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Effects of dietary protein levels on the saimiri sciureus
EXPERIMENTAL
.&ND
Effects
YOLECUL9R
of Dietary
F. A.
DE
The Research
LA
PATHOLOGY
Protein
Inslitufe
Levels E. A.
IGLESIA,~
(1967)
?,182-1%
PORTA,
of The Hospital Received
on the Saimiri
For
&fa~
AND Sick
W.
Children,
S.
Sciurews’
HARTROFT
Toronto,
Canada
18, 1967
Although the production of nut,ritional hepatic injuries in various species of monkeys has been reported (Waisman, 1944; Mann et al., 1952; Cooperman et al., 1945; McCall et al., 1946; Rinehart and Greenberg, 1949; Rinehart and Greenberg, 1951; Rinehart and Greenberg, 1956; Follis, 1957; Wilgram et al., 195s; Gaisford and Zuidema, 1965), the results obtained appear controversial. The composition and proportion of dietary ingredient’s in these studies varied so widely that more experiments would be necessary before considering whet’her the semisynthetic diets employed in previous experiments were, in fact, adequate for a given species of monkeys. Saimiri sciwea (squirrel monkeys from South America) are easy to obtain, to to handle, and do well in capt’ivit’y (Beischer and Furry, 1964). It can be anticipat#ed that X. s&urea will frequently be chosen for nutritional studies. The lack of more specific information on t’he diet’ary requirements for members of this species prompted us to determine the minimal protein requirements of these monkeys which would permit adequate growth as well as normal maintenance of several functional and morphologic hepatic parameters. We have now explored the adequacy of a semi-synt’hetic diet at four different protein levels and the result’s are reported here. MATERIAL
APU’D METHODS a4NIMALS
During an acclimatizat,ion period of one month, 12 young male squirrel monkeys (S. sciureus) were maintained on a stock-pellet diet3 and water ad libitum. Tuberculin tests and parasitic-fecal examinations gave negative results for all animals used in this st#udy. At the end of this initial period when animals’ weights ranged from 514 to 876 gm, they were allot8ed to four groups (A, B, C, and D) which contained three members each. All monkeys were housed in individual cages in airconditioned, humidified (60%) rooms that were supplied during the day with continuous soft music. The animals were weighed twice weekly. 1 This study was supported by the Medical Research Council of Canada (Block grant Ma-1904). 2 Research Fellow of the Medical Research Council of Canada. Present address: WarnerLambert Research Institute of Canada, Sheridan Park, Ontario, Canada. 3 Purina Monkey Chow, Ralston Purina Company, Woodstock, Ontario. 182
DIETARY
PROTEIN
LEVELS
TABLE COMPOSITION
IN
183
MONKEYS
I OF DIETS
A
B
D
C
Ingredients cal o/u cal Soya
proteina
25.00
Corn Beef
oil fat
49.00
gm%
cal 70 cal
32.57
12.50
9.45
gmyO
cal y0 cal
pm%
16.28
9.00
11.73
6.10
7.92
9.45
9.45
9.45
49.00
49.00
18.90
18.90
27.27
16.07 26.00
Vitaminsb Saltsc
cal y0 cal
49.00 18.90
Sucrose Corn starch
grn%
38.50
18.90
29.95
32.55
42.00
44.90
11.29
16.38
18.24
19.49
6.51 5.21
6.51 5.21
6.51 5.21
6.51 5.21
~1 Supplemented with 2.1 9% of methionine. b Complete vitamin diet fortification mixture. Ohio. c Hubbell, Mendel and Wakeman. Nutritional
Nutritional Biochemical
Biochemical Corp.
Corp.
Cleveland,
Cleveland, Ohio.
DIETS
The composition of the semisynthetic diets offered to the different groups is indicated in Table I. Although the amounts of protein in these diets varied, all were isocaloric and supplied 5.2 cal./gm. A purified soya extract4 was employed as the source of protein and was supplemented with 2.1 gm % of methionine to compensate for the diets’ relatively low content of this amino acid. The choline content of this soya protein is 2.09 mg%. The lipotropic value of each diet was different, but all were adequate and provided the same amounts of salts, vitamins, and essential fatty acids. The content of the chosen fat was high in order to improve palatability, to resemble diets widely used in nutritional studies in rats, and to approach the fat content of diets consumed by man in most western cultures. The amounts of vitamin and salt mixtures arbitrarily chosen were 1.20 gm and 1 gm/lOO cal. of the diet. The diets were prepared weekly, stored at 4”C, and were offered daily in known amounts to the animals in the form of small balls of about 15 gm each. These diets plus water were offered ad libitum. The total food intake from which the caloric and protein intakes were calculated was recorded daily. BIOCHEMICAL
DETERMINATIONS
Blood was drawn from the femoral vein of each monkey at the beginning experiment and at the end of weeks 4,8, 12, and 22 for biochemical analyses. protein levels were determined by quantitative electrophoresis (Graham and baum, 1963). Hematograms including hemoglobin concentrations, red and 4 Purified
soya
protein
(Assay
C-l
protein),
Skidmore
Enterprises,
Cincinnati,
of the Serum Grunwhite
Ohio.
IS4
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
TABLE AVER.IGE
Foot,
DIILY
DIFFERENT
PROTEIN, I)IETS
A B c I)
34.59 34.01 31.34 43.9G
* Mean
f
+ + + z!z
S.
HARTROFT
INT.IICES IN FOR 22 WEEKS
1t.k~~
39.41 49.37 47.14 58.71
body
wvt
gm
* 7.63 * G.97 zt 10.42 + 12.71
11.26 5.54 3.68 3.48
FED
THE
Calories
Protein
gm/kg 4.74" 4.22 4.34 9.63
W.
II
.wu CALORIC m LIHITUM
I’ood
&!m
AND
gm/kg
body wt.
12.83 8.04 5.53 4.65
cal 179.87 176.85 162.97 528.59
Cal/kg hod) wt 204.93 256.72 245.13 305.29
SD
blood cell counts and hematocrits were performed. Levels of blood urea nitrogen (&eggs, 1957) and serum alkaline-phosphat’ase activities (Marsh et al., 1959) were also determined. All data were stat’istically analyzed. HISTOLOGIC
STUDIES
Open hepatic biopsies were obtained at the beginning of the experiment and at 4, S, and 22 weeks from all the animals. The monkeys were sedated with acepromazine maleate5, 0.1 ml i. m., and after local anest’hesia wit,h xylocnine, the liver was visualized through a small incision below the right costal margin and specimens were obtained with t#he Menghini needle. Portions of t#hese specimens were fixed in Baker’s solut,ion (Baker, 1945) for light-microscopy studies. Paraffin sections bvere st,ained wit,h hematoxylin-eosin, Oil red 0 (ORO) for detection of ceroid (Wilson, 1950), PAS stain, or with l\Iasson’s trichrome stain (Masson, 1929) to aid in visualizing the connective tissue. Fat in frozen sections was stained wit,h Oil red 0. For electron microscopy, blocks taken from other port,ions of the specimens were fixed by immersion in Dalton’s soh&on (Dalton, 1955) and embedded in Epon 812 (Luft, 1961). Ultrathin sections were stained with lead using Iiarnovsky’s met,hods (Karnovsky, 1961) and photographed in a Philips 200 electron microscope at established initial magnifications. RESULTS MORTALITY
Two monkeys of Group D died of inanit’ion during the experiment; one at week S and t,he other at week 20. Two monkeys of Group B died; one at week 16 (cause: inanition) and t,he ot)her at week 19 (cause: undetermined). One monkey of Group C died at week 19 (also of undetermined cause). None of t’he members of G~OLI~ A died during the course of the experiment. FOOD
INTAKE
The average values of daily food and protein and caloric intakes in the four groups during t#he whole experiment are shown in Table II. The means of absolute values 5 Atravet,
Ayerst,
McKenna
& Harrison
Ltd.,
Montreal,
Quebec.
DIETARY
PROTEIN
LEVELS
IN
155
MONKEYS
between Groups A, B, and C did not differ significantly; however, that of Group D was significant’ly higher than those of groups A (p <0.05), B (p
WEIGHTS
Changes in body weights were expressed in the form of growth curves as percentages of the init’ial values at the beginning of the experiment (Fig. 1). Due to the to the death of animals in Groups B, C, and D, for statistical purposes the only changes considered were those occurring during the first IS weeks of the experiment. Changes in body weight at weeks 3, 6, 9, 12, 15, and 18 are also shown in Table III. ISO-
140-
80 FIG.
weeks
1.
8’
4’
Growth curves
for each
12’
TABLE CHANGES Weeks 3 6 9 12 15 18
16’
20’
24’
group expressed as percent increase in initial body weight. III
IN BODY
WEIGHTS*
A
B
C
27.93 25.59 27.26 19.40 35.55 41.05
15.67 13.66 16.06 11.31 15.04 27.17
8.41 2.93 7.75 8.77 9.17 12.12
D -11.18 -15.49 -7.42 -5.17 -4.49 -5.56
a Average values expressed as percentages of the initial weight.
lS6
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
W.
S.
HARTROFT
While significant increases were observed in Groups A (p
PROTEINS
Levels of total serum proteins of all groups at different periods are included in Table IV. The values between the various groups did not differ except at week 4, when those of Group D were significantly lower Ohan those of Groups A, B, and C (p
ANALYSIS
Mean values and their ranges are shown in Table V. Values for hemoglobin, white blood cells, and hematocrit (in the various groups at different periods) fell within the normal range for t’his species as reported by others (Beischer and Furry, 1964). However, mean values for erythrocytes and their ranges were generally lower than TABLE SERUM Weeks 4
Group
A
Total
proteins
IV
PROTEINS
Albumin
a1
Y
B C n
8.90 9.00 8.97 7.03
* * i *
0.W 0.65 0.57 0.29
4.32 zt 0.25 4.19 f 0.36 4.43 z!z 0.34 2.61 f 0.26
0.29 0.35 0.26 0.26
0.07 0.11 0.04 0.10
0.64 0.65 0.77 0.68
zt f zk f.
0.03 0.12 0.06 0.11
0.56 0.71 0.71 0.52
f * z!z f.
0.06 0.05 0.04 0.07
1.90 2.05 1.47 1.84
f f f f
0.80 0.36 0.22 0.59
8
A B c D
9.00 * 8.80 * 8.63 * 8.30 f
1.51 0.92 0.64 0.71
4.70 z!z 4.02 zt 4.22 i 3.58 *
0.29 + 0.01 0.28 z!c 0.03 0.40 zk 0.03 0.40 * 0.14
0.56 0.63 0.70 0.70
zt zt zk i
0.07 0.10 0.07 0.21
0.86 0.74 0.81 0.62
f f rt z!z
0.26 0.08 0.04 0.04
1.29 1.94 1.21 1.78
+ * f ;t
0.23 0.37 0.23 0.17
12
A B C D
8.40 9.03 7.73 8.80
f. f & f
1.11 0.58 0.22 1.70
4.33 & 0.07 4.34 f 0.25 4.04 * 0.19 3.87 * 0.59
0.23 0.25 0.25 0.25
f f z!c zt
0.02 0.08 0.09 0.01
0.47 0.57 0.64 0.67
zt f zt f
0.03 0.04 0.07 0.03
0.60 0.65 0.76 0.67
f f f zt
0.06 0.03 0.09 0.15
1.78 * 1.10 2.30 + 0.41 1.21 f 0.05 2.47 f 0.74
22
A B C D
7.10 7.90 7.20 7.40
zk 0.42
3.28 f 3.77 3.34 f 2.73
0.26 0.28 0.31 0.35
f
0.05
f
0.21
0.10
0.09
*
0.07
0.46 0.61 0.73 0.79
f
f
0.38 0.46 0.50 0.65
f
0.09
1.96 f 2.11 1.31 * 1.99
a Mean
zk SE.
f
0.50
0.56 0.57 0.27 0.05
1.05 0.09
rt f zk &
B
9
1.15 0.23
DIETARY
PROTEIN
LEVELS
TABLE
Hemoglobin
ANALYSES
Erythrocytes
Leukocytes
Group gm/lOO
ml blood
187
MONKEYS
V
HEMATOLOGICAL Week
IN
l@/cu
mm blood
103/cu
mm blood
Hematocrit ml/100
ml blood
0
A B C D
13.20 12.37 12.77 14.57
(13.1-13.3) (9.7-14.2) (11.7-13.4) (13.6-15.5)
5.97 6.23 5.90 6.20
(5.7-6.3) (5.9-6.8) (5.64.3) (5.8-6.0)
8.63 11.03 9.00 13.70
(5.2-14.0) (8.3-16.3) (7.8-11.3) (11.2-17.6)
4
A B C D
13.77 12.67 10.60 10.83
(12.9-14.5) (11.7-14.3) (9.6-12.3) (10.1-11.7)
7.07 6.33 5.80 5.40
(6.9-7.4) (5.3-7.3) (4.9-6.3) (5.2-5.8)
13.17 15.90 11.27 13.10
(10.7-14.8) (7.6-23.6) (9.1-14.0) (12.1-14.7)
45.0 40.6 38.0 36.6
(4246) (3444) (30-45) (35-39)
8
A B c D
12.17 12.43 11.70 10.85
(10.5-13.9) (10.8-15.0) (10.9-12.4) (9.5-12.2)
4.73 5.77 4.97 4.40
(3.9-5.3) (4.8-6.5) (4.5-5.3) (4.24.6)
10.57 12.50 11.63 10.85
(6.6-13.9) (9.8-14.0) (7.2-16.7) (10.5-11.2)
42.0 44.0 40.6 38.0
(37-46) (41-49) (38-43) (34-42)
12
A B C D
13.77 11.83 12.40 12.40
(13.6-14.0) (10.9-12.8) (8.7-14.7) (11.7-13.1)
5.60 4.83 4.93 4.85
(5.3-5.8) (4.1-5.3) (3.5-5.8) (4.84.9)
9.77 10.63 10.33 10.30
(6.3-10.9) (9.4-13.3) (8.0-14.0) (8.6-12.0)
46.6 37.6 40.6 42.0
(4647) (32-44) (29-48) (4044)
22
A B C D
13.10 12.40 12.05 11.70
(12.6-13.6)
5.45 5.80 5.10 5.40
(4.7-6.2)
11.50 6.10 10.55 9.6
(6.2-06.8) (6.1-11.6) (9.5-11.6)
44.0 40.0 40.0 39.0
(43-45)
(12.0-12.1)
(4.6-5.6)
those obtained in the above cited report; 12. SERUM
ALKALINE
particularly
in Groups
(38-42)
C and D at week
PHOSPHATASE
Values at the beginning of the experiment were not significantly different between groups. Although the values of Groups C and D were generally higher than those of Groups A and B during the other periods of the experiment, the differences were not statistically significant, except bet,ween Groups B and C (p
Levels of BUN in each group varied with time, but no significant differences were found between groups at the different periods studied (Table VII). HISTOLOGIC
FINDINGS
Light-microscopic examinations, before the institution of the different dietary regimens, showed that nine out of twelve monkeys had hepatic fatty changes of various degrees. The predominant lobular location of visible fat in hepatocybes was
1%
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
TABLE VI SERUM ALKALINE-PH~SPH~T.ISE
B
C D
S.
HAIZTIlOFT
ACTIVITY
LVeek
Group A
IV.
0 23.83 27.50 25.67 24.70
+ 8.19” + f f
8
4 29.83 33.50 G-I.25 44.25
0.70 5.43 9.5-l
* Mean h SE; King-Armstrong
f z!z z!z f-
11.30 4.78 8.13 15.20
35.70 16.40 48.60 47.10
z!x f * zt
12 14.78 18.05 10.98 32.28
42.13 51.30 55.07 70.00
It
15.11
z!z 11.71 &
6.71
uuits. TABLE 1’11 BLOW UREI NITROGES
Group ~ A B c D
-0 25.67 16.83 23.07 22.33
f + f f
8
4
8.89” 3.58 6.09 (i.94
21.33 16.67 14.50 16.33
f f I!z +
2.27 1.47 4.95 2.27
25.00 11.33 17.33 23.50
+ 1.41 & 5.21 * 4.71 + G.36
12 37.G7 35.68 40.33 20.00
f
24
1.08
z!z 1.71 zk 8.84 + 12.72
23.00 23.00 19.00 16.00
f
2.83
+
2.83
a Mean + SE.
mainly periportnl. Kupffer cells also had accumukked small droplets of cytoplasmic fat of diffuse lobular distribut’ion. These fatty changes regressedat’ lnt’er stages of the study, particularly in livers of animals from Groups A and B. The hepatic archit’ecture was well preserved in all groups throughout Dhe entire experiment. By eleckon microscopy, the ultrastructural configuration of hcpat,ocytes in the different groups before dietary treatment was characterized by abundant cyt,oplasmic content of glycogen granules which stained deeply with lead (Fig. 2). These granules were gr,ouped to form patchy cytoplasmic areas of erratic location, similar to t’hosefound in normal murine and human hepatocytes. Mitochondria were normal; both rough and smooth endoplasmic reticulum were well developed and normal. Numerous microbodies cont’aining characteristic crystalline cores were found in hepatocyt’es. The ultrastructural appearance of these microbodies as well as their enzymatic (urate oxidase) activity has been reported in det,ail elsewhere (de la Iglesia et al., 1966). Multivesicular bodies and autophagic vacuoles were also present and numerous medium-sized droplets of fat of moderate density were seenthroughout the cytoplasm of hepatocytes in all the animals. Granules of ceroid pigment were occasionally found in Kupffer cells. One interesting finding was the presence of cilia in the epithelial cells of cholangioles. This observation has also been report,ed elsewhere (de la Iglesia and Porta, 1967). In t,he biopsy specimenstaken 4 weeks after the institution of dietary regimens appreciable ultrastructural changes were absent in Groups A (Fig. 3), B, and C But in hepatocytes of Group D, mitochondria mere altered by budding, focal matrical rarefaction, and attenuation of their membranes; in some,the numbers of cristae
DIETARY
FIG. 2. Typical ne acclimatization
ultrastructural period during
PROTEIN
LEVELS
IN
189
MONKEYS
configuration of a hepatocyte which it was fed a commercial
of a squirrel rations. Lead
monkey after stain. X 9,000.
had decreased. In other instances mitochondria had coalesced and their total number appeared generally decreased. Profiles of endoplasmic reticulum were diminished and a few moderately dilated cisternae were seen at cellular peripheries (Figs. 4 and 5). After 8 weeks, although the configuration of hepatocytes in Groups A and B had rkmained unchanged, mitochondri’al alterations did appear in Group C. In general, they were enlarged and the number of cristae had increased. Budding and coalescence of mitochondria, as seen in Group D at 4 weeks, was also now observed at
190
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
W.
S.
HARTROFT
FIG. 3. Ultrastructural configuration of a hepatocyte from Group A (25y0 protein), 4 weeks after commencing the experiment,. Lead stain. X 10,000. FIG. 4. Electron microscopic aspect of a hepatocyte from Group D (6.1yCi; protein) 4 weeks after commencing t,he experiment. Lead stain. X 10,000.
DIETARY
PROTEIN
LEVELS
IN
MONKEYS
191
his time in Group C (Figs. 6 and 7). In addition, shapes of many mitochondria were bizarre, particularly those which were intimately in contact with droplets of fat: Hepatocytes of Group D were altered in much the same way after 4 weeks. ;; In biopsies taken at the end of the experiment, hepatocytes of Group A had not altered appreciably, but the opposite was true for all the other groups. Although ‘ultrastructural configuration of hepatocytes in Group B was almost normal (Fig. S), some of their mitochondria were slightly altered by stacks of parallel-arranged cristae (Figs. 9 and 10). Changes in hepatocytes of Group C did not substantially differ from those of Group B, but particularly in perilobular locations, connective tissue fibers frequently abutted and surrounded them. The most profound changes were seen in hepatocytes of Group D. Enlarged mitochondria often engulfed and enwrapped lipid droplets (Fig. 12) and others had adopted many bizarre shapes (Fig. 11). Most of the cytoplasm in the hepatocytes of this group was occupied by fine granular material which, despite its low electron density and its moderate affinity for lead, still displayed the ultrastructural configuration of glycogen. DISCUSSION The results of this experiment indicated that high-fat diets containing 25 % protein (Cal % Cal) permitted the growth and the normal maintenance of several functional and morphologic parameters in squirrel monkeys. Diets containing only 12.5 % protein (Cal% Cal) supported growth nearly as well as the high-fat diets, and differences between the two groups (A vs. B) were of questionable significance. The protein intake in the latter (Group B) was 5.04 gm/day/kg of body weight and probably represents the borderline level of protein required for the S. sciureus in captivity under our conditions. One would probably be safe in assuming that an adequate protein level would be slightly above that given animals in Group B. Although the protein requirements for the X. sciureus had not previously been established, it has been assumed that an adequate diet for these animals should provide at least 20 % protein (gm). All the commercial diets available in Canada and the USA appear to be formulated on that assumption. These protein levels, however, appear excessive when compared with our results. On the other hand, in the experiments of Wilgram et al. (1958), which used Cebus monkeys, diets containing 15 % protein (10 gm % peanut meal + 10 gm% soya protein) and supplemented with 0.3 gm% choline chloride supported reasonably good rates of growth and livers were histologically normal. The effects of 9.00% and 6.10% protein diets (Cal % Cal) were manifested by poor ponderal growth and by both biochemical and hematologic alterations from normal growth. Although many of these latter changes were not statistically different from values of the groups consuming diets A and B, the small number of monkeys employed prevented any conclusions regarding their significance either statistically or physiologically. At any rate, the consumption of these low-protein diets was associated with ultrastructural alterations of the livers which clearly resembled those previously reported by others for different speciesof monkeys and for rats fed low-protein diets (Svoboda and Higginson, 1964; Svoboda et al., 1966; Racela et al., 1966; Ordy et al., 1966).
FIG. 5. Budding of mitochondria in a monkey from the experiment. Lead stain. X 30,000. FIG. 6. Advanced budding formation in mitochondrion 8. Lead stain. X 30,000. FIG. 7. Bizarre mitochondrial shape suggesting the in a monkey from Group C at the 8th week. Lead stain. FIG. 8. Ultrastructural configuration of a hepatocyte 22. Lead stain. X 12,500. 192
Group
1) 4 weeks
of a monkey
from
aft.er Group
commencing C at week
coalescence of several mitochondria X 30,000. from group B at the end of week
FIGS. 9 and
10. Markedly elongated mitochondria inclusions in hepatocytes of a monkey from FIG. 11. Enlarged mitochondria engulfing droplets Group D at the end of week 22. Lead stain. X 22,500. FIG. 12. Bizarre-shaped mitochondria in hepatocyte end of week 22. Lead stain. X 16,000. crystaline
193
containing in their matrices paragroup B at the end of the experiment. of fat in hepatocyte of a monkey from of a monkey
from
Group
D at the
194
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
W.
S.
HARTROFT
SUMMARY Despite the increasing use of the squirrel monkey in nutritional studies, little is known of the dietary requirements of this species. Twelve young (700 gm initial wt) male squirrel monkeys were allotted to four groups of three animals each, and were offered ad libitum, during 24 weeks, isocaloric diets in which the amounts of prot.ein were 25yo, 12.5’%, 9%, or Gojo. The dietary protein employed was a purified soya protein and was supplemented with 2.1 gm y. of methionine to obtain the same content of this amino acid as present in casein. The diets contained amounts of vitamins and essential food factors in excess of the estimated normal requirements. The results indicate that the diets with 25% protein permit the growth and the normal maintenance of several functional and morphologic parameters studied. An almost similar effect was observed with 12.5% protein diets. The protein intake in monkeys fed this diet was 8 gm/day/kg of body weight, and it appears to be the minimal normal protein requirement for this species in captivity under t,he experimental conditions employed in our work. The effect of 9% and 6yo protein diets was manifested by poor ponderal growth, decrease in albumin/globulin ratio, decrease in hemoglobin concentration, decrease in BUN, and increase in serum alkaline-phosphatase activity. In addition, the livers of animals on the lowprotein diets had moderate to severe ultrastructural alterations. ACKNOWLEDGMENTS The authors express their gratitude to Mrs. C. Manning for their technical assistance.
E. E. Minaker,
Mr.
M.
B. Vanderby
and
Mr.
REFERENCES BAKER, J. R. (1945). Structure and chemical composition of the Golgi element. &art. J. Microscop. Sci. 85, 1-71. BEICHER, D. E., and FURRY, D. E. (1964). Sain%iri scuire[Ls as an experimental animal. Anat. Record 48, 615-624. COOPERMAN, J. M., WaIsuaN, H. A., MCCALL, K.B., and ELVEHJEM, C. A. (1945). Studies on the requirements of the monkey for Riboflavin and a New Factor formed in the liver. J. Nutr. 30, 45-47. DALTON, A. J. (1955). A chrome-osmium fixative for electron microscopy (abstr.). Anat. Record 121, 281-282. DE LA IGLESIA, F. A., PORTA, E. A., and HARTROFT, W. S. (1966). Histochemical urate oxidase and microbodies in non-human primate liver. J. Histochem. Cytochem. 14, 685-687. DE LB IGLESIB, F. A., and PORTA, E. A. (1967). Ciliated biliary epithelial cells in the livers on non-human primates. Ezperientiu 23, 49-51. FOLLIS, R. H. (1957). A Kwashiorkor-like syndrome observed in monkeys fed maize. Proc. Sot. Exptl. Biol. Med. 96, 523-528. GAISFORD, W. D., and ZUIDEM~, G. D. (1965). Nutritional Laennec’s cirrhosis in the lMucuca mulutta monkey. J. Swg. Res. 5, 220-235. A rapid method for microelectrophoresis and GRAHAM, J.L.,and GRUNBAUM, B.W. (1963). quantitation of hemoglobin on cellulose acetate. Am. J. Clin. Puthol. 39, 567-578. KSRNOVKSY, M. J. (1961). Simple methods for “staining with lead” at high pH in electron microscopy. J. Biophya. Biochem. Cytol. 11, 729-732. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. cytoz. 9, 409-414. MANN, G. V., WATSON, P. L., MCN.ZLLY, A., and GODDARD, J. (1952). Primate Nutrition. II. Riboflavin deficiency in the Cebus monkey and its diagnosis. J. Nutr. 4’7, 225-241. MARSH, W., FINGERHUT, B., and KIRSH, E. (1959). Adoption of an alkaline phosphatase method for automatic calorimetric analysis. Clin. Chem. 5, 119-126. MASSON, P. (1929). Some histological methods, trichrome stainings and their preliminary technique. J. Technol. Methods 12, 75-90. MCCALL, K. G., WAISMAN, H. A., ELVEHJEM, C. A., and JONES, E. S. A. (1946). Study of
DIETARY
PROTEIN
LEVELS
IN
MONKEYS
195
pyridoxine and pantothenic acid deficiency in the monkey (Macaca mulatta). J. Nutr. 31, 6885-697. ORDY, J. M., SAMORAJSKI, T., ZIMMERMAN, R. R., and RADY, P. M. (1966). Effects of postnatal protein deficiency on weight gain, serum proteins, enzymes, cholesterol, and liver ultrastructure in a subhuman primate (Macaca mulatta). Am. J. Pathol. 48, 769-791. RACELA, A. A., GRADY, H., HIQGINSON, J., and SVOBODA, D. (1966). Protein deficiency in Rhesus monkeys. Am. J. Pathol. 49, 419-443. RINEHART, J. F., and GREENBERG, L. D. (1949). Arteriosclerotic lesions in pyridoxine deficient monkeys. Am. J. Pathol. 25.481-491. RINEHBRT, J. F., AND GREENBERG, L. D. (1951). Pathogenesis of experimental arteriosclerosis in pyridoxine deficiency with notes to human arteriosclerosis. A. M. A. Arch. Pathol. RINEHART,
51,
12-18.
J. F., and GREENBERG, L. D. (1956). Vitamin B deficiency in the Rhesus monkey with particular reference to the occurrence of atherosclerosis, dental caries and hepatic cirrhosis. Amer. J. Gin. Nutr. 4, 318-328. SKEGGS, L. T. (1957). An automatic method for calorimetric analysis. Am. J. Clin. Pathol. 28, 311-322. SVOBODA, D., and HIGGINSON, J. (1964). Ultrastructural changes produced by protein and related deficiencies in the rat liver. Am. J. Pathol. 45, 353-379. SVOBODA, D., GRADY, H., and HIGGINSON, J. (1966). The effects of chronic protein deficiency in rats. II. Biochemical and ultrastructural changes. Lab. Invest. 15, 731-749. WAISMAN, H. A. (1944). Production of riboflavin deficiency in the monkey. Proc. Sot. Exptl. Biol. &fed. 55, 69-71. WILGRAM, G. F., LUCAS, C. C., and BEST, C. H. (1958). Kwashiorkor-type of fatty liver in primates. J. Exptl. Med. 108, 361-370. WILSON, W. (1950). Trichrome method for staining fat with oil red 0 in frozen sections. Bul. Int. Ass. Med. Mu;. 31.216-220.
.&ND
Effects
YOLECUL9R
of Dietary
F. A.
DE
The Research
LA
PATHOLOGY
Protein
Inslitufe
Levels E. A.
IGLESIA,~
(1967)
?,182-1%
PORTA,
of The Hospital Received
on the Saimiri
For
&fa~
AND Sick
W.
Children,
S.
Sciurews’
HARTROFT
Toronto,
Canada
18, 1967
Although the production of nut,ritional hepatic injuries in various species of monkeys has been reported (Waisman, 1944; Mann et al., 1952; Cooperman et al., 1945; McCall et al., 1946; Rinehart and Greenberg, 1949; Rinehart and Greenberg, 1951; Rinehart and Greenberg, 1956; Follis, 1957; Wilgram et al., 195s; Gaisford and Zuidema, 1965), the results obtained appear controversial. The composition and proportion of dietary ingredient’s in these studies varied so widely that more experiments would be necessary before considering whet’her the semisynthetic diets employed in previous experiments were, in fact, adequate for a given species of monkeys. Saimiri sciwea (squirrel monkeys from South America) are easy to obtain, to to handle, and do well in capt’ivit’y (Beischer and Furry, 1964). It can be anticipat#ed that X. s&urea will frequently be chosen for nutritional studies. The lack of more specific information on t’he diet’ary requirements for members of this species prompted us to determine the minimal protein requirements of these monkeys which would permit adequate growth as well as normal maintenance of several functional and morphologic hepatic parameters. We have now explored the adequacy of a semi-synt’hetic diet at four different protein levels and the result’s are reported here. MATERIAL
APU’D METHODS a4NIMALS
During an acclimatizat,ion period of one month, 12 young male squirrel monkeys (S. sciureus) were maintained on a stock-pellet diet3 and water ad libitum. Tuberculin tests and parasitic-fecal examinations gave negative results for all animals used in this st#udy. At the end of this initial period when animals’ weights ranged from 514 to 876 gm, they were allot8ed to four groups (A, B, C, and D) which contained three members each. All monkeys were housed in individual cages in airconditioned, humidified (60%) rooms that were supplied during the day with continuous soft music. The animals were weighed twice weekly. 1 This study was supported by the Medical Research Council of Canada (Block grant Ma-1904). 2 Research Fellow of the Medical Research Council of Canada. Present address: WarnerLambert Research Institute of Canada, Sheridan Park, Ontario, Canada. 3 Purina Monkey Chow, Ralston Purina Company, Woodstock, Ontario. 182
DIETARY
PROTEIN
LEVELS
TABLE COMPOSITION
IN
183
MONKEYS
I OF DIETS
A
B
D
C
Ingredients cal o/u cal Soya
proteina
25.00
Corn Beef
oil fat
49.00
gm%
cal 70 cal
32.57
12.50
9.45
gmyO
cal y0 cal
pm%
16.28
9.00
11.73
6.10
7.92
9.45
9.45
9.45
49.00
49.00
18.90
18.90
27.27
16.07 26.00
Vitaminsb Saltsc
cal y0 cal
49.00 18.90
Sucrose Corn starch
grn%
38.50
18.90
29.95
32.55
42.00
44.90
11.29
16.38
18.24
19.49
6.51 5.21
6.51 5.21
6.51 5.21
6.51 5.21
~1 Supplemented with 2.1 9% of methionine. b Complete vitamin diet fortification mixture. Ohio. c Hubbell, Mendel and Wakeman. Nutritional
Nutritional Biochemical
Biochemical Corp.
Corp.
Cleveland,
Cleveland, Ohio.
DIETS
The composition of the semisynthetic diets offered to the different groups is indicated in Table I. Although the amounts of protein in these diets varied, all were isocaloric and supplied 5.2 cal./gm. A purified soya extract4 was employed as the source of protein and was supplemented with 2.1 gm % of methionine to compensate for the diets’ relatively low content of this amino acid. The choline content of this soya protein is 2.09 mg%. The lipotropic value of each diet was different, but all were adequate and provided the same amounts of salts, vitamins, and essential fatty acids. The content of the chosen fat was high in order to improve palatability, to resemble diets widely used in nutritional studies in rats, and to approach the fat content of diets consumed by man in most western cultures. The amounts of vitamin and salt mixtures arbitrarily chosen were 1.20 gm and 1 gm/lOO cal. of the diet. The diets were prepared weekly, stored at 4”C, and were offered daily in known amounts to the animals in the form of small balls of about 15 gm each. These diets plus water were offered ad libitum. The total food intake from which the caloric and protein intakes were calculated was recorded daily. BIOCHEMICAL
DETERMINATIONS
Blood was drawn from the femoral vein of each monkey at the beginning experiment and at the end of weeks 4,8, 12, and 22 for biochemical analyses. protein levels were determined by quantitative electrophoresis (Graham and baum, 1963). Hematograms including hemoglobin concentrations, red and 4 Purified
soya
protein
(Assay
C-l
protein),
Skidmore
Enterprises,
Cincinnati,
of the Serum Grunwhite
Ohio.
IS4
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
TABLE AVER.IGE
Foot,
DIILY
DIFFERENT
PROTEIN, I)IETS
A B c I)
34.59 34.01 31.34 43.9G
* Mean
f
+ + + z!z
S.
HARTROFT
INT.IICES IN FOR 22 WEEKS
1t.k~~
39.41 49.37 47.14 58.71
body
wvt
gm
* 7.63 * G.97 zt 10.42 + 12.71
11.26 5.54 3.68 3.48
FED
THE
Calories
Protein
gm/kg 4.74" 4.22 4.34 9.63
W.
II
.wu CALORIC m LIHITUM
I’ood
&!m
AND
gm/kg
body wt.
12.83 8.04 5.53 4.65
cal 179.87 176.85 162.97 528.59
Cal/kg hod) wt 204.93 256.72 245.13 305.29
SD
blood cell counts and hematocrits were performed. Levels of blood urea nitrogen (&eggs, 1957) and serum alkaline-phosphat’ase activities (Marsh et al., 1959) were also determined. All data were stat’istically analyzed. HISTOLOGIC
STUDIES
Open hepatic biopsies were obtained at the beginning of the experiment and at 4, S, and 22 weeks from all the animals. The monkeys were sedated with acepromazine maleate5, 0.1 ml i. m., and after local anest’hesia wit,h xylocnine, the liver was visualized through a small incision below the right costal margin and specimens were obtained with t#he Menghini needle. Portions of t#hese specimens were fixed in Baker’s solut,ion (Baker, 1945) for light-microscopy studies. Paraffin sections bvere st,ained wit,h hematoxylin-eosin, Oil red 0 (ORO) for detection of ceroid (Wilson, 1950), PAS stain, or with l\Iasson’s trichrome stain (Masson, 1929) to aid in visualizing the connective tissue. Fat in frozen sections was stained wit,h Oil red 0. For electron microscopy, blocks taken from other port,ions of the specimens were fixed by immersion in Dalton’s soh&on (Dalton, 1955) and embedded in Epon 812 (Luft, 1961). Ultrathin sections were stained with lead using Iiarnovsky’s met,hods (Karnovsky, 1961) and photographed in a Philips 200 electron microscope at established initial magnifications. RESULTS MORTALITY
Two monkeys of Group D died of inanit’ion during the experiment; one at week S and t,he other at week 20. Two monkeys of Group B died; one at week 16 (cause: inanition) and t,he ot)her at week 19 (cause: undetermined). One monkey of Group C died at week 19 (also of undetermined cause). None of t’he members of G~OLI~ A died during the course of the experiment. FOOD
INTAKE
The average values of daily food and protein and caloric intakes in the four groups during t#he whole experiment are shown in Table II. The means of absolute values 5 Atravet,
Ayerst,
McKenna
& Harrison
Ltd.,
Montreal,
Quebec.
DIETARY
PROTEIN
LEVELS
IN
155
MONKEYS
between Groups A, B, and C did not differ significantly; however, that of Group D was significant’ly higher than those of groups A (p <0.05), B (p
WEIGHTS
Changes in body weights were expressed in the form of growth curves as percentages of the init’ial values at the beginning of the experiment (Fig. 1). Due to the to the death of animals in Groups B, C, and D, for statistical purposes the only changes considered were those occurring during the first IS weeks of the experiment. Changes in body weight at weeks 3, 6, 9, 12, 15, and 18 are also shown in Table III. ISO-
140-
80 FIG.
weeks
1.
8’
4’
Growth curves
for each
12’
TABLE CHANGES Weeks 3 6 9 12 15 18
16’
20’
24’
group expressed as percent increase in initial body weight. III
IN BODY
WEIGHTS*
A
B
C
27.93 25.59 27.26 19.40 35.55 41.05
15.67 13.66 16.06 11.31 15.04 27.17
8.41 2.93 7.75 8.77 9.17 12.12
D -11.18 -15.49 -7.42 -5.17 -4.49 -5.56
a Average values expressed as percentages of the initial weight.
lS6
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
W.
S.
HARTROFT
While significant increases were observed in Groups A (p
PROTEINS
Levels of total serum proteins of all groups at different periods are included in Table IV. The values between the various groups did not differ except at week 4, when those of Group D were significantly lower Ohan those of Groups A, B, and C (p
ANALYSIS
Mean values and their ranges are shown in Table V. Values for hemoglobin, white blood cells, and hematocrit (in the various groups at different periods) fell within the normal range for t’his species as reported by others (Beischer and Furry, 1964). However, mean values for erythrocytes and their ranges were generally lower than TABLE SERUM Weeks 4
Group
A
Total
proteins
IV
PROTEINS
Albumin
a1
Y
B C n
8.90 9.00 8.97 7.03
* * i *
0.W 0.65 0.57 0.29
4.32 zt 0.25 4.19 f 0.36 4.43 z!z 0.34 2.61 f 0.26
0.29 0.35 0.26 0.26
0.07 0.11 0.04 0.10
0.64 0.65 0.77 0.68
zt f zk f.
0.03 0.12 0.06 0.11
0.56 0.71 0.71 0.52
f * z!z f.
0.06 0.05 0.04 0.07
1.90 2.05 1.47 1.84
f f f f
0.80 0.36 0.22 0.59
8
A B c D
9.00 * 8.80 * 8.63 * 8.30 f
1.51 0.92 0.64 0.71
4.70 z!z 4.02 zt 4.22 i 3.58 *
0.29 + 0.01 0.28 z!c 0.03 0.40 zk 0.03 0.40 * 0.14
0.56 0.63 0.70 0.70
zt zt zk i
0.07 0.10 0.07 0.21
0.86 0.74 0.81 0.62
f f rt z!z
0.26 0.08 0.04 0.04
1.29 1.94 1.21 1.78
+ * f ;t
0.23 0.37 0.23 0.17
12
A B C D
8.40 9.03 7.73 8.80
f. f & f
1.11 0.58 0.22 1.70
4.33 & 0.07 4.34 f 0.25 4.04 * 0.19 3.87 * 0.59
0.23 0.25 0.25 0.25
f f z!c zt
0.02 0.08 0.09 0.01
0.47 0.57 0.64 0.67
zt f zt f
0.03 0.04 0.07 0.03
0.60 0.65 0.76 0.67
f f f zt
0.06 0.03 0.09 0.15
1.78 * 1.10 2.30 + 0.41 1.21 f 0.05 2.47 f 0.74
22
A B C D
7.10 7.90 7.20 7.40
zk 0.42
3.28 f 3.77 3.34 f 2.73
0.26 0.28 0.31 0.35
f
0.05
f
0.21
0.10
0.09
*
0.07
0.46 0.61 0.73 0.79
f
f
0.38 0.46 0.50 0.65
f
0.09
1.96 f 2.11 1.31 * 1.99
a Mean
zk SE.
f
0.50
0.56 0.57 0.27 0.05
1.05 0.09
rt f zk &
B
9
1.15 0.23
DIETARY
PROTEIN
LEVELS
TABLE
Hemoglobin
ANALYSES
Erythrocytes
Leukocytes
Group gm/lOO
ml blood
187
MONKEYS
V
HEMATOLOGICAL Week
IN
l@/cu
mm blood
103/cu
mm blood
Hematocrit ml/100
ml blood
0
A B C D
13.20 12.37 12.77 14.57
(13.1-13.3) (9.7-14.2) (11.7-13.4) (13.6-15.5)
5.97 6.23 5.90 6.20
(5.7-6.3) (5.9-6.8) (5.64.3) (5.8-6.0)
8.63 11.03 9.00 13.70
(5.2-14.0) (8.3-16.3) (7.8-11.3) (11.2-17.6)
4
A B C D
13.77 12.67 10.60 10.83
(12.9-14.5) (11.7-14.3) (9.6-12.3) (10.1-11.7)
7.07 6.33 5.80 5.40
(6.9-7.4) (5.3-7.3) (4.9-6.3) (5.2-5.8)
13.17 15.90 11.27 13.10
(10.7-14.8) (7.6-23.6) (9.1-14.0) (12.1-14.7)
45.0 40.6 38.0 36.6
(4246) (3444) (30-45) (35-39)
8
A B c D
12.17 12.43 11.70 10.85
(10.5-13.9) (10.8-15.0) (10.9-12.4) (9.5-12.2)
4.73 5.77 4.97 4.40
(3.9-5.3) (4.8-6.5) (4.5-5.3) (4.24.6)
10.57 12.50 11.63 10.85
(6.6-13.9) (9.8-14.0) (7.2-16.7) (10.5-11.2)
42.0 44.0 40.6 38.0
(37-46) (41-49) (38-43) (34-42)
12
A B C D
13.77 11.83 12.40 12.40
(13.6-14.0) (10.9-12.8) (8.7-14.7) (11.7-13.1)
5.60 4.83 4.93 4.85
(5.3-5.8) (4.1-5.3) (3.5-5.8) (4.84.9)
9.77 10.63 10.33 10.30
(6.3-10.9) (9.4-13.3) (8.0-14.0) (8.6-12.0)
46.6 37.6 40.6 42.0
(4647) (32-44) (29-48) (4044)
22
A B C D
13.10 12.40 12.05 11.70
(12.6-13.6)
5.45 5.80 5.10 5.40
(4.7-6.2)
11.50 6.10 10.55 9.6
(6.2-06.8) (6.1-11.6) (9.5-11.6)
44.0 40.0 40.0 39.0
(43-45)
(12.0-12.1)
(4.6-5.6)
those obtained in the above cited report; 12. SERUM
ALKALINE
particularly
in Groups
(38-42)
C and D at week
PHOSPHATASE
Values at the beginning of the experiment were not significantly different between groups. Although the values of Groups C and D were generally higher than those of Groups A and B during the other periods of the experiment, the differences were not statistically significant, except bet,ween Groups B and C (p
Levels of BUN in each group varied with time, but no significant differences were found between groups at the different periods studied (Table VII). HISTOLOGIC
FINDINGS
Light-microscopic examinations, before the institution of the different dietary regimens, showed that nine out of twelve monkeys had hepatic fatty changes of various degrees. The predominant lobular location of visible fat in hepatocybes was
1%
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
TABLE VI SERUM ALKALINE-PH~SPH~T.ISE
B
C D
S.
HAIZTIlOFT
ACTIVITY
LVeek
Group A
IV.
0 23.83 27.50 25.67 24.70
+ 8.19” + f f
8
4 29.83 33.50 G-I.25 44.25
0.70 5.43 9.5-l
* Mean h SE; King-Armstrong
f z!z z!z f-
11.30 4.78 8.13 15.20
35.70 16.40 48.60 47.10
z!x f * zt
12 14.78 18.05 10.98 32.28
42.13 51.30 55.07 70.00
It
15.11
z!z 11.71 &
6.71
uuits. TABLE 1’11 BLOW UREI NITROGES
Group ~ A B c D
-0 25.67 16.83 23.07 22.33
f + f f
8
4
8.89” 3.58 6.09 (i.94
21.33 16.67 14.50 16.33
f f I!z +
2.27 1.47 4.95 2.27
25.00 11.33 17.33 23.50
+ 1.41 & 5.21 * 4.71 + G.36
12 37.G7 35.68 40.33 20.00
f
24
1.08
z!z 1.71 zk 8.84 + 12.72
23.00 23.00 19.00 16.00
f
2.83
+
2.83
a Mean + SE.
mainly periportnl. Kupffer cells also had accumukked small droplets of cytoplasmic fat of diffuse lobular distribut’ion. These fatty changes regressedat’ lnt’er stages of the study, particularly in livers of animals from Groups A and B. The hepatic archit’ecture was well preserved in all groups throughout Dhe entire experiment. By eleckon microscopy, the ultrastructural configuration of hcpat,ocytes in the different groups before dietary treatment was characterized by abundant cyt,oplasmic content of glycogen granules which stained deeply with lead (Fig. 2). These granules were gr,ouped to form patchy cytoplasmic areas of erratic location, similar to t’hosefound in normal murine and human hepatocytes. Mitochondria were normal; both rough and smooth endoplasmic reticulum were well developed and normal. Numerous microbodies cont’aining characteristic crystalline cores were found in hepatocyt’es. The ultrastructural appearance of these microbodies as well as their enzymatic (urate oxidase) activity has been reported in det,ail elsewhere (de la Iglesia et al., 1966). Multivesicular bodies and autophagic vacuoles were also present and numerous medium-sized droplets of fat of moderate density were seenthroughout the cytoplasm of hepatocytes in all the animals. Granules of ceroid pigment were occasionally found in Kupffer cells. One interesting finding was the presence of cilia in the epithelial cells of cholangioles. This observation has also been report,ed elsewhere (de la Iglesia and Porta, 1967). In t,he biopsy specimenstaken 4 weeks after the institution of dietary regimens appreciable ultrastructural changes were absent in Groups A (Fig. 3), B, and C But in hepatocytes of Group D, mitochondria mere altered by budding, focal matrical rarefaction, and attenuation of their membranes; in some,the numbers of cristae
DIETARY
FIG. 2. Typical ne acclimatization
ultrastructural period during
PROTEIN
LEVELS
IN
189
MONKEYS
configuration of a hepatocyte which it was fed a commercial
of a squirrel rations. Lead
monkey after stain. X 9,000.
had decreased. In other instances mitochondria had coalesced and their total number appeared generally decreased. Profiles of endoplasmic reticulum were diminished and a few moderately dilated cisternae were seen at cellular peripheries (Figs. 4 and 5). After 8 weeks, although the configuration of hepatocytes in Groups A and B had rkmained unchanged, mitochondri’al alterations did appear in Group C. In general, they were enlarged and the number of cristae had increased. Budding and coalescence of mitochondria, as seen in Group D at 4 weeks, was also now observed at
190
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
W.
S.
HARTROFT
FIG. 3. Ultrastructural configuration of a hepatocyte from Group A (25y0 protein), 4 weeks after commencing the experiment,. Lead stain. X 10,000. FIG. 4. Electron microscopic aspect of a hepatocyte from Group D (6.1yCi; protein) 4 weeks after commencing t,he experiment. Lead stain. X 10,000.
DIETARY
PROTEIN
LEVELS
IN
MONKEYS
191
his time in Group C (Figs. 6 and 7). In addition, shapes of many mitochondria were bizarre, particularly those which were intimately in contact with droplets of fat: Hepatocytes of Group D were altered in much the same way after 4 weeks. ;; In biopsies taken at the end of the experiment, hepatocytes of Group A had not altered appreciably, but the opposite was true for all the other groups. Although ‘ultrastructural configuration of hepatocytes in Group B was almost normal (Fig. S), some of their mitochondria were slightly altered by stacks of parallel-arranged cristae (Figs. 9 and 10). Changes in hepatocytes of Group C did not substantially differ from those of Group B, but particularly in perilobular locations, connective tissue fibers frequently abutted and surrounded them. The most profound changes were seen in hepatocytes of Group D. Enlarged mitochondria often engulfed and enwrapped lipid droplets (Fig. 12) and others had adopted many bizarre shapes (Fig. 11). Most of the cytoplasm in the hepatocytes of this group was occupied by fine granular material which, despite its low electron density and its moderate affinity for lead, still displayed the ultrastructural configuration of glycogen. DISCUSSION The results of this experiment indicated that high-fat diets containing 25 % protein (Cal % Cal) permitted the growth and the normal maintenance of several functional and morphologic parameters in squirrel monkeys. Diets containing only 12.5 % protein (Cal% Cal) supported growth nearly as well as the high-fat diets, and differences between the two groups (A vs. B) were of questionable significance. The protein intake in the latter (Group B) was 5.04 gm/day/kg of body weight and probably represents the borderline level of protein required for the S. sciureus in captivity under our conditions. One would probably be safe in assuming that an adequate protein level would be slightly above that given animals in Group B. Although the protein requirements for the X. sciureus had not previously been established, it has been assumed that an adequate diet for these animals should provide at least 20 % protein (gm). All the commercial diets available in Canada and the USA appear to be formulated on that assumption. These protein levels, however, appear excessive when compared with our results. On the other hand, in the experiments of Wilgram et al. (1958), which used Cebus monkeys, diets containing 15 % protein (10 gm % peanut meal + 10 gm% soya protein) and supplemented with 0.3 gm% choline chloride supported reasonably good rates of growth and livers were histologically normal. The effects of 9.00% and 6.10% protein diets (Cal % Cal) were manifested by poor ponderal growth and by both biochemical and hematologic alterations from normal growth. Although many of these latter changes were not statistically different from values of the groups consuming diets A and B, the small number of monkeys employed prevented any conclusions regarding their significance either statistically or physiologically. At any rate, the consumption of these low-protein diets was associated with ultrastructural alterations of the livers which clearly resembled those previously reported by others for different speciesof monkeys and for rats fed low-protein diets (Svoboda and Higginson, 1964; Svoboda et al., 1966; Racela et al., 1966; Ordy et al., 1966).
FIG. 5. Budding of mitochondria in a monkey from the experiment. Lead stain. X 30,000. FIG. 6. Advanced budding formation in mitochondrion 8. Lead stain. X 30,000. FIG. 7. Bizarre mitochondrial shape suggesting the in a monkey from Group C at the 8th week. Lead stain. FIG. 8. Ultrastructural configuration of a hepatocyte 22. Lead stain. X 12,500. 192
Group
1) 4 weeks
of a monkey
from
aft.er Group
commencing C at week
coalescence of several mitochondria X 30,000. from group B at the end of week
FIGS. 9 and
10. Markedly elongated mitochondria inclusions in hepatocytes of a monkey from FIG. 11. Enlarged mitochondria engulfing droplets Group D at the end of week 22. Lead stain. X 22,500. FIG. 12. Bizarre-shaped mitochondria in hepatocyte end of week 22. Lead stain. X 16,000. crystaline
193
containing in their matrices paragroup B at the end of the experiment. of fat in hepatocyte of a monkey from of a monkey
from
Group
D at the
194
F.
A.
DE
LA
IGLESIA,
E.
A.
PORTA,
AND
W.
S.
HARTROFT
SUMMARY Despite the increasing use of the squirrel monkey in nutritional studies, little is known of the dietary requirements of this species. Twelve young (700 gm initial wt) male squirrel monkeys were allotted to four groups of three animals each, and were offered ad libitum, during 24 weeks, isocaloric diets in which the amounts of prot.ein were 25yo, 12.5’%, 9%, or Gojo. The dietary protein employed was a purified soya protein and was supplemented with 2.1 gm y. of methionine to obtain the same content of this amino acid as present in casein. The diets contained amounts of vitamins and essential food factors in excess of the estimated normal requirements. The results indicate that the diets with 25% protein permit the growth and the normal maintenance of several functional and morphologic parameters studied. An almost similar effect was observed with 12.5% protein diets. The protein intake in monkeys fed this diet was 8 gm/day/kg of body weight, and it appears to be the minimal normal protein requirement for this species in captivity under t,he experimental conditions employed in our work. The effect of 9% and 6yo protein diets was manifested by poor ponderal growth, decrease in albumin/globulin ratio, decrease in hemoglobin concentration, decrease in BUN, and increase in serum alkaline-phosphatase activity. In addition, the livers of animals on the lowprotein diets had moderate to severe ultrastructural alterations. ACKNOWLEDGMENTS The authors express their gratitude to Mrs. C. Manning for their technical assistance.
E. E. Minaker,
Mr.
M.
B. Vanderby
and
Mr.
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