Proximal muscle weakness and selenium deficiency

Proximal muscle weakness and selenium deficiency associated with long term parenteral nutrition14 Marilyn R Brown, MD, Harvey J Cohen, MD, PhD, Tom W Curtis, RPh, Barbara Thunberg, RD, MEd, William J Cochran, MD, and William J Klish, MD

Jane

M Lyons,

RN,

MS,

KEY WORDS parenteral nutrition

Proximal

muscle

weakness,

Introduction

Home

parenteral nutrition is a major adthe rehabilitation and prolongation of useful life of people who have intestinal failure, especially those who have lost most of their intestinal absorptive surface and have severe malabsorption. Parenteral nutrition has decreased the mortality and morbidity of such individuals and has returned many to near normal existence (1). These people, however, are prone to develop unusual nutritional deficiency states. Deficiencies have been reported for essential fatty acids, copper, zinc, chromium, biotin, molybdenum, and selenium (2-8). Selenium has been shown to be an essential trace element, necessary for the enzymatic activity of glutathione peroxidase (GSHPx). GSHPX catalyzes the degradation of hydrogen vance

peroxide

in

and

organic

hydroperoxides.

In the

of selenium, GSHPx is not active, and lipid peroxides and free radicals may damage cell membranes. absence

The American Journal of Clinical Nutrition 43: APRIL © 1986 American Society for Clinical Nutrition

selenium

deficiency,

glutathione peroxidase activity,

Selenium deficiency causes muscular dystrophy in sheep and cattle, pancreatic degeneration and exudative diathesis in chickens, and liver necrosis in rats (9, 10, 11). The mechanism by which selenium deficiency results in the development of these diseases is not known. In man, selenium deficiency has been associated with cardiomyopathy or muscle pain. A congestive cardiomyopathy with a high mortality rate that usually affects women of childbearing age and children is endemic to I

From

diatric

the Department

of Pediatrics,

Gastroenterology/Nutrition

and Nutritional

Divisions

of Pc-

and Hematology/OnService, University

Support of Center, Rochester, NY. 2Presented in part at the American College of Nuthtion Meeting, Boston, September, 1984. 3Supported in part by grants from USPHS Al 19656 and AM 33231. 4Address reprint requests to: Marilyn R Brown, MD, Pediatric Gastroenterology/Nutrition, Box 667, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. Received June 17, 1985. Accepted for publication September 17, 1985.

cology Rochester

1986, pp 549-554.

Medical

Printed

in USA

549

Downloaded from www.ajcn.org by guest on July 10, 2011

ABSTRACT A 33-yr-old white female with short bowel syndrome secondary to trauma was maintained on home parenteral nutrition for 4 yr when her plasma, red cell, white cell, and platelet glutathione peroxidase (GSHPx) activities were found to be extremely low, as were her plasma and red cell selenium levels. During her first year on parenteral nutrition she noted the onset of an inability to rise from a squatting position, rapid tiring when stair climbing, and weakness when attempting to lift large or moderately heavy objects. Treatment with 400 g/d of selenious acid intravenously was associated with a disappearance of her symptoms and an increase in proximal muscle strength within 6 wk. The plasma and red cell selenium levels, and the plasma and white cell GSHPx activities rose to normal levels within 6 wk. Red cell GSHPx activity returned to normal by 3 mo. Am iC/in Nuir l986;43:549-554.

550

BROWN

AL

After 4 yr, selenium levels and glutathione peroxidase enzyme activities were measured. Both were profoundly low. The patient had not had muscular pain, but during her first year on home total parenteral nutrition she had become unable to rise from a squatting position and her legs became weak while climbing stairs. She was unable to lift moderately heavy objects and had much less strength in her arms and legs when swimming, She reported that she had not told her physicians or nurses because she thought her weakness was an expected part of her illness. At the time her low selenium level and low GSHPx activity were determined, a complete blood count, SMA 6 and SMA 12 were normal. A Heinz body preparation was negative. To rule out any evidence of a cardiomyopathy, an electrocardiogram, chest radiograph, cardiac sonography, and a multigated cardiac blood pool scan with technetium pertechnate were performed and all were within normal limits. Pulmonary function tests were also within normal limits. A serum vitamin E level was within normal limits. Serum creatine phosphokinase, red cell pyruvate kinase, and glutathione reductase were within normal limits. She declined electromyography and muscle biopsy. No neurological abnormalities were identified cxcept an inability to rise from a squatting position without help, decreased proximal muscle strength to 3+ and 4-, out of 5, in both the upper and lower extremities. The study and treatment protocol was approved by The Commiftee on Investigations Involving Human Subjects of the University ofRochester Medical Center. Afterthese studies were completed, 400 ig ofselenium as selenious acid was added to her daily intravenous solution.

Methods

Case

report

A 33-yr-old female was placed on home total parenteral nutrition at age 29 after she sustained a traumatic loss of her small bowel. The third portion of the duodenum was anastomosed to the cecum. Her intravenous amino aciddextrose solutions contained 1 g/kg protein, minerals, multiple vitamins, and trace minerals which included zinc, copper, manganese, and chromium, but no selenium. The daily intravenous intake of vitamin E was 10 LU. Five hundred ml of a 10% lipid emulsion was infused twice weekly. Medications included daily cimetidine to control diarrhea, and acetaminophen as necessary for headache. She was treated with intravenous antibiotics on two cccasions for central tunneled catheter infections, and the catheter was changed once. Cheilosis developed after 2 yr and responded to an increase in the dose of B and C vitamins. Osteoporosis and rib pain were noted after2#{189} yr. Serum calcium, phosphorus, parathormone, vitamin D, and l,25(OH2) vitamin D levels were normal. Calcium intake was increased to exceed urinary calcium losses, and the rib pain disappeared. Her intake was increased to 470 mg calcium/d, which was approximately twice her prior usual daily intake. An ulcer developed at the margin of the duodenocecal anastomosis after 3 yr, and her abdominal pain responded to an increase in cimetidine with added sucralfate and antacids. Her oral intake varied oonsiderably over the years.Although she would eat normal meals at times and suffer with severe diarrhea, she learned to eat very littleand to drink alcohol sparingly.

Collection

ofblood

Blood samples tubes for isolation Preparation

of

samples (20 ml) were collected in heparinized of blood cells and plasma.

granulocytes

and mononuclear

cells

Blood (12 ml) and 6% dextran (4 ml) were combined in a 20 ml syringe and allowed to sediment. The top layer was extracted into a 50 ml conical centrifuge tube and centrifuged at 1500 RPM for 10 mm at 4#{176}C in a Sorval RC3 refrigerated centrifuge. The pellet was resuspended in 3 ml of phosphate buffered saline (PBS) and layered onto 3 ml of Ficol-Hypaque at room temperature in a 15 ml conical centrifuge tube and spun at 2000 RPM for 30 mm in a Sorval RC-3 centrifuge (DuPont Inc, NJ) maintamed at 20#{176}C. The plasma Ficol interface containing the mononuclear cells was removed with a transfer pipette and the pellet was resuspended in 5 ml PBS. The mononuclear cell fraction and pellet were each centrifuged at 1200 RPM for 10 mm at 4#{176}C and resuspended. Contaminating erythrocytes were removed by adding 10 ml of distilled water to each sample, mixing vigorously, and then adding 0.5 ml of 3.08 M NaCI. The cells were again centrifUged at 1200RPM for 10 mm at 4#{176}C and resuspended in 0.6 ml of PBS. Cell counts were then performed using a hemocytometer. Preparation

of plasma.

platelets.

and red cells

The remaining 8 ml of blood was centrifuged at 800 to 900 RPM for 10 mm at 4#{176}C. The supernatant was care-

Downloaded from www.ajcn.org by guest on July 10, 2011

the Keshan region of China where the soil is low in selenium (12). A similar cardiomyopathy associated with low selenium levels has been reported in a few patients on long-term parenteral nutrition (8, 13). Incapacitating muscular pain, which develo_ in a New Zealand patient on parenteral nutrition for 40 days, disappeared when selenomethionine was added to her intravenous nutrition (14). A 6-yr-old boy on long-term parenteral nutrition developed white fingernails and leg muscle pain and tenderness. These abnormal findings reverted to normal after treatment with selenium (1 5). More recently a 19-yr-old woman with cystic fibrosis developed muscle pain, weakness, and an elevated creatine kinase after 3 mo on total parenteral nutrition not supplemented with selenium. These abnormalities disappeared with treatment (16). This report describes muscle weakness in a patient on total parenteral nutrition associated with marked selenium and glutathione peroxidase deficiency. This weakness responded to selenium supplementation and was related to normalization of plasma and granulocyte GSHPx activities.

ET

TPN,

SELENIUM

DEFICIENCY

fully removed and placed in a 15 ml centrifuge tube and centrifuged at 2000 RPM for 10 mm at 4#{176}C. The pellet was resuspended and 10 ml of distilled water was added. After rapid mixing, 0.5 ml of 3.08 M NaG was added. The platelet pellet was centrifuged again at 2000 RPM for 10 mm and resuspended in 0. 154 M NaCl. The red blood cell pellet from the original centrifugation was washed three times with cold 0.154 M NaCI and after each wash the buffy coat was discarded. Packed red cells (0. 1 ml) were added to 0.001 M potassium phosphate (1.9 ml) buffered at pH 7.4 and centrifuged in a Triac (Clay-Adams, Parsippany, NJ) centrifuge to remove ghosts. The supernatant was used for hemoglobin and enzyme activity. Glutathione

peroxidase

activity

Pyruvate

kinase

and glutazhione

reductase

activities

Pyruvate kinase activity in hemolysates of packed red blood cells was assayed by the method of Beutler (18), eg, the oxidation of NADH was monitored at 37#{176}C in the presence and absence of ADP, utilizing 0.025 ml of hemolysates. Glutathione reductase activity in hemolysates from packed red blood cells was assayed also by the method of Beutler(l9). This assay monitors the oxidation of NADPH at 37#{176}C in the presence and absence of oxidized glutathione utilizing 0.025 ml hemolysates. The assay was performed in the presence and absence of 1 tM flavin adenine dinucleotide (FAD). The activity coefficient was obtained by dividing the activityin the presence of FAD by the activity in the absence of FAD. The hemoglobin (Hgb) concentration in the hemolysates from packed red blood cells was determined utilizing Drabkin’s reagent. Protein analysis utilized the method of Lowry (20) with bovine serum albumin as the standard. Selenium Selenium determinations were performed by the fluorometric technique (21). Selenium levels were measured by Drs Oscar E Olson and Ivan Palmer, Station Biochemistry Section, Chemistry Department, South Dakota State University, Brookings, SD.

Results When

studied

initially,

level of our patient

the plasma

was 5 ng/ml.

selenium

Normal

val-

ues

in our

551

WEAKNESS

area

range

from

60-140

ng/ml.

Erythrocyte selenium level was 22 ng/ml (normal range from 140-250 ng/ml). Her mitial plasma GSPHx activity was 0.02 units/ml (normal values from 0. 19 to 0.33). Erythrocyte GSHPX activity was 1.06 units/g Hgb (normal range from 20-26). All values were approximately one tenth the normal values. After treatment with selenious acid, the plasma selenium levels rose to normal within 6 wk (Table 1). The red blood cell selenium level also rose, but much more slowly. The dose of selenious acid was lowered to 120 zg/day after 6 wk. Plasma, red blood cell, platelet, granulocyte, and mononuclear cell GSHPX activities began to rise within 3 d of treatment with selenious acid (Table 2). The time course for the recovcry of GSHPX activity differed in the various blood elements and is represented in Figure 1. Plasma GSHPX activity responded rapidly; normal values were achieved within 2 wk. The granulocyte levels of GSHPx activity moved into the normal range within 6 wk. Similar time courses were observed for platelets and mononuclear cells. The erythrocyte GSHPX activity, however, appeared to lag for “-6 wk and did not return to the normal range until almost 3 mo had elapsed. The time necessary for normalization is what would have been expected if only newly synthesized red cells contained GSHPx activity (22). After 1 mo of treatment, the patient reported that she was feeling much stronger. She could walk up stairs without difficulty, was able to swim freestyle, and was able not only to push her IV pole, but also the vacuum cleaner and the lawn mower. She also could carry bags of groceries and lift boxes of her total parenteral nutrition supplies. Examination by the physical therapist after 2 mo reTABLE 1 Change in selenium selenium

levels in response

to treatment

with

Selenium

ROC

Baseline 6wk 4mo Normal

(....g

(tMil)

5 117 118 60-120

22 80 111 140-250

Downloaded from www.ajcn.org by guest on July 10, 2011

Glutathione peroxidase activity was measured in plasma, hemolysates, granulocytes, mononuclear cells, and platelets by the method ofBeutler (17). The volume used to assay enzyme activity varied between 0.025 and 0.1 ml for plasma, 0.01 to 0.05 ml for hemolysates, and 0.05 to 0.06 ml for the suspension of platelets, granulocytes, or monocytes. When platelets, granulocytes, or monocytes were used, the final assay contained 0.05% triton X-lOO. This amount of triton did not inhibit glutathione peroxidase activity and was able to solubilize all the enzyme activity from these cells. The rate obtained when a $-mercaptoethanol stabilizing solution was substituted for the sample, was subtracted from the rate obtained with the sample. For red blood cells and plasma, 1 unit was defined as 1 imol of NADPH oxidized per rain; for the other cells, one unit was 1 nmol of NADPH oxidized per mm.

MUSCLE

552

BROWN

TABLE Changes

2 in glutathione

Normal Initial 3days lwk lOdays 2 wk 4 wk 6wk 2mo 2 mo 3 mo

0.26

4mo 6mo

peroxidase activitiesin response

±

±

RBC

Platelet

(snO

(as/gm HGB)

(sM*g PRO)

0.05 (28)t 0.02 0.11 0.15 0.15 0.21 0.22 0.24 0.23 0.21 0.15 0.21

20.6

SD. of control

3.6 (27) 1.1 2.6 2.2 3.1 3.6 4.8 6.0 12.8 19.9 25.1 25.6 28.1

157

±

Discussion

This report describes an association between muscle weakness and severe selenium deficiency in humans. The patient had both a symptomatic and biochemical response to selenium therapy. Except for the addition of selenious acid to her routine intravenous nutrition, other aspects of the patient’s treatment remained the same. Symptoms related to low blood levels of selenium and/or glutathione peroxidase activity are rare. Forty-nine cases of selenium deficiency without recognizable symptoms have been reported when the studies ofBaker, Shils, Baptista, Lane and Fleming and their coproximal

are combined

(23-29).

Although

the

need for degradation ofhydrogen peroxide and organic hydroperoxidases by glutathione peroxidase is important, the absence ofthe activity of this enzyme in selenium-deficient patients does not automatically indicate the danger of developing any particular clinical manifestation, possibly because vitamin E can also prevent peroxidation of tissues at the membrane level. The combination of selenium and vitamin E deficiency may be required

in some

with selenium

83 (22) 11 42 101 75 107 65 38 277 298 237 -

±

Granulocie (u/IO’

1.50

±

Moooauclesr (u/1O’

PM?s9

0.71 (18) 0.22 0.36 0.32 0.88 0.66 1.84 1.18 2.21 3.05

14.1

4.83 4.12

MONO)

2.7 (23) 0.9 4.7 3.9 6.6 15.3 17.9 9.0 3.4 7.7 17.3 15.7

±

samples).

vealed the ability to rise from a squatting position without help, and an increase in houlder girdle and hip girdle muscular strength to 4+ and 5-.

workers

to treatment

patients,

for symptoms

to de-

velop. The serum vitamin tient, however, was within Symptoms

related

E level normal

to selenium

in our limits.

pa-

deficiency,

reported previously in humas, include muscle pain and cardiomyopathy. Lower extremity thigh muscle pain was described in one patient in New Zealand by Van Ru et al in 1979 (14). A 37-yr-old female with enterocutaneous fistulae and intra-abdominal sepsis was noted to have low blood selenium, low whole blood glutathione peroxidase levels, hypoalbuminemia, and low serum iron. Vitamin E levels were not reported. This patient’s muscle pain responded to selenomethionine given intravenously. Other symptoms related to selenium deficiency in the human have involved heart muscle. Keshan disease is a commonly fatal endemic cardiomyopathy that primarily affects women of childbearing age and children living in an area of China in which the soil and staple food have very low selenium levels. Selenium supplementation given to thousands ofchildren decreased the incidence ofthe disease dramatically (12). The features of this cardiomyopathy

are cardiomegaly

and

conges-

tive heart failure, both ofwhich respond poorly to conventional therapy. The pathological features of the heart disease are focal patchy areas ofmyocardial loss and replacement with fibrosis. Two instances of the association of parenteral nutrition, selenium deficiency, and fatal

Downloaded from www.ajcn.org by guest on July 10, 2011

t (Number

AL

Plums

0.23 Mean

ET

TPN,

SELENIUM

DEFICIENCY

PLASMA GSHPx RESPONSE TO SELENIUM

MONTHS

GRANULOCYTE RESPONSE

TO

GSHPx SELENIUM

MUSCLE

553

WEAKNESS

selenium for a short time, but died without apparent response. Collipp and Chen reported a 2-yr-old girl with cardiomyopathy and low selenium levels who responded to selenium supplementation and zinc supplementation (30). Cardiac function in our patient was normal as assessed by echocardiogram and mulitgated cardiac blood pool scan. The 6-yr-old boy who developed leg muscle pain and tenderness, reported by Kien and Ganthen, had elevation in serum activities of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and the MB isozyme of creatine phosphokinase. These findings suggested cardiac muscle injury as well as hepatic and skeletal muscle damage, all of which responded to selenium treatment (1 5). The serum activities ofglutamic oxaloacetic transaminase and creatine phosphokinsae were normal in our patient. Muscle weakness is known to accompany the malnutrition and debilitation that occur with

Had

fully

after

GSHPX

who

require

total

we not questioned

parenteral

our patient

nutri-

care-

determining that her selenium and activities were low, the symptom

might have gone unrecognized. may be missed as a manifestation

This symptom of selenium

deficiency.

Comparison of the rates of recovery of activity in blood components to selenium treatment in our patient revealed three patterns. The plasma enzyme activity increased to normal within 2 wk of treatment. However, the cellular GSHPX activities rose more slowly. This suggests that the blood cells are not the source of the plasma enzyme. The white blood cells and platelet enzyme levels normalized within 1 to 2 mo, coincident with the reversal of clinical symptoms, and may be good indicators of tissue levels of GSHPx. The erythrocyte GSHPx activity remained low for 6 wk and became normal only after 3 mo of treatment. The time required for normalized erythrocyte GSHPx activity in our study was similar to that previously described (22) and probably reflects the time required for the appearance of newly synthesized erythrocytes containing the GSHPx after selenium supplementation. Proximal muscle weakness in a patient receiving long-term home parenteral nutrition was associated with a decrease in plasma and GSHPX

RBC RESPONSE

GSHPx TO SELENIUM

30

E 20 ‘C

0. 10

0

MONTHS

FIG 1. Glutathione peroxidase activities in response to treatment with selenium. Plasma GSHPx activity is depicted in the top graph, granulocyte GSHPx in the middle, and RBC GSHPx activity in the lower graph. Shaded areas represent the range for control samples performed at the same time as the patient samples.

cardiomyopathy have United States, one by other by Fleming et al ported by Johnson and

been reported in the Johnson et al and the (8, 13). The patient recoworkers was treated

Downloaded from www.ajcn.org by guest on July 10, 2011

in patients

tion.

554

BROWN

erythrocyte granulocyte,

selenium, and decreased plasma, mononuclear cell, platelet, and

erythrocyte

glutathione

peroxidase

activities.

with intravenous selenious acid reversed the symptoms and the biochemical abnormalities. The rapid restoration of normal muscle strength and biochemical values during treatment with selenium, with no other change in treatment, strongly suggests that proximal muscle weakness is a clinical feature of selenium deficiency.

ET

14.

Treatment

The authors tance ofMs

wish to acknowledge

Patricia

Liegey

the technical

and Ms Margaret

15.

16.

17.

assis-

Chovaniec.

References

19.

20.

2 1.

22.

23.

24.

25.

26.

27.

28.

29.

30.

EE, Thistle JL. Selenium deficiency and fatal cardiomyopathy in a patient on home parenteral nutrition. Gastroenterology l982;83:689-93. van Rij AM, McKenzie JM, Thomson CD, Robinson MF. Selenium supplementation in total parenteral nutrition. JPEN 198l;5:120-4. Kien CL, Ganthen HE. Manifestations ofchronic selenium deficiency in a child receiving total parenteral nutrition. Am J Gin Nutr l983;37:319-28. Watson RD, Cannon RA, Kurland GS, Cox KL, Frates RC. Selenium responsive myositis during prolonged home total parenteral nutrition in cystic fibrosis. JPEN 1985;9:58-.60. Beutler E. Red cell metabolism: a manual of biochemical methods. 2nd ed. New York: Grune and Stratton, 1975:71. Beutler E. Red cell metabolism: a manual of biochemical methods. 2nd ed. New York: Grune and Stratton, 1975:60. Beutler E. Red nell metabolism: a manual of biochemical methods. 2nd ed. New York: Grune and Stratton, 1975:69. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J BiolChem l95l;193:265. Olson OE, Palmer IS, Cary EE. Modification of the official fluorometric method for selenium in plants. J Assoc OffAnal Chem 1975;58:l 17-9. Cohen Hi, Chovaniec ME, MiStretta D, Baker SS. Selenium repletion and glutathione peroxidase-differential effects on plasma and red blood cell enzyme activity. Am J Clin Nutr 1985;4l:735-47. Baker SS, King WWK, Michel L, Wood WC, Malt RA, Cohen HJ. Reversal of biochemical and functional abnormalities in erythrocytes secondary to selenium deficiency. JPEN l983;7:293-6. Baker SS, Lerman RH, Krey SH, Crocker KS, Hirsh EF, Cohen HJ. Selenium deficiency with total parenteral nutrition: reversal of biochemical and functional abnormalities by selenium supplementation: a case report. Am J Gin Nutr l983;38:769-74. Shils ME, LevanderOA, Alcock NW. Selenium levels in long term TPN patients. Am J Gin Nutr 1982;35: 838 (abstr). Baptista RJ, Bistrian BR, Blackburn GL, Miller DG, Champagne CD, Buchanan L Utilizingselenious acid to reverse selenium deficiency in total parental nutrition patients. Am J Gin Nutr l984;39:816-20. Lane HW, Dudrick 5, Warren DC. Blood selenium levels and glutathione peroxidase activities in university and chronic intravenous hyperalimentation subjects. Proc Soc Exp Biol Med 1981 ;l67:383-90. Lane HW, Barroso AO, Englert D, et al. Selenium status in seven chronic intravenous hyperalimentation patients. JPEN l982;6:426-3l. Fleming CR, McCall JT, O’Brien iF, Forsman RW, Ilstrup DM, Petz J. Selenium status in patients receiving home parenteral nutrition. JPEN l984;8:25862. Collipp PJ, Chen SY. Cardiomyopathy and selenium deficiency in a two year old girl. N Engl J Med 198 l;304: 1304-5.

Downloaded from www.ajcn.org by guest on July 10, 2011

1. Fleming CR, Beast RW, Berkner 5, et al. Home parenteral nutrition for management of the severely malnourished adult patient. Gastroenterology l980;79: 11-8. 2. Riella MC. Essential fatty acid deficiency in human adults during total parenteral nutrition. Ann Intern Med 1975;83:796-9. 3. Fleming CR, Hodges RE, Hurley LS. A prospective study of serum copper and zinc levels in patients receiving total parenteral nutrition. Am J Clin Nutr l976;29:70-7. 4. Kay RG, Tasman-Jones C, Pybus J, et al. A syndrome of acute zinc deficiency during total parenteral alimentation in man. Ann Surg 1976;183:331-40. 5. Freund H, Atamian S. Fischer JE. Chromium deficiency during total parenteral nutrition. JAMA l979;241’496-8. 6. lnnis SM, Allardyce DB. Possible biotin deficiency in adults receiving long-term total parenteral nutrition. Am J Gin Nutr l983;37:185-7. 7. Abumrad NN, Schneider AJ, Steel D, Rogers LS. Amino acid intolerance during prolonged total parenteral nutrition reversed by molybdate therapy. Am J Clin Nutr l98l;34:2551-9. 8. Johnson RA, Baker SS, Fallon JT, et al. An occidental case of cardiomyopathy and selenium deficiency. N EnglJ Med l98l;304:l210-2. 9. Muth OH, Oldfield JE, Schubert JR, Remmert LF. White muscle disease (myopathy) in lambs and calves. VI. Effects of selenium and vitamin E on lambs. Am J Vet Res 1959;75:231-3. 10. Burk RF. Selenium in nutrition. World Rev Nutr Diet l978;30:88-l06. 11. Schwartz K, Foltz CM. Selenium as an integral part of factor3 against dietary necrotic liver degeneration. J Am Chem Soc l957;79:3292-3. 12. Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Observations on the effect of sodium selenite in prevention of Keshan Disease. Chinese Med J l979;93:47l-6. 13. Fleming CR, Lie JT, McCall JT, O’Brien JF, Baillie

18.

AL