Dietary and metabolite effects on trivalent chromium retention and distribution in rats

OCopyright 1995 by Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/95/5002-97 $06.40

Dietary and Metabolite Effects on Trivalent Chromium Retention

and Distribution in Rats

RICHARD A. ANDERSON* AND MARILYN M. POLANSKY

Vitamin and Mineral Nutrition Laboratory, Beltsville Human Nutrition Research Center, US Department of Agriculture,

ARS, Beltsville, MD 20705-2350

Received April 1, 1994; Revised July 14, 1994; Accepted August 3, 1994

ABSTRACT

The purpose of this study was to determine if diet or various metabolites alter chromium (Cr) uptake and distribution in rats. Radioactively labeled Cr was detected within 15 min of oral administration to rats, and the total amount retained remained relatively constant from 1 to 24 h. Dietary Cr intake did not alter Cr retention or distribution. The majority of the Cr was retained in the carcass. However, when the amount of labeled Cr was expressed per gram of tissue, the highest amounts of Cr were found in the kidneys, spleen, and pancreas. Pharmacological doses of insulin, epinephrine, glucagon, and dibutyryladenosine-3' -5'cyclic monophosphate, prostaglandins A1, A2, B1, B2, El, E2, FIR, and F20~ did not significantly influence Cr retention. Glucose, sucrose, nicotinic acid, glutathione, and other metabolites administered orally in conjunction with labeled Cr also did not significantly alter Cr retention. These data indicate that most nutrients and metabolites do not alter Cr retention and distribution. The regulation of Cr homeostasis appears to be at the level of excretion.

Index Entries: Chromium absorption; tissue chromium distribution; trace elements; insulin.

INTRODUCTION

C h r o m i u m (Cr) is an essential trace e lement required for no rma l car- bohydra t e and lipid metabol i sm (1-4) . In humans , low levels of d ie tary

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 97 Vol. 50, 1995

98 Anderson and Polansky

Cr can lead to fasting hyperglycemia, impaired glucose tolerance, elevated circulating insulin, and hypoglycemic symptoms (5).

Cr absorption for humans from normal diets ranges from 0.4 to 2% and is inversely related to dietary intake at daily intakes of < 40 ~tg. Above 40 ~tg, Cr absorption remains constant at roughly 0.4% (6). Absorption of radioactively labeled Cr chloride by healthy young and old human subjects and maturity-onset diabetics is similar, whereas Cr absorption of insulin requiring diabetes is fourfold higher (7). Insulin- requiring diabetics also excrete more Cr in the urine than control subjects. Diabetics appear to have homeostatic mechanisms that sense a need for additional Cr, but appear to be unable to utilize Cr once it is absorbed. Lactating women also have greater urinary Cr losses than control subjects, but the overall percentage of Cr absorbed appears to be similar since dietary intake is also greater (8).

The precise mechanism of Cr absorption has not been determined. In the rat, the jejunum is the most diffusible segment of the small intestine followed by the ileum and the duodenum (9). Plasma proteins, including albumin and transferrin, are involved in Cr uptake and transport, but do not alter Cr retention in the small intestine (10). Cr absorption in rats declines with age (11) and may explain the progressive age-associated decrease observed in tissue Cr of humans. Cr absorption is elevated in insulin-requiring diabetics (7), but the effect on absorption in rats has not been delineated, nor have the effects of related hormones been evaluated. Prostaglandins have been reported to increase zinc absorption (12) and zinc affects Cr absorption (13), but the effects of prostaglandins on Cr absorption and retention have not been evaluated.

The present study was conducted to determine the effects of dietary factors, metabolites, and effectors on Cr retention in the rat. The effects of insulin, glucagon, and related effectors, such as thyroxine, epinephrine, and prostaglandins, were also evaluated. It is postulated that knowledge of factors that alter Cr retention in the rat will shed light on the factors that affect absorption, retention, and urinary Cr losses in humans.

MATERIALS AND METHODS

Cr-deficient and Cr-supplemented rats (weanling CD Sprague Daw- ley rats, Charles River, WI) were raised on a low Cr diet in a metal-free environment on a 12-h light-dark cycle (14). Low Cr diet has been described (15) and contained < 0.67 ~tmol of Cr /kg of diet. Cr-supplemented animals were given 38.5 ~tM of Cr as Cr chloride/L of drinking water. Animals designated as stock were fed from weaning a commercial stock diet (Agway, Charles River, WI) containing roughly 7.6 _+ 1 ~tmol of Cr/kg. Weights of animals at the time of Cr administration are noted in figure and table legends.

Biological Trace Element Research Vol. 50, 1995

Chromium Absorption 99

Stock rats weighing roughly 200 g were made chemically diabetic by injection of streptozotocin (Sigma Chemical Co., St. Louis, MO), 0.19 mmol/kg body wt. Streptozotoxin, 0.19 mmol/L, was dissolved in 0.01 mol/L sodium citrate, pH 4.5, and injected ip. Animals were allowed to stabilize for 2 wk prior to the Cr retention studies.

Methods of Cr analysis and use of standard reference materials have been reported (16). A purified rat diet (0.050 +_ 0.009 ~tg of Cr/g) was analyzed with each set of samples as an internal control. A composite human diet sample, Reference Material 8431 (National Institute of Stan- dards and Technology, Gaithersburg, MD), recommended Cr concentration 0.102 _+ 0.006 ~tg/g, was employed as an accuracy check. The Cr concentration of the reference material was 0.092 +_ 0.006 ~tg/g under our conditions.

Retention of labeled Cr at designated times was ascertained by determining the radioactivity that remained in the rat after removal of the entire gastrointestinal tract. Rats were given 51Cr as CrC13 (Dupont NEN, Boston, MA) either in feed (radioactive Cr mixed with I g of feed) or stomach-tubed in 1 mL of saline as noted. Animals were sacrificed by decapitation at times noted and counted in a Nuclear Chicago Model 825 Whole Body Counter (Des Plaines, IL). Individual organs were counted in a Searle 1185 7-counter (Des Plaines, IL). Reagent-grade chemicals tested were obtained commercially (Sigma Chemical Co). Insulin was a generous gift from Eli Lilly Co. (Indianapolis, IN).

Data were analyzed by analysis of variance followed by Duncan's multiple-range test. Values are means _+ SEM unless stated.

RESULTS

The percent of total Cr retained was similar in rats given from 25,000 to 1 million counts of carrier-free radioactive Cr as Cr chloride (Table 1). Since there appeared to be a plateau in Cr retention, we chose 600,000 cpm (30 ~tCi) as our standard dose range for subsequent studies. (The total amount of Cr administered, even at the highest dose, was < 1 ~tg). We did not choose lower doses, since the amount of Cr in individual tissues was so low that it was difficult to obtain good counting statistics.

Cr retention is independent of dietary Cr intake as shown in Table 2. Rats fed a low Cr diet from weaning to 200-250 g had similar retention to animals fed the same diet supplemented with Cr in the drinking water. Animals fed a stock diet also had similar Cr absorption.

Since animals fed a low Cr diet plus or minus supplemented Cr or a stock diet retained similar amounts of Cr, stock animals were used in the remaining studies. The effects of fasting or feeding on Cr retention are shown in Table 3. Animals that were fasted, gavaged, and fasted retained significantly more Cr than other groups tested. However, these animals would have an increased transit time coupled with an increase



Table 1 Effect of Amount of Radioactive Cr Administered on Retention

CPM administered Retention (%)

25,000 0.42 +_ 0.105

100,000 0.32 __+ 0.10 b

600,000 0.31 + 0.065

1,000,000 0.41 +_ 0.035

5,000,000 0.09 + 0.03 a

Rats (five per group) weighing 190-225 g were fasted 18 h, gavaged with the designated amounts of carrier-free 51Cr as Cr chloride in 1 mL of saline, given free access to their diet, and sacrificed after 24 h. Retention is defined in Materials and Methods. Numbers with different superscripts are significantly different from other groups tested (P < .05).

Table 2 Effect of Diet on Cr Retention

Diet Retention (%)

Low Cr diet (12)

Low Cr diet plus Cr (12)

Stock (15)

0.58 _+ 0.11

0.42 + 0.07

0.37 + 0.05

Rats weighing 200-250 g were fasted for 18 h, gavaged with the 30 ~tCi (600,000 cpm) of carrier-free 51Cr as Cr chloride in mL of saline, given free free access to their diet, and sacrificed after 24 h. Cr content of the diet and retention are defined in Materials and Methods. Number in parentheses denotes number of animals. Values are not significantly different.

in coprophagy compared to nonfasted animals. Mixing the labeled Cr wi th feed and al lowing rats to eat normal ly rather than gavaging d id not significantly alter Cr retention.

The percentages of the adminis tered dose of Cr in the s tomach, the first 15 cm of the small intestine, and the GI tract are shown in Fig. 1.


Chromium Absorption

Table 3 Effect of Feeding and Mode of Administration on Cr Retention

101

T r e a t m e n t Retention (~

Fasted - Cr in feed - fasted

Fasted - Cr in feed - nonfasted

Fasted - gavaged - nonfasted

Fasted - gavaged - fasted

Nonfasted - gavaged - nonfasted

0.23 + 0.03 a

0.15 + 0.01 a

0.38 + 0.13 ~

0.82 + 0.25 b

0.10 + 0.02 a

Rats weighing 190-225 g were given 30 ~tCi of Cr added to 1 g of feed and allowed to eat normally, or Cr was di luted into 1 mL of saline and administered via a stomach tube. Animals designated as fasted prior to Cr administrat ion were depr ived of food for 18 h. All rats were sacrificed 24 h after administrat ion of Cr. Each value represents an average of five rats. Values with different superscripts are significantly different at p < 0.05.

The majority of the radioactive Cr passed through the s tomach and the first 15 cm of the small intestine in 1 h or less wi th over 50% of the labeled Cr in the lower por t ion (past the first 15 cm of the small intestine) wi thin 20 min. The radioactivity was not b o u n d to the GI tract since f lushing wi th saline removed essentially all of the labeled Cr. Hahn and Evans (13) reported that in zinc-deficient rats, Cr remained b o u n d to the lining of the GI tract.

The majority of the counts were in the carcass (muscle and bone wi th skin and head removed) (Table 4). The radioactivity in the carcass, skin and hair, heart, lungs, spleen, and pancreas declined over the 24 h of the study. The counts in the liver, kidney, and testes declined in the first 30--60 min, bu t increased in the remaining 23 h. Values in Table 5 are on a per-gram basis. These are from a separate set of animals from Table 4. On a per-gram basis, 51Cr in carcass, skin and hair, heart, and lungs declined over the dura t ion of the study. Counts per g ram in the pancreas, spleen, liver, kidney, testes, and fat pads decl ined in the first 30-60 min and then increased wi th the greatest retention after 24 h. Al though the carcass had the greatest total n u m b e r of counts (Table 4), on a per-gram basis, the Cr concentrat ion of the carcass was significantly lower than that of the kidneys, spleen, and pancreas after 24 h.

In Table 6 are shown a number of hormones , prostaglandins, dietary components , and other effectors that d id not improve Cr retention. At



I T A / A -

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Fig. 1. Percent distribution of orally administered radioactive Cr in the gastrointestinal tract with time. Rats were gavaged at zero time with 30 ~tCi of carrier- free Cr chloride, sacrificed at times indicated, and the gastrointestinal tract was divided into three sections: (0) stomach, (�9 15-cm section of the small intestine, (A) remainder of GI tract. Each data point is mean _+ SEM for 10 rats. Percent denotes % of radioactivity in each section of the GI tract compared to the total number of counts administered. Total counts in GI were constant in the first 240 min.

least five rats were sacrificed at 1, 4, and 24 h. Effectors that appeared to have more than a 10% effect were retested, but none of the substances listed in Table 6 consistently altered Cr retention, either positively or neg- atively.

DISCUSSION

These data clearly show that regardless of the intake of dietary Cr, its mode of administration, presence of hormones, or dietary components or effectors, the retention of Cr is largely unchanged. Earlier studies also reported that Cr distribution was independent of dietary Cr when Cr was administered iv (17,18). However, Jain et al. (19) reported that dietary Cr alters exchangeability of iv injected Cr.

Studies using injected Cr (17-20) demonstrated at least three exchangeable pools for Cr. When Cr is injected iv, forms of Cr that would not enter the circulation if given orally, i.e., olated forms that would likely be retained in the reticulo-endothelial system if given iv (21), would greatly alter tissue distribution. Obviously, all injected Cr would


Chromium Absorption 103

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Chromium Absorption

Table 6 Substances Tested That Did Not Enhance Cr Retention

105

Amount/rat Insulin (subcutaneous, (S.C.))

or intraperitoneal - control and 0.6 - 500 mU diabetic rats

Glucagon (S.C.) 1.5 - 3 mg

Dibutyryl adenosine-3' -5'cyclic 9 - 12 mg monophosphate (S.C.)

Epinephrine (S.C.) 1.2 mg

Thyroxin (S.C.) 0.8 mg

ACTH (S.C.) I00 IU

Prostaglandins: (oral) 0.00085 - 0.25 mg A1 & B1 B2 E1 E2 F1 F2

10% Corn oil (oral) 1 ml

95% Ethanol (oral 1 ml

1% Glutathione (GSH) (oral) 1 ml

10% Sucrose (oral) 1 ml

10% Dextrose (oral) 1 ml

1% Ascorbic acid (oral) 1 ml

1% Nicotinic acid (NA) (oral) 1 ml

1% Histidine (oral) 1 ml

Milk (oral) 1 ml

Rats weighing 200-250 g were fasted for 18 h, gavaged with 30 ~tCi of radioactive 51Cr at zero time, and at least five rats were sacrificed at 1, 4, and 24 h. Hormones and prostaglandins were added to saline prior to oral administration. Remaining compounds were diluted with water. Retention for all of the compounds tested was roughly 1% or less. Hormones were administered at times ranging from simultaneous injection of hormone to 24 h before gavage of labeled Cr. S.C. denotes subcutaneous injection.



enter the circulation, whereas only the Cr in specific soluble forms would enter the circulation via the oral route. Because of the low absorption of oral Cr (usually < 2%), the comparisons of orally and iv injected Cr may be questionable.

Fasting and refeeding appeared to alter Cr retention (Table 3), but these differences may be the result of changes in coprophagy and transit time. Animals that were fasted and not refed would likely have the longest transit times and would also practice a high degree of coprophagy. We conducted studies to limit coprophagy using rat restrainers, but the results were inconclusive. Restrainers prevent coprophagy, but do not alter changes in transit time owing to fasting and refeeding.

The significant retention of Cr in < 15 min. (Tables 4 and 5) may suggest that Cr may be absorbed via the stomach. However, because of the rapid movement of Cr through the GI tract (Fig. 1), the absorption of Cr via the small intestine cannot be eliminated. Ligation of the pyloric sphincter blocked Cr absorption and transit, which suggests minimal absorption via the stomach (22). Oberleas et al. (22) also reported rapid transit of Cr with 60% of the labeled Cr past the ileum within 4 h of intu- bation.

The extremely rapid (5 min) absorption of Cr is difficult to explain. There appears to be a nonspecific diffusion process occurring. Although Cr values in selected tissues are high, values in blood are relatively low compared to later values. Tissue Cr is not in equilibrium with blood Cr (18). Values do not appear to be the result of nonspecific contamination, since tissues throughout the animal were high at the early time-points.

Insulin-requiring diabetics excrete higher quantities of orally administered Cr in their urine than control subjects (7). Cr retention of iv injected 51Cr is reduced in diabetic rats and partially restored to normal by insulin administration (23). Injections of 0.5-500 mU of insulin did not affect Cr retention at 1, 4, or 24 h in nondiabetic or chemically diabetic rats (Table 6).

Thyroparathyroidectomized rats displayed a significant increase in iv injected 51Cr that was reversed to normal by thyroxin administration (24). Thyroxin did not alter Cr absorption of control rats (Table 6).

Prostaglandins have also been postulated to affect trace element absorption (12), but oral administration of a number of prostaglandins had no significant effect on Cr absorption (Table 6).

Chelating agents have been reported to alter Cr absorption. Oxalate was reported to increase and phytate to decrease Cr absorption in rats. Citrate and EDTA did not alter Cr absorption (9). Later studies reported no effect of phytate or bran on Cr absorption (25). Antacids, containing calcium carbonate (26), zinc (13), iron (27), and vanadium (28) inhibit Cr absorption. Dietary amino acids improve Cr uptake and transport when added to the perfusion media, but not above normal basal levels (10).

Cr homeostasis appears to be controlled primarily at the excretion levels with minimal effects on Cr absorption. Cr is the only essential trace


C h r o m i u m A b s o r p t i o n 107

metal whose absorption is not increased when dietary intake is low. There are very few metabolites that improve Cr absorption. Factors that alter Cr absorption, such as chelators, bind Cr and make it less available, and other metals compete with Cr at the site of absorption from the GI tract or transport. In studies using perfused intestinal preparations, it has been shown that factors that improve Cr absorption, such as amino acids, added to perfusion media do not increase Cr absorption above normal levels, but rather removal of these will decrease absorption (10). The best example of a control of Cr nutrition at the absorption level for humans is the inverse relationship of Cr absorption with dietary intake (6). However, this relationship is no longer valid at intakes above 40 ~tg. This inverse relationship of Cr intake and absorption does not appear to be present in rats. In contrast to absorption, numerous factors have been shown to alter Cr losses, including high sugar diets, exercise, and physical trauma (5).

In summary, these data suggest that Cr absorption is rapid with significant retention within 15 min of administration. Cr retention is independent of dietary intake, and few if any dietary components or hormones increase Cr absorption above basal levels. Body Cr stores appear to be regulated at the excretion stage, rather than at the level of absorption.

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(1981). 20. C. Onkelinz, Am. J. Physiol. Endocrinol. Metab. Gastrointest. Physiol. 1, E478

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Biol. Med. 84, 610 (1953). 22. D. Oberleas, Y.-C. Li, B. J. Stoecker, S. A. Henley, K. S. Keim, and J. C. Smith,

Jr. Nutr. Res. 10, 1189 (1990). 23. J. L. Kraszeski, S. Wallach, and R. L. Verch, Endocrinology 104, 881 (1979). 24. M. L. Lifschitz, S. Wallach, R. A. Peabody, R. L. Verch, and R. Agrawal, Am.

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