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Serum Cholesterol Levels in Rats Fed Thirteen Trace Elements1


									Serum Cholesterol Levels in Rats Fed
Thirteen Trace Elements1
                            HENRY A. SCHROEDER*
                            Department of Physiology, Dartmouth Medical School, Hanover,
                            New Hampshire, and Brattleboro Memorial Hospital,
                            Brattleboro, Vermont

       ABSTRACT          To ascertain differences in serum cholesterol levels as possibly affected
       by trace elements in the drinking water, rats were given soluble salts of zirconium,
       vanadium, niobium, chromium, nickel, cadmium, germanium, tin, lead, arsenic, and
       antimony at 5 ppm and selenium and tellurium at 2 ppm from the time of weaning
       until 11 to 30 months of age. The feeding of chromium at 1 ppm was associated with
       suppressed levels in males but not in females; 5 ppm appeared to be required for this
       effect in females. The lowest serum cholesterol levels were observed in groups given
       niobium, chromium and nickel, the highest in groups fed tellurium, with all animals
       receiving 1 ppm chromium. Significant differences in values of the 2 sexes appeared
       for 7 elements. It is possible that chromium, nickel and niobium exert anti-cholestero-

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       genic properties, whereas tellurium may be cholesterogenic.

    Mean circulating cholesterol levels in           tunity to measure serum cholesterol and
human beings tend to rise with age. This             glucose levels. The present report con
 tendency is exaggerated in persons living in        cerns 13 trace elements fed to rats from
industrialized countries and is less evi             the time of weaning. Differences in serum
 dent in more primitive societies. The pos           cholesterol associated with element and
 sibility arose that some abnormal trace             age are shown.
element to which civilized man is exposed
 and which accumulates in his tissues with                             METHODS
age might influence the synthesis of cho                 The environmental conditions in the lab
lesterol or interfere with its catabolism.           oratory, the precautions taken to avoid
Conversely, some modern nutritional prac             metallic contamination, the low metal diet
tices might promote marginal deficiency of           and the double-deionized drinking water
 an essential trace element on which ho-             used have been reported in detail (6, 7).
meostasis of cholesterol depends.                    The trace element content of the diet,
    Curran ( 1) showed that rat liver, in vi         which was composed of seed rye, powdered
tro and in vivo, synthesized cholesterol             skim milk and corn oil, and the estimated
less well in the presence of vanadyl ions            total intakes, are shown in table 1. To the
 and better in the presence of trivalent chro        basic drinking water, which contained zinc
mium and manganous ions than liver un-                (50 ppm), manganese (10 ppm), copper
exposed to trace elements. Other trace                (5 ppm), cobalt (1 ppm), molybdenum
metals in the first transitional series were          (1 ppm) and chromium (1 ppm), was
relatively inert. Curran's rats were fed a           added one of the following at 5 ppm ele
commercial ration, which at that time was            ment; zirconium sulfate, sodium niobate,
marginally deficient in chromium (2). The            antimony potassium tartrate, sodium ger-
action of chromium feeding in stabilizing            manate, stannous chloride, sodium arsen-
serum cholesterol at a low level in male             ite, vanadyl sulfate, nickelous acetate,
rats has been demonstrated (3, 4); asso              cadmium acetate, lead acetate. Sodium
ciated with this effect was virtual abolition
of spontaneous aortic plaques and accumu                Received for publication November 1, 1967.
                                                        1Supported by Public Health Service Research Giant
lation of aortic lipids (4).                         no. HE-05076 from the National Heart Institute; Con
    Studies on the Ufe-term effects of a num         tract DA 2595 from the U.S. Army; CIBA Pharma
                                                     ceutical Products, Inc.; and the Selenium-Tellurium
ber of trace elements given in low doses to          Development Association.
                                                        2Present address : 9 Beunont Avenue, Brattleboro,
rats and mice (5) have provided an oppor             Vermont 05301.
476                                     HENRY    A.    SCHROEDER

                     TABLE 1                       Berkeley Medical Instrument spectropho-
Trace element content of diet and drinking water   tometer;5 a few were measured by the
          and estimated daily intakes 1J!
                                                   method of AbeU et al. (11). Duplicate and
                                                   replicate analyses agreed within 3%.
                                                      Analyses of the diet for trace elements
                                    body           were made by a number of standard meth
                                                   ods, colorimetrie for zirconium, vanadium,
                                                   niobium, chromium, nickel, germanium,
                                                   arsenic, tin, lead and selenium, and atomic
                                                   absorption spectrophotometric            for chro
                                                  mium, cadmium, antimony and tellurium.
                                                          Serum cholesterol levels in rats given
                                                       various amounts of chromium are shown
                                                       in table 2. Sex differences appeared. Males
                                                       fed 1.0 to 5.0 ppm in drinking water had
   'Based on 6 g food and 7 g water Ingested/100 g     lower mean values than those given none

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body weight/day.                                       in water. Females given 5.0 ppm in water
   2These intakes are approximate but are comparable
Gams in weight of young rats were similar, weights     or about 2 ppm in food and water had
of adult animals remained fairly constant, and varia   lower values than those given 1.0 ppm or
tions among groups were not observed. The intake of
water was measured on control rats for one year-       less. The difference in male and female
marked variations in other groups were not evident
   3Not detected.                                      values at 1.0 ppm was significant (P <
                                                       0.001). According to these data, female
selenate and sodium tellurite were added               rats apparently require more chromium
at 2 ppm element; sodium selenite at                   than males in order that cholesterol in
3 ppm.3 In addition, the basic water was
                                                       serum be suppressed. Fourth generation
given without chromium or with 5 ppm as                chromium-deficient      young rats of both
the acetate.                                           sexes had higher values than second gen
   Random-bred pregnant female rats were               eration young rats which were assumed to
purchased from a supplier4 and their off
                                                       be less deficient.
spring weaned at 21 to 23 days. Groups                    In table 3 are the mean serum choles
of 52 or more of each sex, four to a cage,             terol levels of rats fed 13 trace elements in
were given one of the trace elements until             drinking water, arranged according to the
natural death occurred. Each group was                 values of males. Again sex differences ap
placed in separate wooden racks, and cages             peared in 7 cases. Male values were higher
were not interchanged between groups.                  in the groups given vanadium, chromium,
   In addition, 4 generations of rats were             selenium (P < 0.025), and in the controls
bred without exposure to chromium other                without chromium (P < 0.05). Female
than the small amount in the diet, as re               values were higher in those given cad
ported (8). The second and fourth genera               mium (P < 0.05), germanium and tin (P
tions were used in these experiments.                  < 0.001). More or less consistently low
Thirty brood females obtained from the                 values were noted in the groups of both
supplier, which had been fed a diet con
taining adequate chromium, were also                      ' In a previous publication on the effects of selenite
studied. Their offspring were not believed             and selenate on mice and rats (14), the concentration
                                                       of selenium in the selenite given in water was errone
to be deficient in chromium according to               ously reported as 2 ppm. The supplier of sodium
                                                       selenite (Nutritional Biochemicals Corporation, Cleve
analyses of tissues of similarly bred ani              land) stated in a letter that this product was
mals (9).                                              Na2SeO3'5H2O. Nearly 2 years later the compound
                                                       was found by D. V. Frost, in our laboratory, to be
   Blood was obtained from the tail of the             anhydrous, and was then so admitted to be by the
warmed rat by cutting with a razor blade,              supplier. As the hydrated form contains 45.7%
                                                       selenium and the hydrated form 30%, the rats and
and centrifuged.   Serum cholesterol was               mice given selenite actually received 3 ppm selenium.
                                                         4 Blue Spruce Farms, Altamont, New York.
measured by the method of Huang et al.                   5Berkeley Medical Instruments. New England X-Ray
(10), using a premixed reagent and a                   and Electronic Equipment, Brookline, Massachusetts
                           CHOLESTEROL         IN   RATS    FED   TRACE   ELEMENTS                          477

                                                  TABLE 2
                              Serum cholesterol levels in rats given chromium

                                           ±3.9»114.0                               ±
                                           ±5.0«122.9                         494.5 ± 2.7

                                                         ±8.277.5                          ±11.2116.0±
                                                         2.1s86.2                           6.0108.6
                                                                                             772.1 ± 1.7
                                                         ±3.2»77.0                               ±
                                                             ±6.6sNo.12101219730128Females        ± 4.2«
        l Male and female values differ significantly:   2.0 ppm group, P < 0.025; 1.0 ppm group, P < 0.001;
     5.0 ppm groups, P < 0.025.
       * Fa and Fi are rats bred from increasingly deficient mothers.
       3 SE.
       «Differs from Fs group, P < 0.001.
       5Differs from mature group, no chromium, P < 0.001.

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       *Breeders purchased while pregnant, food containing chromium.
       * Differs from mature groups, 1.0 ppm 660 and 689 days old, P < 0.001.
       8 Differs from 2.0 ppm group, P < 0.01.
       » iffers from 2.0 ppm group, P < 0.001.

                                                    TABLE 3
                 Serum cholesterol      levels in mature rats given various trace elements
                        value                            'mg/100
                                ml63.4                                             ml107.6±75.3
                                ±75.3           0.001nsus—ns<
                                ±75.7                                              ±78.6
                                ±77.5                                             ±116.0±105.3
         (Cr                    ±82.5
                                ±86.2                                              ±72.
                                ±86.6           0.02ns<                           1±103.7±113.0±100.7
                                ±89.7           0.05<0.01<
                                ±91.0±91.6±97.6                                 ±109.5
                                                           0.05<                    ±67.9
                                                           0.005<                   ±97.0
                                      ±109.6±1           0.001<                   ±90.4
(VI)Selenium                                               0.001<                   ±90.2
(IV)TelluriumControls                                                              ±110.4±94.5
                                     11.4                0.001<
          (no Cr)No.121212231212141012121110121212Agedays557342849662520405695750893804761790445405761P
  1Significance of differences from controls.
  *Not significant.
  «Methodof Abell et al. (11), previously reported (4),for comparison.

sexes given niobium, chromium,          and                     Female control levels were the highest
nickel, and high values in those fed tel                     of any group. Other elevated values (>
lurium.                                                      100 mg/100 ml) occurred in the groups
   Compared with the controls given 1.0                      given zirconium, cadmium, germanium,
ppm chromium, values for males were es                       tin, lead, arsenic and tellurium. Low val
pecially elevated in groups fed tellurium,                   ues (< 80 mg/100 ml) were found in the
selenium, antimony and no chromium and                       groups given vanadium, niobium, chro
slightly elevated in the groups fed zirco                    mium and nickel.
nium and vanadium. Cholesterol levels in                        Because two of these elements, chro
males given germanium were suppressed.                       mium and vanadium, have been shown to
478                                       HENRY       A.   SCHROEDER

affect cholesterol metabolism in vitro and                    values were higher in rats given all of the
in vivo ( 1, 4 ), comparisons were made as                    elements except niobium and nickel.
to significant differences in circulating cho                    Comparisons of levels of male rats fed
lesterol between rats fed these metals and                    vanadium with those given other elements
other elements (table 4). In relation to the                  showed significantly elevated values only
chromium-fed group, levels were higher in                     in rats fed selenium and tellurium. Signifi
male rats given antimony, selenium and                        cantly depressed values were observed in
tellurium and lower in those fed niobium,                     those given niobium, nickel and germa
nickel and germanium. In females, mean                        nium. In females, all values, except those
                                                    TABLE 4
Significance of differences     in serum cholesterol levels of rats given various trace elements compared
                                  with those given chromium and vanadium
                                           Males                                            Females

                                    P          Penee                                  P                         P
                          enee ¡ value          2+ i         value        enee 1
                                                                             *+     value         enee '
                                                                                                    2+        value
Controls, no             0.001- <          0.001- <               0.05+ <             0.05+ <

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          1 ppm                 <
                         0.02ns»ns-              <
                                           0.005ns-                                   0.001+<
                                                                        <                   <
                            0.01- <                0.005- <
                            0.01ns-<                      <
                                                                       0.001+ <             0.001+ <
                                    <                    <
                                                   0.001nsnsnsns+      0.001+<              0.001+<
                            nsnsns+                                    0.001+<              0.001+ <
                                                                       0.001+ <             0.005+ <
                                                                       0.001+<              0.001+<
                            0.02+ <                                    0.01+ <              0.02+ <
                            0.001+ <            0.001+ <               0.01+ <              0.02+ <
                                   < 0.005VanadiumDiffer- 0.005ChromiumDiffer- < 0.001VanadiumDiffer- 0.005
  1 Plus sign indicates that the value is larger; minus sign, smaller    than that for chromium or vanadium.
  2 Significance of difference in the value from that of chromium        or vanadium (see table 2).
  3 Not significant.

                                              TABLE 5
Changes with age in serum cholesterol in rats fed zirconium,            niobium, antimony,      lead and cadmium

                                              '< value                                                      value

                                  ± 3.8
                            »89.7±                                       ±1.8100.7±9.081.1±3.778.6
                             5.969.3            0.005ns'ns<                                    0.01nsnsnsnsns
                             2.776.7                                        ±4.886.3
                             2.497.6                                        ±4.492.6
                                  ±            0.001nsnsns<               ±5.6103.4
                             6.4886.6                                      ±7.12103.7
                             ±10.0376.1                                    ±8.5397.0
                             2.367.6                                        ±8.986.5
                             2.8188.6                                      ±9.80113.0
                             8.7091.6±10.591.0±10.1P                          ±8.98P

  i Significance of difference from first value in each group.
  * SE.
  » ot significant.
  * For comparison of repetitive analyses on different rats of same group.
                     CHOLESTEROL    IN RATS     FED   TRACE   ELEMENTS                  479

of animals given niobium and nickel, were        those on the right of the Table were more
significantly elevated.                         likely to be associated with high serum
   Changes in circulating cholesterol levels     cholesterol levels than were the transi
with age are shown in table 5. No increase       tional metals on the left. There was no
was found in animals fed niobium, lead or        significant correlation with atomic num
cadmium, nor in females given antimony.         ber. In the experience of this laboratory,
Increases occurred in rats of both sexes        which will be reported, the elements on
fed zirconium and in males fed antimony.         the right of the Table were more likely
Data on chromium (table 1) show some             to exert innate or overt toxicity in mice
decrease with age, especially in females.        and rats than were the transitional metals.
                                                    The data on changes in serum choles
                DISCUSSION                       terol with age are incomplete. They sug
   The rat is a poor animal for the study        gest, however, that age-linked increases
of experimental     hypercholesteremia,    re   can occur in marginal chromium defi
quiring special diets and the feeding of        ciency, in rats of both sexes fed zirconium
cholesterol and saturated fats to develop        and in males fed antimony. They suggest
high serum levels. The present study con         that such increases may not occur in rats
cerns adequate diets and unsaturated fats        given niobium, lead and cadmium.
 (corn oil), and high serum levels were             When the groups fed tellurium were

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only noted occasionally.        Nevertheless,   compared with the others, as in table 3,
trends and tendencies appeared which            significant differences appeared largely in
might turn out to be significant in more        males. Probabilities (P) of the differences
susceptible mammals.                            being due to chance of the order of <0.005
   On this basis, these data indicate that      occurred in the zirconium, niobium, chro
trivalent chromium given to rats in drink       mium, nickel, germanium, tin, lead and
ing water was associated with lowered           control groups, of < 0.01 in the cadmium
serum cholesterol levels, as shown previ        group, of < 0.025 in the antimony group
ously (4), that chromium deficiency in          and < 0.05 in the arsenic group, all values
young rats was associated with elevated         being smaller. Only the selenium group
levels, and that female rats appeared to        did not so differ. In females, the vana
require larger doses for this effect to ap      dium, niobium, chromium, and nickel
pear than males. The data show that, in         groups differed (P < 0.005), as possibly
general, higher levels occurred in male         did the selenium group (P < 0.05), values
rats fed the metalloid or nonmetallic ele       being smaller than those of the tellurium
ments in the "A" groups of the Periodic         group.
Table — arsenic, antimony, selenium and           Because it was possible that differences
tellurium — than in those fed metals in       in cholesterol levels might be related to
the transitional or "B" groups — vana         factors other than the trace elements fed
dium, niobium, nickel and cadmium —or         to the animals, attempts were made to
heavy metals in the "A" groups —tin and       avoid unknown influences as far as practi
lead. Female rats, however, also had            cable. Males and females were strictly
higher levels when they were given those        comparable.     Several groups of animals
elements situated in the right of the Peri      were bred and observed simultaneously as
odic Table, beginning with cadmium, and         follows: Controls (no chromium), chro
lower levels when fed elements situated in      mium (5 ppm), cadmium and lead; con-
the left or "B" groups.                         trals (1 ppm chromium), arsenic, germa
   To test this impression, the elements        nium and tin; lead (males), zirconium,
were arranged in order of position in the       niobium and antimony;         chromium     (5
Periodic Table from left to right and as        ppm), vanadium, nickel, selenium and
signed numbers from 2 (zirconium) to 27         tellurium.    Each experiment required 4
(tellurium).   Paired rank correlations with    years for completion (5). As facilities were
serum cholesterol levels were calculated.       limited to 1,000 rats, additional groups
For males, r = +0.486 (P ~ 0.05); for           were bred and started, usually in the fall
females, r = +0.622 (P < 0.01). Although        or winter, when previous groups were
the list of elements studied is incomplete,      partly depleted through death. Analyses
480                              HENRY   A.   SCHKOEDER

of sera were made during the summers on       noted in samples from the adult population
several groups at weekly intervals. The       of the United States (9) and the excess of
diet was as uniform as feasible, although     tellurium (12, 13) deserve study.
annual variations in the trace element con
tent of seed rye obtained from the same                       LITERATURE CITED
area each year were possible, owing to          1. Curran, G. L. 1954 Effect of certain transi
annual variations in rainfall. No consist          tion group elements on hepatic synthesis of
                                                   cholesterol in the rat. J. Biol. Chem., 230: 765.
ent changes were found among the groups         2. Mertz, W. 1967 Biological role of chro
analyzed at each interval which could be           mium. Federation Proc., 26: 186.
attributed to unknown factors.                  3. Schroeder, H. A., W. H. Vinton, Jr. and J. J.
   The values obtained by the method of            Balassa 1962 Effect of chromium, cad
                                                   mium and lead on serum cholesterol of rats.
Abell et al. (Il) in the first series (4)          Proc. Soc. Exper. Biol. Med., 109: 859.
were compared with those obtained by that       4. Schroeder, H. A., and J. J. Balassa 1965
of Huang et al. (10) which was an essen            Influence of chromium, cadmium and lead
tially similar but more simple method.             on rat aortic lipids and circulating choles
                                                   terol. Amer. J. Physiol., 209: 433.
Mature males fed lead in the first series       5. Schroeder, H. A., J. J. Balassa and W. H.
(4) had 79.3 ±6.5 mg cholesterol/100              Vinton, Jr. 1965 Chromium, cadmium and
ml; those in the second had 86.6 ± mg6.5          lead in rats : Effects on life span, tumors and
/100 ml. Mature males fed chromium had             tissue levels. J. Nutr., 86: 51.

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                                                6. Schroeder, H. A., W. H. Vinton, Jr. and J. J.
77.0 ±  6.6 mg/100 ml in the first series         Balassa 1963 Effects of chromium, cad
and 86.2 ±   3.2 mg/100 ml in the second.         mium and lead on the growth and survival
Apparently the 2 methods gave values in            of rats. J. Nutr., 80: 819.
the same ranges for 2 sets of animals fed       7. Schroeder, H. A., W. H. Vinton, Jr. and J. J.
                                                   Balassa 1963 Effect of chromium, cad
the same metals.                                   mium and other trace metals on the growth
    Because all these animals, except those        and survival of mice. J. Nutr., 80: 39.
in one control group, were given chro           8. Schroeder, H. A. 1966 Chromium deficiency
                                                   in rats: A syndrome simulating diabetes mel-
mium, it is evident that if an element in          litus with retarded growth. J. Nutr., 88: 439.
fluenced serum cholesterol levels, the ef       9. Schroeder, H. A., J. J. Balassa and I. H.
fect was either additive to or antagonistic        Tipton 1962 Abnormal trace metals in man.
to that of chromium. Additive effects in           Chromium. J. Chron. Dis., 15: 941.
one sex might be postulated for niobium        10. Huang, R. C., C. P. Chen, V. Wefler and A.
                                                   Raftery 1961 A stable reagent for the
and nickel; antagonistic effects for tellu         Liebermann-Burchard reaction. Clin. Chem.,
rium, selenium, antimony, vanadium and             7: 542.
zirconium.                                     11. Abell, L. L., B. B. Levy, B. B. Brodie and
    Therefore, it is possible that a common        F. E. Kendall 1952 A simplified method
                                                   for the estimation of total cholesterol in
property of nickel, niobium and chromium           serum and demonstration of its specificity.
may be to influence serum cholesterol of           J. Biol. Chem., 195: 357.
rats at low levels, and that tellurium in      12. Schroeder, H. A., J. Buckman and J. J.
some way may raise it. The lowest stan             Balassa 1967 Abnormal trace metals in
                                                   man: tellurium. J. Chron. Dis., 20: 147.
dard deviations, and therefore individual      13. International Commission on Radiological
variations, were found among analyses of           Protection 1960 Report of Committee II
sera from rats fed these metals. Tissue            on Permissible Dose for Internal Radiation
levels of the various elements will be or           (1959). Pergamon Press, Oxford, England.
                                               14. Schroeder, H. A. 1967 Effects of selenate,
have been reported (5). Application of              selenite and tellurite on the growth and early
these data to the chromium deficiency              survival of mice and rats. J. Nutr., 92: 334.

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