectrophotometry with a PerkinElmer 420 [14]. The coefficient of variation was 2.7% for serum and 3.3% for urine. Hair samples were cut close to the scalp in the occi pital region of the head, roughly 1 cm2 for the surface and 5 cm for length, and the distal portion was re jected. Hair samples were washed in individual plastic containers with a nonionic detergent solution by shak ing during 8 min. followed by several rinses with dis tilled-deionized water. Subsequently, the hair sample was blotted on filter paper, dried in an oven at 4 0 °C. stored in a desiccator and weighed for analysis. The samples were dry-ashed in a muffle furnace at 450 °C. Ash was dissolved in a CIH/NO3H/H2O solution ( 1: 1:2). and then diluted samples were analyzed for zinc by flame atomic absorption spectrophotometry. The betwcen-batch precision of the method was 4.7%. Bovine liver (Standard Reference Material 1577a: Na tional Bureau of Standards, Gaithersburg. Md., USA) was used as reference sample. The zinc content was 126 ± 5 gg/g (certified value 123 ± 8 gg/g). ALP (EC 3.1.3.1) and LDH (EC 1.1.1.27) were determined by an autoanalvzer RA-100-Assist (Technicon). These tests were measured in a double-blind study. Immunological Measurements. Evaluation of im mune response capacity was carried out by delayed hypersensitivity skin test response to 7 recall antigens, which was assessed using the Mcricux Multitest CMI
Varcla/Marcos/Navarro
Zinc Status in Anorexia nervosa
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 11:14:36 PM
It was recently noted [8] that zinc is the nutrient best characterized with respect to its ability to influence immune functions. Zn de ficiency in humans and/or experimental ani mals can result in thymic involution and thy mocyte depletion and depression of delayed dermal hypersensitivity. T lymphocyte num ber. T cell mitogen responses. T helper func tion. natural killer function and cytotoxic killer cell activity [9-11], Basic anthropometric measurements, se rum. urine and hair zinc levels as well as serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and T cell function throughout delayed dermal hypersensitivity were tested in patients suffering from an orexia nervosa and in healthy control sub jects, in order to further investigate the zinc nutritive state in anorexia nervosa.
Results At the time of the study (table 1) the age and the height of both groups (controls and anorectic patients) were similar, whereas weight means were lower in the patients. Weight values for controls were roughly 95% of IBW, whereas patient weights only reached 75% of IBW. which is linked to a low BMI value (16% lower than controls). The data obtained for the clinical chemical parameters are summarized in table 2 (a and b). The patients with anorexia nervosa had much higher zinc levels in serum (123%), hair (85%) and urine (215%) than controls, whereas the concentrations of ALP and LDH in serum were 38 and 21%, respectively, lower than those from the control group. Table 3 summarizes the response to the delayed hypersensitivity skin test. The nor mal range of these parameters must be taken into account. Thus, the number of positive responses to the 7 antigens has to be 3 at least, and for the score normal levels should exceed
Table 1. A nthropom etric param eters in controls and anorectic patients (m eans ±SE M )
Age. years Height, cm Weight, kg IBW. % BMI. kg/m2 Evolutive period of the illness, years
Controls ( n - 10)
Patients (n= 10)
18.7 ± 0.7 162.1 ±1.4 53.5 ±1.6 94.1 ±13.2 20.4±0.4
17.6 ± 1.1 159.8 ±1.8 43.3 ±2.1*' 74.6 ±4.5* 17.2 ±1.3*
-
3.8 ±0.5
* p =£ 0.05: significant differences between the two groups.
10 mm as cited above. As showed table 3, the patients were unable to reach those normal values, and they showed lower levels both in the number of positive responses to 7 antigens and in the score in comparison with controls (39 and 56%, respectively). Moreover, all young controls showed a normal response to the skin test. On the con trary. half of the patients presented hvpoergy. and less than 50% of them were able to show a normal response.
Discussion Control group weights (table 1) are within the normal range adjusted for age and height, according to the Metropolitan Life Insurance tables [13]. As cited above patients presented 75% of IBW. which fits into one of the main characteristic signs in anorexia nervosa. BMI means were normal for the controls, but the values for the anorectic group are considered as ‘low weight', according to the classification of Llewellyn-Jones and Abraham [ 18]. On the other hand, in spite of the fact that some authors have pointed out decreased [ 19]
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skin test antigen applicator (Merieux Institute Inc., Miami. Fla., USA) [15). The 7 antigens administered simultaneously by this applicator were tetanus, diph theria, streptococcus, tuberculin, Candida, protcus and trichophyton, as well as a control (glycerin) injection. Reactions were assessed 48 ± 2 h after injection by measuring mean induration diameter (in millimeters). Induration of 2 mm or greater was considered a posi tive reaction. Data are reported as the number of posi tive responses to the 7 antigens administered and the sum of induration for positive responses (score). The classification established by Jaurrieta et at. [16] for females was followed in this study: anergv when the value of the score is 0. since there are no positive responses: relative anergv. when there is only one posi tive response: hvpoergy, when the value of the score is less than 5 mm: low response when the score is be tween 5 and 10 mm: normal response, when the score is equal to or higher than 10 mm. Statistical evaluation of results was done by Stu dent’s t test [ 17). Differences were considered to be sta tistically significant if p < 0.05.
Table 2a. Zinc in samples from anorectic patients (single results)
1 2
Serum zinc pg/ml
Hair zinc fig/g 205
9
1.54 2.59 2.04 2.17 1.45 1.50 2.15 1.89 1.87
10
2.22
X ± SEM
1.94 ±0.12*
3 4 5
6 7
8
*p groups.
1.076 318 1,068 175 363 378 217 858 265 910 215 950 710 925 317 368 184 842 769 ±99* 285 ±53.75*
202
Controls X ± SEM 0.87 ±0.07
154 ±11.15
Table 2b. Enzyme concentrations in samples from controls and anorectic patients (means ± SEM)
Serum ALP, U/l Scrum L.DH, U/l
Patients (n = 10)
35.10 ± 2.24* 56.50 ±3.43 358.40± 13.57 283.09 ±7.27*
* p < 0.05: significant differences between the two groups.
or similar [5, 20], zinc levels in plasma, urine and hair in patients suffering from anorexia nervosa, the results found in this study were opposite (table 2a). Therefore, serum, urine and hair zinc determinations were tested twice in a blind study as cited above. Thus, serum zinc levels, urinary zinc output and hair zinc stores were much higher in anorectic patients than in controls, although zinc intake
200
Patients (n = 10)
Number of positive responses 4.78 ±0.49 to 7 antigens 2.90±0.48* 14.42 ± 1.49 6.30 ±1.26* Score, mm 0 Relative anergy. % 10 Hypocrgy. % 0 50 Low response, % 0 10 30 Normal response. % 100 *p groups.
0.05: significant differences between the two
244 ±25
0.05: significant differences between the two
Controls (n = 10)
Controls (n = 10)
Urine zinc pg/24 h
had been much lower for the patients [21]. In this respect. Pekarek et al. [22] observed simi larly high hair zinc levels in a severely zincdeprived patient, and increases in hair zinc content during zinc deprivation have been found in monkeys [23], These results might be due to a high zinc release from tissues, since it is well known that 'stress’ is sufficient to cause tissue catabo lism. Moreover, severe muscle catabolism leads to a substantial loss of zinc in the urine [24]. Furthermore, an increased absorption efficiency may contribute to such a loss of zinc, since Milman et al. [25] had pointed out that hyperzincuria appearing as a conse quence of nutritive deficiencies may be due to a corresponding increase in zinc absorption from the gastrointestinal tract. Nevertheless, the results found of zinc metabolism under malnutrition conditions are controversial. Therefore, Fell et al. [26] and Henry and Elmer [27] have also discussed the problems of determining zinc levels in conditions of tis sue breakdown. Furthermore, these authors reported that in catabolic conditions blood and urinary zinc levels may not be sensitive indicators of a deficiency as both may remain
Varela/Marcos/Navarro
Zinc Status in Anorexia nervosa
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 11:14:36 PM
Patients
Table 3. Delayed derm al hypersensitivity response in control and anorectic patients (m eans ± SEM)
stable or may be found higher despite chronic zinc losses. On the other hand. Golden and Golden [28. 29] have pointed out that children recov ering from severe malnutrition had a dramati cal fall in their plasma zinc concentration and that the metabolic state of the children with regard to anabolism or catabolism seems to be a major determinant of the plasma zinc con centration. Although zinc levels shown at the present study were high for the anorectic patients, a decrease in the zinc metalloenzymes tested (ALP and LDH) was observed in comparison with controls (table 2b). In this sense, the activities of many zinc-dependent enzymes have been shown to be adversely affected in nutritive deficiencies. Thus. ALP is notable for its rapid loss of activity following the induction of zinc deficiency [30]. It is difficult to explain these clinical fea tures (high zinc levels and low zinc metalloenzyme values) on the basis of tissue-typical zinc deficiencies, since no lack of tissue zinc was found in these patients. However, one must take into account that these patients were suffering not only from a low Zn intake but from most nutrients as well, and as a con sequence they underwent a series of metabolic changes among which the low enzyme levels found. These changes would lead to protein and energy saving.
Delayed dermal hypersensitivity (table 3) was chosen in this study, since it is the single most important method for clinically evaluat ing cellular immune responses [31], It tests the competence of the afferent, central and efferent links of the immune response, as well as the functional integrity of the nonspecific inflammatory response and is therefore a use ful monitor of in vivo cellular immunity [32], although criticized as being a nondefinitive screening procedure. The high zinc level found in this study might be one of the causes for a depletion of the immune system in anorectic patients, since both zinc deficiency [9, 10, 33] and excessive intake of zinc that is sufficient to promote high serum zinc levels [34] impair immune responses. Nevertheless, further research is required, since patients suffering from anorexia ner vosa showed physiological damage due to nu tritional disorders involved in this syndrome, affecting substrate availability in order to or ganize both enzymatic and immune systems.
Acknowledgments We acknowledge the assistance given by Dr. G. Morandc. We would also like to express out heart-felt appreciation to the young female controls and patients who made this study possible. Thanks arc due to Mrs. I. Orvav for the invaluable help.
1 Bruch H: Anorexia nervosa and its differential diagnosis. J Nerv Mem Dis 1965:141:55-60. 2 Selhulb J. Rosenberg TH: Assess ment of vitamin depiction: in Wright RA. Heynsfield S. McManus CB (eds): Nutritional Assessment. Bos ton. Blackwell Scientific Publica tions. 1984, pp 209-236.
3 Casper RC. Kirschner B, Sandstead HH, et al: An evaluation of trace metals, vitamins and taste function in anorexia nervosa. Am J Clin Nutr 1980:33:1801-1808. 4 Bryce-Smith D: Anorexia, depres sion and zinc deficiency, l ancet 1984:ii: I 162.
5 Bryce-Smith D. Simpson RID: Case of anorexia nerv osa responding to zinc sulphate. Lancet I984:ii:350. 6 Safai-Kutti S. Kutti J: Zinc therapy in anorexia nervosa. Am J Psychia try 1986:143:1059. 7 Norris D: Zinc and cutaneous in flammation. Arch Dermatol 1985; 121:985-989.
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References
18 Llewellyn-Jones D, Abraham SF: Quetelet index in anorexia nervosa. Br Med J 1984:288:1800-1808. 19 Dowd PS. Kelleher J. Walker BE. Guillon PJ: Nutritional and immu nological assessment of patients with anorexia nervosa. Clin Nutr 1983:2:79-83. 20 Casper RC. Kirchner B, Sandstead HH. et a): An evaluation of trace metals, vitamins, and taste function in anorexia nervosa. Am J Clin Nutr 1980:33:1801-1808. 21 Moreiras-Varela O. Carbajal A. Núñez C, ct al: Ingesta de energía y zinc y respuesta inmune celular en un grupo de mujeres con anorexia nerviosa. III Congreso Luso-Espa ñol de Bioquímica (Acta), Santiago de Compostela, 1988, pp 13-18. 22 Pekarek R. Sandstead HH. Jacob R. Barcome D: Abnormal cellular im mune responses during acquired zinc deficiency. Am J Clin Nutr 1979:32:1466-1471. 23 Sandstead HH. Strobel DA, Logan GM. Marks EO. Jacob RA: Zinc de ficiency in pregnant rhesus mon keys: Effects on behaviour of in fants. A m J Clin Nutr 1978:31:844849. 24 Cuthbertson DP, Fell GS, Smith CM, Tilstone WJ: Metabolism after injury. 1. Effects of severity, nutri tion and environmental tempera ture on protein, potassium, zinc and creatine. Br J Surg 1972:59:925— 931. 25 Milman N, Jacobscn-Hvid K. Hegnhoj J. Sorensen SS: Zinc absorption in patients with compensated alco holic cirrhosis. Scand J Gastroen terol 1983;18:871-875.
26 Fell GS, Cuthbertson DP. Horrison C. Flecic A. Queen K, Besset RG. Husain SL: Urinary zinc levels as indications of muscle catabolism. Lancet l972;i:280-282. 27 Henry1RW. Elmer ME: Plasma zinc in acute starvation. Br Med J 1975: iv:625—626. 28 Golden BE, Golden MHN: Plasma zinc, rate of weight gain and the energy cost of tissue deposition in children recovering from severe malnutrition on a cow's milk or soya protein based diet. Am J Clin Nutr 1981:34:892-899. 29 Golden MHN, Golden BE: Effect of zinc supplementation on the dietary intake, rate of weight gain, and en ergy' cost of tissue deposition in chil dren recovering from severe malnu trition. Am J Clin Nutr I981;34: 900-908. 30 Rothbaum RJ. Maur PR. Farrell MK: Scrum alkaline phosphate and zinc undernutrition in infants. Am J Clin Nutr 1982;35:595-598. 31 Jensen TG, Englert DM. Dudrick SJ, et al: Delayed hypersensitivity skin testing: Response rates in a sur gical population. J Am Diet Assoc 1983:82:17-23. 32 Bogden JD, Oleske JM. Munves EM. et al: Zinc and immunocompé tence in the elderly: Baseline data on zinc nutritive and immunity in un supplemented subjects. Am J Clin Nutr 1987;46:101-109. 33 Prasad AS: Clinical spectrum and diagnostic aspects of human zinc de ficiency: in Prasad AS(ed): Essential and Toxic Trace Elements in Hu man Health and Disease. New York. Liss, 1988. pp 3-53. 34 Chandra RK: Excessive intake of zinc impairs immune responses. JAMA 1984;252:1443-1446.
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8 Fraker PJ. Gershwin MF.. Good RA, Prasad AS: interrelationships be tween zinc and immune function. Fed Proc 1986;45:1474-1479. 9 Good RA, Fernandes G. Garofalo JA, Cunningham-Rundles C, Iwata T. West A: Zinc and immunity; in Prasad AS (ed): Clinical, Biochemi cal and Nutritional Aspects of Trace Elements. New York, Liss. 1982, pp 189-202. 10 Castillo-Duran C, Hcresi G, Fisberg M, Uvay R: Controlled trial of zinc supplementation during recovery from malnutrition: Effects on growth and immune function. Am J Clin Nutr 1987;45:602-608. 11 Beisel WR: Single nutrients and im munity. Am J Clin N utr 1982:35: 417-468. 12 Feighner JP. Robins E, Guze SB: Diagnostic criteria for use in psy chiatric research. Arch Gen Psychia try 1972;26:57-63. 13 Metropolitan Life Insurance Com pany: New weight standards for men and woman. Stat Bull 1959:40:1 -3. 14 Perkin Elmer Handbook: Analytical Methods for Atomic Absorption Spectrophotometry. Norwalk, Per kin Elmer Co. 1979. 15 Kaminski MV, Pinchcofky-Devin GD. McCormick DC: DDH testing as a nutritional assessment tool. Nutr Support Serv 1985;5:21-23. 16 Jaurrieta E. Sitges A, Sánchez JM. Sitges A: Desnutrición e inmunodepresión preoperatorias: factores de riesgo en cirugía mayor. Cir Esp 1981:35:81-86. 17 Sokal RR. Rohlf FJ: Estimación y test de hipótesis; in Blumc H (ed): Biometría: Principios y Métodos Es tadísticos en la Investigación Bio lógica. Madrid. Héroes. 1979, pp 145-194.
Pilar Varda
Ascensión Marcos María Pilar Navarro
Zinc Status in Anorexia nervosa
Instituto de Nutrición y Bromatología (CSIC), Facultad de Farmacia, Ciudad Universitaria, Madrid, España
Abstract Some aspects of zinc nutritive status in patients suffering from anorexia nervosa were evaluated. Basic anthropometric mea surements, Zn levels in serum, urine and hair as well as serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH; Zn-dependent enzymes) and delayed dermal hypersensitivity were tested both in patients and in control subjects. The patients showed higher Zn levels in serum (123%), hair (85%) and urine (215%) than controls, whereas ALP and LDH val ues were 38 and 21 %, respectively, lower than those from the control group. Half of the patients showed hypoergy, and less than 50% of them were able to show a normal immune response. These results suggest that Zn-dependent functions may be impaired in anorexia nervosa as a consequence of Zn unavailability.
Introduction Anorexia nervosa is a psychophysiological disorder predominantly occurring in adoles cent girls that is characterized by behavior directed toward weight loss, resulting in ema ciation, hypermotility, fear of becoming obese and distortions in the body image [1], The restricted dietary intake of these patients in creased the risk of developing marginal or severe deficient nutritive status [2], Disturbed zinc metabolism has been sug gested to be a potential factor in the etiology
Received: May 21, 1991 Accepted: April 2. 1992
of anorexia nervosa and to explain some of the symptoms of this syndrome, such as skin lesions, alopecia, nausea, hypogeusia, depres sion, irritability and anorexia [3,4], Oral sup plementation with zinc sulfate has been re ported to be successful in the treatment of anorexia nervosa [5, 6], Zinc is a cofactor in over 100 metalloen zymes in different species. It is essential for the normal functioning of cells and the spe cialized enzymatic function of cells in many organs [7],
Pilar Varela Instituto de Nutrición y Bromatología (CSIC) Facultad de Farmacia Ciudad Universitaria E-28040 Madrid (Spain)
© 1992 S. Kargcr AG. Basel 0250-6807/92/ 0364—0 197S2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 11:14:36 PM
Key Words Zinc status Anorexia nervosa I mmunocompetence Metalloenzymes
Subjects and Methods Subjects The study involved 10 female patients (15-21 years of age) suffering from anorexia nervosa. They were admitted for treatment at the Children Psychiat ric Service of the Red Cross Hospital in Madrid. The patients satisfied the diagnostic criteria of Feighner et al. [12]: they were characterized by a distorted attitude towards food, eating and weight, a greater than 25% loss in body weight, a persistence in an idealized slim self-image, amenorrhea and an absence of other medi cal and psychiatric illness. The control group (10 females) comprised students and was matched by age and sociocultural level. Consent for venipuncture, urine collection and hair samples was obtained from the patients and the medi cal staff after the purpose and nature of the study had been explained. The control population studied was drawn from a public secondary school in Madrid on the official school list and of intermediate socioeconomic status. The socioeconomic status of the families w'as as sessed using the father’s working position or, when there w'as no father in the family, information concern ing the mother’s occupation.
198
Methods Anthropometric Measurements. Height, weight, percentage of ideal body weight (I BW) according to the Metropolitan Life Insurance tables [ 13] and body mass index [BMI: weight (kg)/height- (m2)] w'ere taken in both groups (control and anorectic) by the same trained investigator using standard techniques. The procedures followed were in accord with the Helsinki Declaration as updated in Tokyo, Japan, in 1975. Biochemical Measurements. After an overnight fast all subjects were seen in the outpatient clinic for the sampling of blood. Urine collections were begun at 10 h a.m. for the next 24 and 48 h. Urine was voided directly into plastic containers in acid medium, kept under refrigeration (4 °C), the volumes were recorded and the samples aliquoted were frozen until analysis. Collection syringes, needles, tubes and urine con tainers were determined to be essentially free of zinc and thus did not contaminate the serum and urine. All chemicals used for serum, urine and hair sam ples were of high purity, and distilled-deionized water was used throughout. Duplicate samples of serum and urine, diluted as necessary', were analyzed directly for Zn by flame atomic absorption spectrophotometry with a PerkinElmer 420 [14]. The coefficient of variation was 2.7% for serum and 3.3% for urine. Hair samples were cut close to the scalp in the occi pital region of the head, roughly 1 cm2 for the surface and 5 cm for length, and the distal portion was re jected. Hair samples were washed in individual plastic containers with a nonionic detergent solution by shak ing during 8 min. followed by several rinses with dis tilled-deionized water. Subsequently, the hair sample was blotted on filter paper, dried in an oven at 4 0 °C. stored in a desiccator and weighed for analysis. The samples were dry-ashed in a muffle furnace at 450 °C. Ash was dissolved in a CIH/NO3H/H2O solution ( 1: 1:2). and then diluted samples were analyzed for zinc by flame atomic absorption spectrophotometry. The betwcen-batch precision of the method was 4.7%. Bovine liver (Standard Reference Material 1577a: Na tional Bureau of Standards, Gaithersburg. Md., USA) was used as reference sample. The zinc content was 126 ± 5 gg/g (certified value 123 ± 8 gg/g). ALP (EC 3.1.3.1) and LDH (EC 1.1.1.27) were determined by an autoanalvzer RA-100-Assist (Technicon). These tests were measured in a double-blind study. Immunological Measurements. Evaluation of im mune response capacity was carried out by delayed hypersensitivity skin test response to 7 recall antigens, which was assessed using the Mcricux Multitest CMI
Varcla/Marcos/Navarro
Zinc Status in Anorexia nervosa
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 11:14:36 PM
It was recently noted [8] that zinc is the nutrient best characterized with respect to its ability to influence immune functions. Zn de ficiency in humans and/or experimental ani mals can result in thymic involution and thy mocyte depletion and depression of delayed dermal hypersensitivity. T lymphocyte num ber. T cell mitogen responses. T helper func tion. natural killer function and cytotoxic killer cell activity [9-11], Basic anthropometric measurements, se rum. urine and hair zinc levels as well as serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and T cell function throughout delayed dermal hypersensitivity were tested in patients suffering from an orexia nervosa and in healthy control sub jects, in order to further investigate the zinc nutritive state in anorexia nervosa.
Results At the time of the study (table 1) the age and the height of both groups (controls and anorectic patients) were similar, whereas weight means were lower in the patients. Weight values for controls were roughly 95% of IBW, whereas patient weights only reached 75% of IBW. which is linked to a low BMI value (16% lower than controls). The data obtained for the clinical chemical parameters are summarized in table 2 (a and b). The patients with anorexia nervosa had much higher zinc levels in serum (123%), hair (85%) and urine (215%) than controls, whereas the concentrations of ALP and LDH in serum were 38 and 21%, respectively, lower than those from the control group. Table 3 summarizes the response to the delayed hypersensitivity skin test. The nor mal range of these parameters must be taken into account. Thus, the number of positive responses to the 7 antigens has to be 3 at least, and for the score normal levels should exceed
Table 1. A nthropom etric param eters in controls and anorectic patients (m eans ±SE M )
Age. years Height, cm Weight, kg IBW. % BMI. kg/m2 Evolutive period of the illness, years
Controls ( n - 10)
Patients (n= 10)
18.7 ± 0.7 162.1 ±1.4 53.5 ±1.6 94.1 ±13.2 20.4±0.4
17.6 ± 1.1 159.8 ±1.8 43.3 ±2.1*' 74.6 ±4.5* 17.2 ±1.3*
-
3.8 ±0.5
* p =£ 0.05: significant differences between the two groups.
10 mm as cited above. As showed table 3, the patients were unable to reach those normal values, and they showed lower levels both in the number of positive responses to 7 antigens and in the score in comparison with controls (39 and 56%, respectively). Moreover, all young controls showed a normal response to the skin test. On the con trary. half of the patients presented hvpoergy. and less than 50% of them were able to show a normal response.
Discussion Control group weights (table 1) are within the normal range adjusted for age and height, according to the Metropolitan Life Insurance tables [13]. As cited above patients presented 75% of IBW. which fits into one of the main characteristic signs in anorexia nervosa. BMI means were normal for the controls, but the values for the anorectic group are considered as ‘low weight', according to the classification of Llewellyn-Jones and Abraham [ 18]. On the other hand, in spite of the fact that some authors have pointed out decreased [ 19]
199
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skin test antigen applicator (Merieux Institute Inc., Miami. Fla., USA) [15). The 7 antigens administered simultaneously by this applicator were tetanus, diph theria, streptococcus, tuberculin, Candida, protcus and trichophyton, as well as a control (glycerin) injection. Reactions were assessed 48 ± 2 h after injection by measuring mean induration diameter (in millimeters). Induration of 2 mm or greater was considered a posi tive reaction. Data are reported as the number of posi tive responses to the 7 antigens administered and the sum of induration for positive responses (score). The classification established by Jaurrieta et at. [16] for females was followed in this study: anergv when the value of the score is 0. since there are no positive responses: relative anergv. when there is only one posi tive response: hvpoergy, when the value of the score is less than 5 mm: low response when the score is be tween 5 and 10 mm: normal response, when the score is equal to or higher than 10 mm. Statistical evaluation of results was done by Stu dent’s t test [ 17). Differences were considered to be sta tistically significant if p < 0.05.
Table 2a. Zinc in samples from anorectic patients (single results)
1 2
Serum zinc pg/ml
Hair zinc fig/g 205
9
1.54 2.59 2.04 2.17 1.45 1.50 2.15 1.89 1.87
10
2.22
X ± SEM
1.94 ±0.12*
3 4 5
6 7
8
*p groups.
1.076 318 1,068 175 363 378 217 858 265 910 215 950 710 925 317 368 184 842 769 ±99* 285 ±53.75*
202
Controls X ± SEM 0.87 ±0.07
154 ±11.15
Table 2b. Enzyme concentrations in samples from controls and anorectic patients (means ± SEM)
Serum ALP, U/l Scrum L.DH, U/l
Patients (n = 10)
35.10 ± 2.24* 56.50 ±3.43 358.40± 13.57 283.09 ±7.27*
* p < 0.05: significant differences between the two groups.
or similar [5, 20], zinc levels in plasma, urine and hair in patients suffering from anorexia nervosa, the results found in this study were opposite (table 2a). Therefore, serum, urine and hair zinc determinations were tested twice in a blind study as cited above. Thus, serum zinc levels, urinary zinc output and hair zinc stores were much higher in anorectic patients than in controls, although zinc intake
200
Patients (n = 10)
Number of positive responses 4.78 ±0.49 to 7 antigens 2.90±0.48* 14.42 ± 1.49 6.30 ±1.26* Score, mm 0 Relative anergy. % 10 Hypocrgy. % 0 50 Low response, % 0 10 30 Normal response. % 100 *p groups.
0.05: significant differences between the two
244 ±25
0.05: significant differences between the two
Controls (n = 10)
Controls (n = 10)
Urine zinc pg/24 h
had been much lower for the patients [21]. In this respect. Pekarek et al. [22] observed simi larly high hair zinc levels in a severely zincdeprived patient, and increases in hair zinc content during zinc deprivation have been found in monkeys [23], These results might be due to a high zinc release from tissues, since it is well known that 'stress’ is sufficient to cause tissue catabo lism. Moreover, severe muscle catabolism leads to a substantial loss of zinc in the urine [24]. Furthermore, an increased absorption efficiency may contribute to such a loss of zinc, since Milman et al. [25] had pointed out that hyperzincuria appearing as a conse quence of nutritive deficiencies may be due to a corresponding increase in zinc absorption from the gastrointestinal tract. Nevertheless, the results found of zinc metabolism under malnutrition conditions are controversial. Therefore, Fell et al. [26] and Henry and Elmer [27] have also discussed the problems of determining zinc levels in conditions of tis sue breakdown. Furthermore, these authors reported that in catabolic conditions blood and urinary zinc levels may not be sensitive indicators of a deficiency as both may remain
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Patients
Table 3. Delayed derm al hypersensitivity response in control and anorectic patients (m eans ± SEM)
stable or may be found higher despite chronic zinc losses. On the other hand. Golden and Golden [28. 29] have pointed out that children recov ering from severe malnutrition had a dramati cal fall in their plasma zinc concentration and that the metabolic state of the children with regard to anabolism or catabolism seems to be a major determinant of the plasma zinc con centration. Although zinc levels shown at the present study were high for the anorectic patients, a decrease in the zinc metalloenzymes tested (ALP and LDH) was observed in comparison with controls (table 2b). In this sense, the activities of many zinc-dependent enzymes have been shown to be adversely affected in nutritive deficiencies. Thus. ALP is notable for its rapid loss of activity following the induction of zinc deficiency [30]. It is difficult to explain these clinical fea tures (high zinc levels and low zinc metalloenzyme values) on the basis of tissue-typical zinc deficiencies, since no lack of tissue zinc was found in these patients. However, one must take into account that these patients were suffering not only from a low Zn intake but from most nutrients as well, and as a con sequence they underwent a series of metabolic changes among which the low enzyme levels found. These changes would lead to protein and energy saving.
Delayed dermal hypersensitivity (table 3) was chosen in this study, since it is the single most important method for clinically evaluat ing cellular immune responses [31], It tests the competence of the afferent, central and efferent links of the immune response, as well as the functional integrity of the nonspecific inflammatory response and is therefore a use ful monitor of in vivo cellular immunity [32], although criticized as being a nondefinitive screening procedure. The high zinc level found in this study might be one of the causes for a depletion of the immune system in anorectic patients, since both zinc deficiency [9, 10, 33] and excessive intake of zinc that is sufficient to promote high serum zinc levels [34] impair immune responses. Nevertheless, further research is required, since patients suffering from anorexia ner vosa showed physiological damage due to nu tritional disorders involved in this syndrome, affecting substrate availability in order to or ganize both enzymatic and immune systems.
Acknowledgments We acknowledge the assistance given by Dr. G. Morandc. We would also like to express out heart-felt appreciation to the young female controls and patients who made this study possible. Thanks arc due to Mrs. I. Orvav for the invaluable help.
1 Bruch H: Anorexia nervosa and its differential diagnosis. J Nerv Mem Dis 1965:141:55-60. 2 Selhulb J. Rosenberg TH: Assess ment of vitamin depiction: in Wright RA. Heynsfield S. McManus CB (eds): Nutritional Assessment. Bos ton. Blackwell Scientific Publica tions. 1984, pp 209-236.
3 Casper RC. Kirschner B, Sandstead HH, et al: An evaluation of trace metals, vitamins and taste function in anorexia nervosa. Am J Clin Nutr 1980:33:1801-1808. 4 Bryce-Smith D: Anorexia, depres sion and zinc deficiency, l ancet 1984:ii: I 162.
5 Bryce-Smith D. Simpson RID: Case of anorexia nerv osa responding to zinc sulphate. Lancet I984:ii:350. 6 Safai-Kutti S. Kutti J: Zinc therapy in anorexia nervosa. Am J Psychia try 1986:143:1059. 7 Norris D: Zinc and cutaneous in flammation. Arch Dermatol 1985; 121:985-989.
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