nutrient
Metabolism
Incorporation of 3H-Label from Folie Acid is Tissue-Dependent in Folate-Deficient Rats1»2 BERNARD
H. EISENGA,
TIMOTHY D. COLLINS AND KENNETH
E. McMARTIN3
Most studies in animals (6-8) have shown little (10-20%) or no decrease in brain folate content after consumption of folate-deficient diets for 4-11 mo, at which times hepatic folate levels were less than half of those of control animals. In contrast, Hakim et al. (9) observed marked depletion of both brain and liver folate concentrations in rats fed folate-deficient diets for 70 d. These differences have probably resulted from the different folate-deficient diets used, which probably contained various small but physiologically significant amounts of folate (2). Folate homeostasis includes several adaptive mech anisms that would conserve folate and help to cir cumvent the development of tissue folate depletion. The diminished urinary excretion of folate in states of decreased folate intake would result in greater body retention of folate (1). Maintenance of tissue folate stores could also result from increased absorption and/or distribution from exogenous dietary sources (10). Redistribution of folate from the liver is not a likely mechanism (11), but increased uptake and re tention of folate by individual organs could occur in states of reduced folate intake. Increased uptake of folate occurs in vitro in studies with cultured cells exposed to media containing low levels of folate (12, 13), although no studies have reported such changes in vivo. A more efficient incorporation of plasma folate by tissues would contribute to a greater body retention of folate and to more rapid clearance of exogenous folate from the plasma in folate-deficient
ABSTRACT To study the tissue-specificity of folate deficiency, male Sprague-Dawley rats were fed folatereplete or folate-deficient diets with and without sulfonamide for 16 wk, and then injected with |3H]folic acid (1.5 nmol/kg). Rats were killed after 24 h, and the blood, urine and various organs were prepared for analysis of endogenous and ^H-labeled folate. Endog enous folate levels decreased due to folate deficiency to the greatest extent in the urine and plasma, followed by liver, kidney and other tissues (spleen, testis, lung and intestine), but no decrease was noted in the brain. Of all tissues of folate-deficient rats, the brain showed the greatest increase in incorporation of 3H-label from folate relative to folate-replete rats, with the largest effect in rats that were most deficient in plasma folate. Incorpo ration of label was increased due to folate deficiency in a number of tissues, with an inverse correlation with the tissue folate concentration. In contrast, hepatic [3Hlfolate incorporation was lower in folate-deficient rats than in folate-replete rats, with a direct correlation be tween endogenous folate concentration and the incorpo ration of labeled folate. These results show that the brain and other organs adapt to the development of folate deficiency because of greater incorporation of folate from exogenous sources. The lower incorporation by the liver of folate-deficient rats may result from the greater incorporation by other tissues. J. Nutr. 122: 977-985, 1992. INDEXING KEY WORDS:
•folate deficiency •rats •brain •folate uptake •liuer
Previous studies of animals fed folate-deficient diets have shown a differential loss of folate among various tissues (1, 2). Folate depletion has been shown to occur in a sequential manner (3, 4), being rapidly evident in urine by as early as 2 wk (1) and in plasma by 4 wk of dietary treatment (3). Depletion of folates in tissues such as the liver and kidney generally re quires consumption of a folate-deficient diet for about 2-4 mo (1, 5). In comparison, the brain has been shown to be relatively resistant to folate depletion. 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
'Supported
in part by U.S. Public Health Service grant RO1-
AA05308. 2Presented in preliminary
form at the annual meeting of the
Federation of American Societies for Experimental Biology, April 1990, Washington, DC [McMartin, K. E., Collins, T. D., Eisenga, B. H. & Bhandari, S. D. (1990) Increased folate uptake prevents dietary development of folate deficiency in the rat brain. FASEB J. 4: A673 labs.)]. 3To whom correspondence and reprint requests should be ad dressed.
Received 18 July 1991. Accepted 22 November 977
1991.
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Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, LA 71130-3932
978
EISENGA ET AL.
subjects (14). Differences in the degree to which various tissues adapt to dietary folate insufficiency could be related to the differential loss of folate among tissues. The present study was designed to examine the ability of various tissues of control and folate-deficient animals to incorporate [3H]folate from exogenous
sources.
Animals and reagents. Male Sprague-Dawley rats weighing 125-150 g were obtained from HarÃ-an Sprague Dawley (Houston, TX) and quarantined for 14 d, during which time they were fed a nonpurified diet (Purina Laboratory Chow #5001, Ralston-Purina, St. Louis, MO). Subsequently the rats were housed in wire-mesh cages with a daily light cycle from 0600-1800 h and controlled temperature (23 ±TC) and humidity (45-55%). These studies were approved by the Animal Resources Advisory Committee of Louisiana State University Medical Center in Shreveport. [3',5',7,9-3H]Folic acid (PteGlu)4 (1.5 x IO12 Bq/ mmol) was purchased from Moravek Biochemicals (Brea, CA). Radiochemical purity was determined to be that supplied by Moravek (99%) by HPLC analysis (15, 16). Lactobacillus casei (ATCC 7469a) was ob tained from the American Type Culture Collection (Rockville, MD). Folie Acid Casei Assay medium and Lactobacilli broth AOAC were obtained from Difco Laboratories (Detroit, MI). Complete Counting Cocktail 3a70B was purchased from Research Products International (Mount Prospect, IL). Econofluor-2 and Protosol were purchased from NEN Research Products, Du Pont (Boston, MA). All other reagents used in these investigations were from com mercial sources. Experimental diets. A preliminary study of tissue incorporation of folate (17) was done in conjunction with previous studies of ethanol and folate deficiency (1). The present study was performed to follow up on the initial results. As such, this study was conducted using the liquid diets as previously described (1). Briefly, rats were fed liquid diets obtained from Dyets (Bethlehem, PA) with an energy distribution of 35% fat, 18% protein and 47% carbohydrate. A thorough description of the diet composition has been reported by Yamada et al. (18).5 Succinylsulfathiazole (3 g/4.2 MJ) was added to two of the diets to reduce the production of folate by intestinal bacteria (19). After quarantine, rats were acclimated for 3 d to the control (folate-containing) diet. On d 1 of wk 1, rats were divided into four groups and fed the following experi mental diets: C (control diet with folate in the vi tamin mix); CS (control diet plus sulfonamide); FD (folate-deficient diet, no folate in the vitamin mix); FDS (folate-deficient diet plus sulfonamide). The folate contents of the diets, determined by L. casei
power, followed by 5 s at 100% power (Tissumizer, Tekmar, Cincinnati, OH) and then adjusted to a 25% (wt/v) homogenate with the addition of 1.5 mol/L 2-mercaptoethanol. Feces were weighed and boiled in 10 volumes of 1.5 mol/L 2-mercaptoethanol and then homogenized. The outer layer of the carcass (skin and hair) was removed and the remainder was weighed and prepared as a 33% (wt/v) homogenate with deionized water (Millipore, Bedford, MA) using a blender. Aliquots (10 mL) were removed and 2mercaptoethanol was added to produce a 25% wt/v mixture in 1.5 mol/L 2-mercaptoethanol. These homogenates were then heated for 5 min at 95°Cand rehomogenized. Tissue and fecal homogenates were centrifuged at 15,000 x g for 20 min at 4°C.Supernatants were used for analysis of endogenous and la beled folate content. All samples were stored at -20°C
4Abbreviations used: C, control diet; CS, control diet with sul fonamide; FD, folate-deficient diet; FDS, folate-deficient diet with sulfonamide; r^PteGlu, tetrahydrofolic acid (and its derivatives]; PteGlu, folie acid. •"Controldiets were the same as the Liquid B diet of Yamada et al. (18| with the following exceptions: Î)vitamin-free casein was used rather than edible casein,- 2} xanthum gum at 3.0 ug/4.2 Mf was substituted for sodium carrageenate; 3| vitamin mix provided 200 ug of menadione sodium bisulfite per 4.2 MJ diet instead of 25 \ig; 4) mineral mix provided (g/kg mix); manganous sulfate, 4.6, and ferrous sulfate, 4.95, as replacement for manganous carbonate and ferrous citrate.
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MATERIALS AND METHODS
assay, were 910 and 42 nmol (0.43 and 0.02 mg) folate/4.2 MJ for the control and folate-deficient diets, respectively. Tissue incorporation of labeled folate. Rats were fed the experimental diets for 16 wk and then injected intraperitoneally with a solution containing [3H]PteGlu in a dose of 1.5 nmol/kg body wt. In a previous study of rats injected with a tracer dose of [3H]PteGlu (15), the distribution of 3H-labeled folate and of endogenous folate forms in the liver (converted to the monoglutamyl forms before HPLC analysis) were similar at 24 h after dosing. These data suggest that, by 24 h, incorporation of label into the onecarbon tetrahydrofolate derivatives in tissues had oc curred. As such, the tissues in the present study were similarly collected to assess the incorporation of 3H-label from folie acid. Animals were placed into metabolic chambers that permitted the complete separation of urine and feces. Urine was directed into beakers containing 0.15 mL of neat 2mercaptoethanol (adjusted after collection at 6-h in tervals to 0.05 mL/mL urine, equal to 0.7 mol/L). At the end of 24 h, rats were anesthetized and exsangui nated via abdominal aortic puncture. The blood was centrifuged and plasma was preserved with 1.5 mol/L 2-mercaptoethanol (16). Organs (liver, kidneys, brain, testes, lungs, spleen, jejunum and ileum) were rapidly removed, weighed, minced and boiled in 1.5 mol/L 2-mercaptoethanol (2 mL/g wet tissue) (15). All tissue samples (at 4°C)were homogenized for 30 s at 30%
TISSUE FOLATE INCORPORATION
the various significance. group mean enous folate tissues were PHARM/PCS Philadelphia, as the level
groups, with P < 0.05 as the level of Values cited in the text represent the ±SEM. Individual data points of endog level and of 3H-label content for various analyzed by linear regression using the program (Microcomputer Specialists, PA) to test for correlation, with P < 0.05 of significance.
RESULTS In our previous study (1) using these liquid diets, neither the lack of folate nor the presence of sulfonamide in the diet affected body weight gain or food consumption for 12 wk. Similar results were observed in this group of rats fed these diets for 16 wk. Body weights during wk 15 were 359 ±3, 371 ±6, 361 ±8 and 360 ±4 g for rats fed diets C, FD, CS and FDS, respectively. Similarly, mean daily food consump tions during wk 14 were 58 ±2, 58 ±1, 60 ±0 and 57 ±2 mL of diet, respectively (1 mL diet provides 4.2 KJ). Daily folate consumption was therefore -150 nmol/kg body wt for rats fed the control diets and ~7 nmol/kg body wt for rats fed the folate-deficient diets. Previous studies with these diets (1) showed that depletion of urinary folates occurred within as little as 14 d. Subsequently, the folate-deficient diets produced significant reductions in plasma folate levels by 4 wk (the first collection period) and in tissue folate content (RBC, liver and kidney) by 8 wk.
979
These decreases were marked (70-95%) in rats fed the folate-deficient diet containing sulfonamide,- in rats fed the folate-deficient diet without sulfonamide, folate depletion occurred more slowly and to a lesser extent. In the latter rats, however, tissues were signif icantly folate depleted by 12 wk. Endogenous folate concentrations were monitored at 2-4-wk intervals in the present study and showed a temporal progression to lower folate levels similar to that previously ob served (1). Because the purpose of this study was to examine how folate deficiency would affect the incor poration of [3H]folate from exogenous sources, rats were fed the diets for 16 wk before [3H]PteGlu dosing to ensure that a severe folate deficiency was present. The data in Figures 1-3 confirm that endogenous folate concentrations were decreased by 16 wk. Data in Figure 1 show the plasma folate concentra tions 24 h after administration of a tracer dose of [3H]PteGlu. Endogenous plasma folate was signifi cantly lower in rats chronically fed folate-deficient diets. This reduction was exacerbated by the addition of sulfonamide and was similar to results previously published (1, 19). There were no differences in the plasma retention of labeled folate derivatives within corresponding diet groups (C vs. FD, CS vs. FDS). A tendency towards reduction due to folate deficiency was evident, but was significant only between control (C) and folate-deficient sulfonamide diet groups (FDS). Figure 2 shows data obtained from analysis of liver folate concentrations. The incorporation of labeled folate derivatives in the liver was very similar to that seen in the plasma, with a significant difference only between control and folate-deficient sulfonamide diet groups. In contrast to the results in the liver and the plasma, significantly greater incorporation of 3H-label was seen (Fig. 3) in the kidney of rats fed folatedeficient sulfonamide diets (CS vs. FDS). Other tissues (jejunum, ileum, lung, testis and spleen) showed the same pattern of dietary effects, i.e., an increase in the incorporation of 3H-label in sulfonamide-treated, folate-deficient rats, as was ob served in the kidney (data not shown). As is indicated in Figure 4, chronic diet treatment did not significantly lower endogenous brain folate concentration. Yet, the incorporation of labeled folate by the brain was significantly higher in rats fed folatedeficient diets. This effect was noted for both groups of diets (C vs. FD, CS vs. FDS) and was further augmented by the addition of sulfonamide to the diet (FD vs. FDS). As seen in Table 1, there were no differences among diet groups in either the urinary or fecal ex cretion of 3H-label, suggesting that folate deficiency does not affect excretion within 24 h of exogenous folate administration. Endogenous urinary folate ex cretion was, however, markedly reduced in 24-h urine collections at 16 wk (7.9 ±2.5, 0.9 ±0.5, 2.4 ±0.5 and 0.2 ±0.1 nmol/24 h for C, FD, CS and FDS diets,
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until analyzed. Approximately l g of skin (containing hair) was prepared by cutting random samples from throughout the outer layer and was mixed with 15 volumes of Protosol. This mixture was incubated at 37°Cuntil the tissue was dissolved. Determination of tissue folate and 3H-label con tent. Endogenous folate content was determined using a Lactobacillus casei microtiter growth assay (20). For measurement of total folate content, tissue and fecal samples were pretreated with hog kidney folylpolyglutamate hydrolase (15) and then analyzed with L. casei. All tissues were analyzed for endog enous folate content except the carcass and the skin. The quantities of individual folate derivatives were determined in urine samples by HPLC as previously described (16, 21). Fractions eluted from HPLC were analyzed for folate derivatives by L. casei assay and for 3H-label by scintillation analysis. Total radioactivity was determined by liquid scin tillation analysis of 10-uL aliquots of urine or 100-uL aliquots of all other samples in 4 mL of 3a70B counting cocktail. Radioactivity in the skin was de termined by adding 2 mL of the skin-Protosol mixture to 10 mL of Econofluor-2. Statistics. Group data were analyzed by ANOVA using a general linear models procedure with Dun can's test to test for differences among the means of
IN FOLATE DEFICIENCY
EISENGA ET AL.
980
3H- Ldb.f.d
Endogenous 160 U3 120-£
TabTabTT
5!1
im.1a.eT
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1 FD
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1
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CS
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FIGURE l Endogenous and 3H-labeled folate levels in the plasma of rats fed control and folate-deficient diets. Bars represent the group mean, expressed in nmol folate or pmol folate equivalence/L plasma, ±SEM (n = 6). Bars with letters in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folate-deficient diet with sulfonamide.
respectively), as previously reported (1). The HPLC analysis of the urine samples in the present study (Table 2) showed that the 3H-labeled products were primarily a nonfolate compound (21), 5-formiminotetrahydrofoÃ-ate and 5- and 10-formyl-tetrahydrofolates. These folate-derived compounds made up >75% of the total 3H-label in the urine. There were no differences among the treatment groups in terms of distribution of the derivatives (Table 2). These folate-derived compounds are not well reabsorbed by the kidney, as was shown previously (21). No effect of folate deficiency on total 3H-labeled excretion in the urine was seen in the present study, probably because the 3H-labeled products were those forms that are not conserved by the kidney.
As noted for tissue 3H-label content (Fig. 2), the hepatic recovery of labeled folate (expressed as the percentage of dose in Table 1) was significantly lower in rats fed folate-deficient sulfonamide diets. In con trast to the liver, the recovery of labeled folate in the kidney and the carcass was significantly greater in rats fed folate-deficient sulfonamide diets. Recovery of 3H-label in the brain and other organs showed a more complex pattern among the diets. In these tis sues, labeled folate recovery was significantly greater in rats fed folate-deficient diets (FD vs. C). The ad dition of sulfonamide to folate-deficient diets further increased the recovery of labeled folate (FDS vs. FD). Figure 5 shows the relationship between endog enous folate concentrations and the resulting incorpo-
'H-Labcted
Endog«nous 125ai 30
30-,a aTaÕT25-»20-I
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FDS CS FDS FD FD CS FIGURE 2 Endogenous folate level and 3H-labeled folate incorporation in the liver of rats fed control and folate-deficient diets. Bars represent the group mean value, expressed in nmol folate or pmol folate equivalence/g wet wt, ±SEM (n = 6). Bars with letters above in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folatedeficient diet with sulfonamide.
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fo £3 o.400-aTbTibT280-200\
TISSUE FOLATE INCORPORATION IN FOLATE DEFICIENCY
981
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FIGURE 3 Endogenous folate level and ^H-labeled folate incorporation in the kidney of rats fed control and folate-deficient diets. Bars represent the group mean value, expressed in nmol folate or pmol folate equivalence/g wet wt, ±SEM(n - 6). Bars with letters above in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folatedeficient diet with sulfonamide.
ration of 3H-label in specific tissues. Except for the liver and the brain, the degree of labeled folate incor poration in tissue was inversely related to the degree of folate depletion, i.e., the more severe the folate deficiency, the greater the incorporation of labeled folate. In contrast, the liver showed a significant positive correlation between endogenous folate con centration and incorporation of labeled folate. Finally, because endogenous folate concentration in the brain did not change (Fig. 4), there was no statistically significant correlation between folate content and the incorporation from labeled folate. In all other cases, these correlations were statistically significant (P < 0.05).
DISCUSSION Chronic folate deficiency occurs by a time-ordered depletion of tissue folate stores. Certain tissues are particularly vulnerable to diminished folate intake, whereas other tissues require a long time for de velopment of folate depletion. As noted previously by others (6, 7), even after 16 wk of consumption of a folate-deficient plus sulfonamide diet, the brain was resistant to the development of folate deficiency. The present study has suggested that this resistance was probably due to an increased incorporation of available folate. Although the brain did not become folate deficient per se, labeled folate incorporation in
SH-üib«l*d 0.20 T
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FIGURE 4 Endogenous folate level and 3H-labeled folate incorporation in the brain of rats fed control and folate-deficient diets. Bars represent the group mean value, expressed in nmol folate or pmol folate equivalence/g wet wt, ±SEM(n = 6). Bars with letters above in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folatedeficient diet with sulfonamide.
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*-*4-o.baTaTa
*~*O52s 3]. There were no significant differences among values for diet groups for any of the derivatives. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folate-deficient diet with sulfonamide. 2This derivative eluted before any of the standard folate derivatives and did not support the growth of L. casei. It was not a folate form and has been termed nonfolate (21). Nonfolate25-Formimino-H4PteGlu5-
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Liver Kidney Brain Organs2 Carcass3Total15.43
CS
TISSUE FOLATE INCORPORATION
IN FOLATE DEFICIENCY
LJVER
983
KIDNEY
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INCORPORATION . (pmd/g)o
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ENDOGENOUS FOLATE LEVEL (nmol/g)
FIGURE 5 Correlations between endogenous folate levels (abscissas) and incorporation of 3H-labeled folate (ordinales) in tissues of rats fed control and folate-deficient diets. Each point represenis the values from one rat, with values from all diet groups included. The correlations were statistically significant for all tissues (except brain), P < 0.05. (Liver: y = 2.3x + 1466, r = +0.51; kidney: y = -3.9x + 1474, r = -0.45; jejunum: y = -9.8x + 623, r = -0.59; testis: y = -4.Ox + 146, r = -0.43; lung: y -5.6x + 194, r = -0.60.)
could have been taken up by the mesenteric circula tion, placing the PteGlu directly into the portal circu lation (whereas that entering as small amounts via the enterocyte would be 5-CH3-H4PteGlu). The liver seems to have separate systems for the uptake of PteGlu and 5-CH3-H4PteGlu (23) with a higher Km for PteGlu. The limiting factor for hepatic uptake of PteGlu from the portal circulation might then be the capacity of the carrier for PteGlu. The PteGlu passing through the liver would reach the other tissues, where uptake would be controlled by the relative depletion of tissue folate stores.
In the folate-deficient rats treated with sulfonamide, the plasma retained less of the labeled folate. The decrease in plasma retention might have resulted from the increased tissue incorporation of 3H-labeled folate. Therefore, in states of folate deficiency, dietary or exogenous folate seemed to be shunted from the liver, to be preferentially taken up and retained by other tissues, especially the brain. These studies support the report by Weir et al. (11), who suggested that tissue folate levels are not regulated by supply from liver stores during states of folate deprivation. In their study, the livers of deficient rats did not show
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OS-
984
EISENGA ET AL.
plasma membrane and retention within the cell. Intracellular uptake of folate probably involves seques tration by the folate-binding protein and transport across the membrane (28). A highly specific folatebinding protein has been identified in the choroid plexus of the brain (29) and in other tissues (30, 31). Recent evidence has shown that folate uptake in certain cells is related to the folate-binding protein activity in the cell membrane (12, 13). Cells cultured in folate-deficient medium have increased amounts of the folate-binding protein (13) and enhanced uptake of labeled folate (12). Although there have been no studies linking these changes in vivo, cellular adaptive changes during states of folate deficiency (increased folate-binding protein or enhanced trans port) might explain the increased amount of labeled folate found in the tissues of these animals. Alterna tively, the increased uptake could have resulted from a change in the intracellular compartmentation of folate. Intracellular folate exists mostly in the protein-bound state and this is not as rapidly depleted during folate deficiency as is unbound folate (22). Therefore, a decrease in the unbound fraction would tend to increase folate uptake from extracellular sources. As an alternative to increased cellular uptake, the increased incorporation of exogenous folate by various tissues could result from increased retention of cellular folate. In general, folates are retained in the cell by conversion to the polyglutamate form by the enzyme folylpolyglutamate synthetase (32). Recent research (24) has shown that an increase in folate glutamate chain length occurs in tissues of folatedeficient rats. An increase in polyglutamylation may explain the increased tissue incorporation of labeled folate in the folate-deficient tissues in the present study. However, because the tissue samples were not properly prepared for analysis of polyglutamate chain length (they were treated initially with folyl polyglutamate hydrolase to allow for measurement of total folate content by L. casei], the respective contri butions of increased uptake vs. increased retention by polyglutamylation towards the increased incorpo ration cannot be determined from these results. In the present study, the amount of 3H-labeled folate incorporation in tissues was indirectly deter mined, i.e., whether the label in the tissue samples was PteGlu or another folate form was not directly measured (except in the urine). Nevertheless, the 3H-label in the tissues at 24 h must have resulted from tissue incorporation of a labeled dose of folie acid. The purity of the [3H]PteGlu was assessed before dosing, so that the initial label was entirely folie acid. Hence, the 3H-label in the tissue resulted from an initial uptake of PteGlu, followed by retention of folie acid or its metabolite(s). For instance, in a previous study (33), at l h after intravenous dosing with [3H]PteGlu, >82% of the 3H-label recovered in the
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an accelerated excretion of folate via the enterohepatic circulation (for distribution to other tissues) as compared with controls,- thus the liver did not act as a reservoir of folate. Previous studies of animals fed folate-deficient diets showed many variations in effects on brain folate content. Short-term treatment (30 d) of rats with folate-deficient sulfonamide-containing diets produced no reduction in brain folate content (24). In rats fed a folate-deficient, sulfonamide-containing diet for 9.5 mo, Fehling et al. (7) observed only a 16% (but significant) reduction in brain folate content. Similar rats in our studies showed about a 20% decrease in brain folate content (not significant). In rats fed folatedeficient diets without the addition of sulfonamide for 11 mo, a significant reduction in liver folate, but not in brain folate levels, was observed (6). Other studies in rats fed folate-deficient, sulfonamidesupplemented diets for periods of 4 mo (8) and 70 d (9) showed significant reductions in both hepatic and brain folate concentrations. One explanation for these differences could be that those studies that showed large reductions in brain folate concentrations after relatively short feeding regimens also showed a signif icant weight loss in folate-deficient animals. The weight loss [which did not occur in the present study, nor in other studies (6, 7)] could enhance the re duction of brain folate. The most probable expla nation for the lack of brain folate depletion in some studies could be that the diets were not "sufficiently deficient." In a recent definitive dietary study, Walzern and Clifford (2) showed that casein-based, folate-deficient diets (as in the present study) con tained a small amount of folate (intakes of -1 (¿g/d) that allowed normal weight gain, but did not produce maximal tissue folate depletion. Instead they pro posed the use of defined amino acid diets, from which severe tissue folate deficiency, reduced weight gain, and hématologie effects were produced in 5 wk. Un fortunately, brain folate content was not reported in their studies. Sparing of the brain from deficiency of needed sub strates or other nutrients is not a new concept (25). In contrast to many substrates, there is only a limited blood-brain barrier for folate (26). Folate may be a vital compound for normal brain function, because studies have shown correlations between states of folate deficiency and different types of dysfunctions of the central nervous system (27). These studies have shown that, in many instances, correction of the folate deficiency improves the mental status of the subject (27). It seems plausible that there should be adaptive mechanisms during states of folate defi ciency to prevent the loss of folate from the brain (as well as other vital tissues). These adaptations most likely involve changes in the incorporation of folate by the tissues, which would comprise two processes: uptake across the
TISSUE FOLATE INCORPORATION
14.
15.
16.
17.
ACKNOWLEDGMENTS Special thanks go to Tammy Fortney and Tim Tyler for their technical assistance, to Julie Woods, Michelle Cooper and James Gulledge for their assis tance in the care and feeding of the animals, and to Sharon Farrar for preparation of this manuscript.
18.
19.
20.
LITERATURECITED 21. 1. McMartin, K. E., Collins, T. D., Eisenga, B. H., Fortney, T., Bates, W. R. & Bairnsfather, L. (1989) Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat. f. Nutr. 119: 1490-1497. 2. Walzern, R. L. & Clifford, A. f. (1988) Folate deficiency in rats fed diets containing free amino acids or intact proteins. J. Nutr. 118: 1089-1096. 3. Siddons, R. C. (1974) Experimental nutritional folate defi ciency in the baboon. Br. J. Nutr. 32: 579-587. 4. Herbert, V. (1962) Experimental nutritional folate deficiency in man. Trans. Assoc. Am. Physicians 75: 307-320. 5. Herbert, V. (1990) Development of human folate deficiency. In: Folie Acid Metabolism in Health and Disease (Picciano, M. F., Stokstad, E.L.R. & Gregory, J. F., eds.), pp. 195-210. Wiley-Liss, New York, NY. 6. Thomson, A. D., Frank, O., DeAngelis, B. & Baker, H. (1972) Thiamin depletion induced by dietary folate deficiency in rats. Nutr. Rep. Int. 6: 107-110. 7. Fehling, C., Jagerstad, M., Lindstrand, K. & Elmquist, D. (1976) Reduction of folate levels in the rat: difference in depletion between the central and the peripheral nervous system. Z. Ernaehrungswiss. 15: 1-8. 8. Akesson, B., Fehling, C., Jagerstad, M. & Stenram, U. (1982) Effect of experimental folate deficiency on lipid metabolism in liver and brain. Br. J. Nutr. 47: 505-520. 9. Hakim, A. M., Arrieta, M. J., Cooper, B. A. & Pappius, H. M. (1984) Effect of folate deficiency on local cerebral glucose utilization in the rat. J. Neurochem. 42: 1582-1587. 10. Whitehead, V. M. (1986) Pharmacokinetics and physiological disposition of folate and its derivatives. In: Folates and Pterins (Blakley, R. L. & Whitehead, V. M., eds.), vol. 3, pp. 177-205. John Wiley and Sons, New York, NY. 11. Weir, D. G., McGing, P. G. & Scott, J. M. (1985) Folate metabolism, the enterohepatic tirculation and alcohol. Biochem. Pharmacol. 34: 1-7. 12. Kamen, B. A. & Capdevila, A. (1986) Receptor-mediated folate accumulation is regulated by the cellular folate content. Proc. Nati. Acad. Sci. U.S.A. 83: 5983-5987. 13. Henderson, G. B., Tsuji, J. M. & Kumar, H. P. (1988) Mediated uptake of folate by a high-affinity binding protein in sublines
22.
23.
24.
25. 26. 27.
28.
29.
30.
31.
32.
33.
985
of L1210 cells adapted to nanomolar concentrations of folate. J. Membr. Biol. 101: 247-258. Chanarin, L, Mollin, D. L. & Anderson, B. B. (1958) The clearance from the plasma of folie acid injected intravenously in normal subjects and patients with megaloblastic anaemia. Br. I. Haematol. 4: 435-446. McMartin, K. E., Virayotha, V. &. Tephly, T. R. (1981) Highpressure liquid chromatography separation and determination of rat liver folates. Arch. Biochem. Biophys. 209: 127-136. McMartin, K. E. (1984) Increased urinary folate excretion and decreased plasma folate levels in the rat after acute ethanol treatment. Alcohol. Clin. Exp. Res. 8: 172-178. McMartin, K. E., Collins, T. D., Eisenga, B. H. & Bhandari, S. D. (1990) Increased folate uptake prevents dietary development of folate deficiency in the rat brain. FASEB J. 4: A673 (abs.). Yamada, S., Wilson, I. S. & Lieber, C. S. (1985) The effects of ethanol and diet on hepatic and serum yglutamyltranspeptidase activities in rats. J. Nutr. 115: 1285-1290. Thenen, S. W. (1978) Blood and liver folacin activity, formiminoglutamic acid excretion, growth and hematology in guinea pigs fed a folacin-deficient diet with and without sulfonamides. J. Nutr. 108: 836-842. Home, D. W. & Patterson, D. (1988) Lactobacillus casei microbiological assay of folie acid derivatives in 96-well microtiter plates. Clin. Chem. 34: 2357-2359. Eisenga, B. H., Collins, T. D. & McMartin, K. E. (1989) Effects of acute ethanol on urinary excretion of 5methyltetrahydrofolic acid and folate derivatives in the rat. J. Nutr. 119: 1498-1505. Zamierowski, M. M. & Wagner, C. (1977) Effect of folacin deficiency on folacin-binding proteins in the rat. J, Nutr. 107: 1937-1945. Hörne,D. H., Briggs, W. T. & Wagner, C. (1979) Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol. Arch. Biochem. Biophys. 196: 557-565. Ward, G. J. & Nixon, P. F. (1990) Modulation of pteroylpolyglutamate concentration and length in response to altered folate nutrition in a comprehensive range of rat tissues, f. Nutr. 120: 476-484. Spector, R. (1989) Micronutrient homeostasis in mammalian brain and cerebrospinal fluid. J. Neurochem. 53: 1667-1674. Botez, M. I. & Bachevalier, J. (1981) The blood-brain barrier and folate deficiency. Am. J. Clin. Nutr. 34: 1725-1730. Young, S. N. & Ghadirian, A. M. (1989) Folie acid and psychopathology. Prog. Neuro-Psychopharmacol. & Biol. Psy chiatry 13: 841-863. Rothberg, K. G., Ying, Y., Kolhouse, J. F., Kamen, B. A. & Anderson, R.G.W. (1990) The glycophospholipid-linked folate receptor internalizes folate without entering the clathrincoated pit endocytic pathway. J. Cell Biol. 110: 637-649. Spector, R. & Lorenzo, A. V. (1975) Folate transport by the choroid plexus in vitro. Science (Washington, DC) 187: 540-542. Selhub, }. & Franklin, W. A. (1984) The folate-binding protein of rat kidney. Purification, properties, and cellular distribu tion. J. Biol. Chem. 259: 6601-6606. DaCosta, M. & Rothenberg, S. P. (1988) Characterization of the folate-binding proteins associated with the plasma mem brane of rat liver. Biochim. Biophys. Acta 939: 533-541. McGuire, J. J. & Berlino, I. R. (1981) Enzymatic synthesis and function of folylpolyglutamates. Mol. Cell. Biochem. 38: 19-48. Eisenga, B. H., Collins, T. D. & McMartin, K. E. (1988) Differ ential effects of acute ethanol on urinary excretion of folate derivatives in the rat. J. Pharmacol. Exp. Ther. 248: 916-922.
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liver was [3H]PteGlu and the rest of the label occurred as H4PteGlu derivatives only. The present study was designed to assess the effects of folate deficiency on the in vivo incorporation of folate, i.e., on the end result of the processes of uptake and retention in the cell. Conclusions cannot be made in terms of specific cellular metabolic changes due to folate deficiency. However, we can conclude that folate deficiency alters the tissue incorporation of 3H-label from folie acid, with different effects in different tissues.
IN FOLATE DEFICIENCY
Metabolism
Incorporation of 3H-Label from Folie Acid is Tissue-Dependent in Folate-Deficient Rats1»2 BERNARD
H. EISENGA,
TIMOTHY D. COLLINS AND KENNETH
E. McMARTIN3
Most studies in animals (6-8) have shown little (10-20%) or no decrease in brain folate content after consumption of folate-deficient diets for 4-11 mo, at which times hepatic folate levels were less than half of those of control animals. In contrast, Hakim et al. (9) observed marked depletion of both brain and liver folate concentrations in rats fed folate-deficient diets for 70 d. These differences have probably resulted from the different folate-deficient diets used, which probably contained various small but physiologically significant amounts of folate (2). Folate homeostasis includes several adaptive mech anisms that would conserve folate and help to cir cumvent the development of tissue folate depletion. The diminished urinary excretion of folate in states of decreased folate intake would result in greater body retention of folate (1). Maintenance of tissue folate stores could also result from increased absorption and/or distribution from exogenous dietary sources (10). Redistribution of folate from the liver is not a likely mechanism (11), but increased uptake and re tention of folate by individual organs could occur in states of reduced folate intake. Increased uptake of folate occurs in vitro in studies with cultured cells exposed to media containing low levels of folate (12, 13), although no studies have reported such changes in vivo. A more efficient incorporation of plasma folate by tissues would contribute to a greater body retention of folate and to more rapid clearance of exogenous folate from the plasma in folate-deficient
ABSTRACT To study the tissue-specificity of folate deficiency, male Sprague-Dawley rats were fed folatereplete or folate-deficient diets with and without sulfonamide for 16 wk, and then injected with |3H]folic acid (1.5 nmol/kg). Rats were killed after 24 h, and the blood, urine and various organs were prepared for analysis of endogenous and ^H-labeled folate. Endog enous folate levels decreased due to folate deficiency to the greatest extent in the urine and plasma, followed by liver, kidney and other tissues (spleen, testis, lung and intestine), but no decrease was noted in the brain. Of all tissues of folate-deficient rats, the brain showed the greatest increase in incorporation of 3H-label from folate relative to folate-replete rats, with the largest effect in rats that were most deficient in plasma folate. Incorpo ration of label was increased due to folate deficiency in a number of tissues, with an inverse correlation with the tissue folate concentration. In contrast, hepatic [3Hlfolate incorporation was lower in folate-deficient rats than in folate-replete rats, with a direct correlation be tween endogenous folate concentration and the incorpo ration of labeled folate. These results show that the brain and other organs adapt to the development of folate deficiency because of greater incorporation of folate from exogenous sources. The lower incorporation by the liver of folate-deficient rats may result from the greater incorporation by other tissues. J. Nutr. 122: 977-985, 1992. INDEXING KEY WORDS:
•folate deficiency •rats •brain •folate uptake •liuer
Previous studies of animals fed folate-deficient diets have shown a differential loss of folate among various tissues (1, 2). Folate depletion has been shown to occur in a sequential manner (3, 4), being rapidly evident in urine by as early as 2 wk (1) and in plasma by 4 wk of dietary treatment (3). Depletion of folates in tissues such as the liver and kidney generally re quires consumption of a folate-deficient diet for about 2-4 mo (1, 5). In comparison, the brain has been shown to be relatively resistant to folate depletion. 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
'Supported
in part by U.S. Public Health Service grant RO1-
AA05308. 2Presented in preliminary
form at the annual meeting of the
Federation of American Societies for Experimental Biology, April 1990, Washington, DC [McMartin, K. E., Collins, T. D., Eisenga, B. H. & Bhandari, S. D. (1990) Increased folate uptake prevents dietary development of folate deficiency in the rat brain. FASEB J. 4: A673 labs.)]. 3To whom correspondence and reprint requests should be ad dressed.
Received 18 July 1991. Accepted 22 November 977
1991.
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Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, LA 71130-3932
978
EISENGA ET AL.
subjects (14). Differences in the degree to which various tissues adapt to dietary folate insufficiency could be related to the differential loss of folate among tissues. The present study was designed to examine the ability of various tissues of control and folate-deficient animals to incorporate [3H]folate from exogenous
sources.
Animals and reagents. Male Sprague-Dawley rats weighing 125-150 g were obtained from HarÃ-an Sprague Dawley (Houston, TX) and quarantined for 14 d, during which time they were fed a nonpurified diet (Purina Laboratory Chow #5001, Ralston-Purina, St. Louis, MO). Subsequently the rats were housed in wire-mesh cages with a daily light cycle from 0600-1800 h and controlled temperature (23 ±TC) and humidity (45-55%). These studies were approved by the Animal Resources Advisory Committee of Louisiana State University Medical Center in Shreveport. [3',5',7,9-3H]Folic acid (PteGlu)4 (1.5 x IO12 Bq/ mmol) was purchased from Moravek Biochemicals (Brea, CA). Radiochemical purity was determined to be that supplied by Moravek (99%) by HPLC analysis (15, 16). Lactobacillus casei (ATCC 7469a) was ob tained from the American Type Culture Collection (Rockville, MD). Folie Acid Casei Assay medium and Lactobacilli broth AOAC were obtained from Difco Laboratories (Detroit, MI). Complete Counting Cocktail 3a70B was purchased from Research Products International (Mount Prospect, IL). Econofluor-2 and Protosol were purchased from NEN Research Products, Du Pont (Boston, MA). All other reagents used in these investigations were from com mercial sources. Experimental diets. A preliminary study of tissue incorporation of folate (17) was done in conjunction with previous studies of ethanol and folate deficiency (1). The present study was performed to follow up on the initial results. As such, this study was conducted using the liquid diets as previously described (1). Briefly, rats were fed liquid diets obtained from Dyets (Bethlehem, PA) with an energy distribution of 35% fat, 18% protein and 47% carbohydrate. A thorough description of the diet composition has been reported by Yamada et al. (18).5 Succinylsulfathiazole (3 g/4.2 MJ) was added to two of the diets to reduce the production of folate by intestinal bacteria (19). After quarantine, rats were acclimated for 3 d to the control (folate-containing) diet. On d 1 of wk 1, rats were divided into four groups and fed the following experi mental diets: C (control diet with folate in the vi tamin mix); CS (control diet plus sulfonamide); FD (folate-deficient diet, no folate in the vitamin mix); FDS (folate-deficient diet plus sulfonamide). The folate contents of the diets, determined by L. casei
power, followed by 5 s at 100% power (Tissumizer, Tekmar, Cincinnati, OH) and then adjusted to a 25% (wt/v) homogenate with the addition of 1.5 mol/L 2-mercaptoethanol. Feces were weighed and boiled in 10 volumes of 1.5 mol/L 2-mercaptoethanol and then homogenized. The outer layer of the carcass (skin and hair) was removed and the remainder was weighed and prepared as a 33% (wt/v) homogenate with deionized water (Millipore, Bedford, MA) using a blender. Aliquots (10 mL) were removed and 2mercaptoethanol was added to produce a 25% wt/v mixture in 1.5 mol/L 2-mercaptoethanol. These homogenates were then heated for 5 min at 95°Cand rehomogenized. Tissue and fecal homogenates were centrifuged at 15,000 x g for 20 min at 4°C.Supernatants were used for analysis of endogenous and la beled folate content. All samples were stored at -20°C
4Abbreviations used: C, control diet; CS, control diet with sul fonamide; FD, folate-deficient diet; FDS, folate-deficient diet with sulfonamide; r^PteGlu, tetrahydrofolic acid (and its derivatives]; PteGlu, folie acid. •"Controldiets were the same as the Liquid B diet of Yamada et al. (18| with the following exceptions: Î)vitamin-free casein was used rather than edible casein,- 2} xanthum gum at 3.0 ug/4.2 Mf was substituted for sodium carrageenate; 3| vitamin mix provided 200 ug of menadione sodium bisulfite per 4.2 MJ diet instead of 25 \ig; 4) mineral mix provided (g/kg mix); manganous sulfate, 4.6, and ferrous sulfate, 4.95, as replacement for manganous carbonate and ferrous citrate.
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MATERIALS AND METHODS
assay, were 910 and 42 nmol (0.43 and 0.02 mg) folate/4.2 MJ for the control and folate-deficient diets, respectively. Tissue incorporation of labeled folate. Rats were fed the experimental diets for 16 wk and then injected intraperitoneally with a solution containing [3H]PteGlu in a dose of 1.5 nmol/kg body wt. In a previous study of rats injected with a tracer dose of [3H]PteGlu (15), the distribution of 3H-labeled folate and of endogenous folate forms in the liver (converted to the monoglutamyl forms before HPLC analysis) were similar at 24 h after dosing. These data suggest that, by 24 h, incorporation of label into the onecarbon tetrahydrofolate derivatives in tissues had oc curred. As such, the tissues in the present study were similarly collected to assess the incorporation of 3H-label from folie acid. Animals were placed into metabolic chambers that permitted the complete separation of urine and feces. Urine was directed into beakers containing 0.15 mL of neat 2mercaptoethanol (adjusted after collection at 6-h in tervals to 0.05 mL/mL urine, equal to 0.7 mol/L). At the end of 24 h, rats were anesthetized and exsangui nated via abdominal aortic puncture. The blood was centrifuged and plasma was preserved with 1.5 mol/L 2-mercaptoethanol (16). Organs (liver, kidneys, brain, testes, lungs, spleen, jejunum and ileum) were rapidly removed, weighed, minced and boiled in 1.5 mol/L 2-mercaptoethanol (2 mL/g wet tissue) (15). All tissue samples (at 4°C)were homogenized for 30 s at 30%
TISSUE FOLATE INCORPORATION
the various significance. group mean enous folate tissues were PHARM/PCS Philadelphia, as the level
groups, with P < 0.05 as the level of Values cited in the text represent the ±SEM. Individual data points of endog level and of 3H-label content for various analyzed by linear regression using the program (Microcomputer Specialists, PA) to test for correlation, with P < 0.05 of significance.
RESULTS In our previous study (1) using these liquid diets, neither the lack of folate nor the presence of sulfonamide in the diet affected body weight gain or food consumption for 12 wk. Similar results were observed in this group of rats fed these diets for 16 wk. Body weights during wk 15 were 359 ±3, 371 ±6, 361 ±8 and 360 ±4 g for rats fed diets C, FD, CS and FDS, respectively. Similarly, mean daily food consump tions during wk 14 were 58 ±2, 58 ±1, 60 ±0 and 57 ±2 mL of diet, respectively (1 mL diet provides 4.2 KJ). Daily folate consumption was therefore -150 nmol/kg body wt for rats fed the control diets and ~7 nmol/kg body wt for rats fed the folate-deficient diets. Previous studies with these diets (1) showed that depletion of urinary folates occurred within as little as 14 d. Subsequently, the folate-deficient diets produced significant reductions in plasma folate levels by 4 wk (the first collection period) and in tissue folate content (RBC, liver and kidney) by 8 wk.
979
These decreases were marked (70-95%) in rats fed the folate-deficient diet containing sulfonamide,- in rats fed the folate-deficient diet without sulfonamide, folate depletion occurred more slowly and to a lesser extent. In the latter rats, however, tissues were signif icantly folate depleted by 12 wk. Endogenous folate concentrations were monitored at 2-4-wk intervals in the present study and showed a temporal progression to lower folate levels similar to that previously ob served (1). Because the purpose of this study was to examine how folate deficiency would affect the incor poration of [3H]folate from exogenous sources, rats were fed the diets for 16 wk before [3H]PteGlu dosing to ensure that a severe folate deficiency was present. The data in Figures 1-3 confirm that endogenous folate concentrations were decreased by 16 wk. Data in Figure 1 show the plasma folate concentra tions 24 h after administration of a tracer dose of [3H]PteGlu. Endogenous plasma folate was signifi cantly lower in rats chronically fed folate-deficient diets. This reduction was exacerbated by the addition of sulfonamide and was similar to results previously published (1, 19). There were no differences in the plasma retention of labeled folate derivatives within corresponding diet groups (C vs. FD, CS vs. FDS). A tendency towards reduction due to folate deficiency was evident, but was significant only between control (C) and folate-deficient sulfonamide diet groups (FDS). Figure 2 shows data obtained from analysis of liver folate concentrations. The incorporation of labeled folate derivatives in the liver was very similar to that seen in the plasma, with a significant difference only between control and folate-deficient sulfonamide diet groups. In contrast to the results in the liver and the plasma, significantly greater incorporation of 3H-label was seen (Fig. 3) in the kidney of rats fed folatedeficient sulfonamide diets (CS vs. FDS). Other tissues (jejunum, ileum, lung, testis and spleen) showed the same pattern of dietary effects, i.e., an increase in the incorporation of 3H-label in sulfonamide-treated, folate-deficient rats, as was ob served in the kidney (data not shown). As is indicated in Figure 4, chronic diet treatment did not significantly lower endogenous brain folate concentration. Yet, the incorporation of labeled folate by the brain was significantly higher in rats fed folatedeficient diets. This effect was noted for both groups of diets (C vs. FD, CS vs. FDS) and was further augmented by the addition of sulfonamide to the diet (FD vs. FDS). As seen in Table 1, there were no differences among diet groups in either the urinary or fecal ex cretion of 3H-label, suggesting that folate deficiency does not affect excretion within 24 h of exogenous folate administration. Endogenous urinary folate ex cretion was, however, markedly reduced in 24-h urine collections at 16 wk (7.9 ±2.5, 0.9 ±0.5, 2.4 ±0.5 and 0.2 ±0.1 nmol/24 h for C, FD, CS and FDS diets,
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until analyzed. Approximately l g of skin (containing hair) was prepared by cutting random samples from throughout the outer layer and was mixed with 15 volumes of Protosol. This mixture was incubated at 37°Cuntil the tissue was dissolved. Determination of tissue folate and 3H-label con tent. Endogenous folate content was determined using a Lactobacillus casei microtiter growth assay (20). For measurement of total folate content, tissue and fecal samples were pretreated with hog kidney folylpolyglutamate hydrolase (15) and then analyzed with L. casei. All tissues were analyzed for endog enous folate content except the carcass and the skin. The quantities of individual folate derivatives were determined in urine samples by HPLC as previously described (16, 21). Fractions eluted from HPLC were analyzed for folate derivatives by L. casei assay and for 3H-label by scintillation analysis. Total radioactivity was determined by liquid scin tillation analysis of 10-uL aliquots of urine or 100-uL aliquots of all other samples in 4 mL of 3a70B counting cocktail. Radioactivity in the skin was de termined by adding 2 mL of the skin-Protosol mixture to 10 mL of Econofluor-2. Statistics. Group data were analyzed by ANOVA using a general linear models procedure with Dun can's test to test for differences among the means of
IN FOLATE DEFICIENCY
EISENGA ET AL.
980
3H- Ldb.f.d
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FIGURE l Endogenous and 3H-labeled folate levels in the plasma of rats fed control and folate-deficient diets. Bars represent the group mean, expressed in nmol folate or pmol folate equivalence/L plasma, ±SEM (n = 6). Bars with letters in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folate-deficient diet with sulfonamide.
respectively), as previously reported (1). The HPLC analysis of the urine samples in the present study (Table 2) showed that the 3H-labeled products were primarily a nonfolate compound (21), 5-formiminotetrahydrofoÃ-ate and 5- and 10-formyl-tetrahydrofolates. These folate-derived compounds made up >75% of the total 3H-label in the urine. There were no differences among the treatment groups in terms of distribution of the derivatives (Table 2). These folate-derived compounds are not well reabsorbed by the kidney, as was shown previously (21). No effect of folate deficiency on total 3H-labeled excretion in the urine was seen in the present study, probably because the 3H-labeled products were those forms that are not conserved by the kidney.
As noted for tissue 3H-label content (Fig. 2), the hepatic recovery of labeled folate (expressed as the percentage of dose in Table 1) was significantly lower in rats fed folate-deficient sulfonamide diets. In con trast to the liver, the recovery of labeled folate in the kidney and the carcass was significantly greater in rats fed folate-deficient sulfonamide diets. Recovery of 3H-label in the brain and other organs showed a more complex pattern among the diets. In these tis sues, labeled folate recovery was significantly greater in rats fed folate-deficient diets (FD vs. C). The ad dition of sulfonamide to folate-deficient diets further increased the recovery of labeled folate (FDS vs. FD). Figure 5 shows the relationship between endog enous folate concentrations and the resulting incorpo-
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fo £3 o.400-aTbTibT280-200\
TISSUE FOLATE INCORPORATION IN FOLATE DEFICIENCY
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FIGURE 3 Endogenous folate level and ^H-labeled folate incorporation in the kidney of rats fed control and folate-deficient diets. Bars represent the group mean value, expressed in nmol folate or pmol folate equivalence/g wet wt, ±SEM(n - 6). Bars with letters above in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folatedeficient diet with sulfonamide.
ration of 3H-label in specific tissues. Except for the liver and the brain, the degree of labeled folate incor poration in tissue was inversely related to the degree of folate depletion, i.e., the more severe the folate deficiency, the greater the incorporation of labeled folate. In contrast, the liver showed a significant positive correlation between endogenous folate con centration and incorporation of labeled folate. Finally, because endogenous folate concentration in the brain did not change (Fig. 4), there was no statistically significant correlation between folate content and the incorporation from labeled folate. In all other cases, these correlations were statistically significant (P < 0.05).
DISCUSSION Chronic folate deficiency occurs by a time-ordered depletion of tissue folate stores. Certain tissues are particularly vulnerable to diminished folate intake, whereas other tissues require a long time for de velopment of folate depletion. As noted previously by others (6, 7), even after 16 wk of consumption of a folate-deficient plus sulfonamide diet, the brain was resistant to the development of folate deficiency. The present study has suggested that this resistance was probably due to an increased incorporation of available folate. Although the brain did not become folate deficient per se, labeled folate incorporation in
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FIGURE 4 Endogenous folate level and 3H-labeled folate incorporation in the brain of rats fed control and folate-deficient diets. Bars represent the group mean value, expressed in nmol folate or pmol folate equivalence/g wet wt, ±SEM(n = 6). Bars with letters above in common were not significantly different from each other with a protection level of 0.95 over all comparisons. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folatedeficient diet with sulfonamide.
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*-*4-o.baTaTa
*~*O52s 3]. There were no significant differences among values for diet groups for any of the derivatives. Diet abbreviations: C, control diet; FD, folate-deficient diet; CS, control diet with sulfonamide; FDS, folate-deficient diet with sulfonamide. 2This derivative eluted before any of the standard folate derivatives and did not support the growth of L. casei. It was not a folate form and has been termed nonfolate (21). Nonfolate25-Formimino-H4PteGlu5-
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Liver Kidney Brain Organs2 Carcass3Total15.43
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TISSUE FOLATE INCORPORATION
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14
ENDOGENOUS FOLATE LEVEL (nmol/g)
FIGURE 5 Correlations between endogenous folate levels (abscissas) and incorporation of 3H-labeled folate (ordinales) in tissues of rats fed control and folate-deficient diets. Each point represenis the values from one rat, with values from all diet groups included. The correlations were statistically significant for all tissues (except brain), P < 0.05. (Liver: y = 2.3x + 1466, r = +0.51; kidney: y = -3.9x + 1474, r = -0.45; jejunum: y = -9.8x + 623, r = -0.59; testis: y = -4.Ox + 146, r = -0.43; lung: y -5.6x + 194, r = -0.60.)
could have been taken up by the mesenteric circula tion, placing the PteGlu directly into the portal circu lation (whereas that entering as small amounts via the enterocyte would be 5-CH3-H4PteGlu). The liver seems to have separate systems for the uptake of PteGlu and 5-CH3-H4PteGlu (23) with a higher Km for PteGlu. The limiting factor for hepatic uptake of PteGlu from the portal circulation might then be the capacity of the carrier for PteGlu. The PteGlu passing through the liver would reach the other tissues, where uptake would be controlled by the relative depletion of tissue folate stores.
In the folate-deficient rats treated with sulfonamide, the plasma retained less of the labeled folate. The decrease in plasma retention might have resulted from the increased tissue incorporation of 3H-labeled folate. Therefore, in states of folate deficiency, dietary or exogenous folate seemed to be shunted from the liver, to be preferentially taken up and retained by other tissues, especially the brain. These studies support the report by Weir et al. (11), who suggested that tissue folate levels are not regulated by supply from liver stores during states of folate deprivation. In their study, the livers of deficient rats did not show
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plasma membrane and retention within the cell. Intracellular uptake of folate probably involves seques tration by the folate-binding protein and transport across the membrane (28). A highly specific folatebinding protein has been identified in the choroid plexus of the brain (29) and in other tissues (30, 31). Recent evidence has shown that folate uptake in certain cells is related to the folate-binding protein activity in the cell membrane (12, 13). Cells cultured in folate-deficient medium have increased amounts of the folate-binding protein (13) and enhanced uptake of labeled folate (12). Although there have been no studies linking these changes in vivo, cellular adaptive changes during states of folate deficiency (increased folate-binding protein or enhanced trans port) might explain the increased amount of labeled folate found in the tissues of these animals. Alterna tively, the increased uptake could have resulted from a change in the intracellular compartmentation of folate. Intracellular folate exists mostly in the protein-bound state and this is not as rapidly depleted during folate deficiency as is unbound folate (22). Therefore, a decrease in the unbound fraction would tend to increase folate uptake from extracellular sources. As an alternative to increased cellular uptake, the increased incorporation of exogenous folate by various tissues could result from increased retention of cellular folate. In general, folates are retained in the cell by conversion to the polyglutamate form by the enzyme folylpolyglutamate synthetase (32). Recent research (24) has shown that an increase in folate glutamate chain length occurs in tissues of folatedeficient rats. An increase in polyglutamylation may explain the increased tissue incorporation of labeled folate in the folate-deficient tissues in the present study. However, because the tissue samples were not properly prepared for analysis of polyglutamate chain length (they were treated initially with folyl polyglutamate hydrolase to allow for measurement of total folate content by L. casei], the respective contri butions of increased uptake vs. increased retention by polyglutamylation towards the increased incorpo ration cannot be determined from these results. In the present study, the amount of 3H-labeled folate incorporation in tissues was indirectly deter mined, i.e., whether the label in the tissue samples was PteGlu or another folate form was not directly measured (except in the urine). Nevertheless, the 3H-label in the tissues at 24 h must have resulted from tissue incorporation of a labeled dose of folie acid. The purity of the [3H]PteGlu was assessed before dosing, so that the initial label was entirely folie acid. Hence, the 3H-label in the tissue resulted from an initial uptake of PteGlu, followed by retention of folie acid or its metabolite(s). For instance, in a previous study (33), at l h after intravenous dosing with [3H]PteGlu, >82% of the 3H-label recovered in the
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an accelerated excretion of folate via the enterohepatic circulation (for distribution to other tissues) as compared with controls,- thus the liver did not act as a reservoir of folate. Previous studies of animals fed folate-deficient diets showed many variations in effects on brain folate content. Short-term treatment (30 d) of rats with folate-deficient sulfonamide-containing diets produced no reduction in brain folate content (24). In rats fed a folate-deficient, sulfonamide-containing diet for 9.5 mo, Fehling et al. (7) observed only a 16% (but significant) reduction in brain folate content. Similar rats in our studies showed about a 20% decrease in brain folate content (not significant). In rats fed folatedeficient diets without the addition of sulfonamide for 11 mo, a significant reduction in liver folate, but not in brain folate levels, was observed (6). Other studies in rats fed folate-deficient, sulfonamidesupplemented diets for periods of 4 mo (8) and 70 d (9) showed significant reductions in both hepatic and brain folate concentrations. One explanation for these differences could be that those studies that showed large reductions in brain folate concentrations after relatively short feeding regimens also showed a signif icant weight loss in folate-deficient animals. The weight loss [which did not occur in the present study, nor in other studies (6, 7)] could enhance the re duction of brain folate. The most probable expla nation for the lack of brain folate depletion in some studies could be that the diets were not "sufficiently deficient." In a recent definitive dietary study, Walzern and Clifford (2) showed that casein-based, folate-deficient diets (as in the present study) con tained a small amount of folate (intakes of -1 (¿g/d) that allowed normal weight gain, but did not produce maximal tissue folate depletion. Instead they pro posed the use of defined amino acid diets, from which severe tissue folate deficiency, reduced weight gain, and hématologie effects were produced in 5 wk. Un fortunately, brain folate content was not reported in their studies. Sparing of the brain from deficiency of needed sub strates or other nutrients is not a new concept (25). In contrast to many substrates, there is only a limited blood-brain barrier for folate (26). Folate may be a vital compound for normal brain function, because studies have shown correlations between states of folate deficiency and different types of dysfunctions of the central nervous system (27). These studies have shown that, in many instances, correction of the folate deficiency improves the mental status of the subject (27). It seems plausible that there should be adaptive mechanisms during states of folate defi ciency to prevent the loss of folate from the brain (as well as other vital tissues). These adaptations most likely involve changes in the incorporation of folate by the tissues, which would comprise two processes: uptake across the
TISSUE FOLATE INCORPORATION
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ACKNOWLEDGMENTS Special thanks go to Tammy Fortney and Tim Tyler for their technical assistance, to Julie Woods, Michelle Cooper and James Gulledge for their assis tance in the care and feeding of the animals, and to Sharon Farrar for preparation of this manuscript.
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LITERATURECITED 21. 1. McMartin, K. E., Collins, T. D., Eisenga, B. H., Fortney, T., Bates, W. R. & Bairnsfather, L. (1989) Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat. f. Nutr. 119: 1490-1497. 2. Walzern, R. L. & Clifford, A. f. (1988) Folate deficiency in rats fed diets containing free amino acids or intact proteins. J. Nutr. 118: 1089-1096. 3. Siddons, R. C. (1974) Experimental nutritional folate defi ciency in the baboon. Br. J. Nutr. 32: 579-587. 4. Herbert, V. (1962) Experimental nutritional folate deficiency in man. Trans. Assoc. Am. Physicians 75: 307-320. 5. Herbert, V. (1990) Development of human folate deficiency. In: Folie Acid Metabolism in Health and Disease (Picciano, M. F., Stokstad, E.L.R. & Gregory, J. F., eds.), pp. 195-210. Wiley-Liss, New York, NY. 6. Thomson, A. D., Frank, O., DeAngelis, B. & Baker, H. (1972) Thiamin depletion induced by dietary folate deficiency in rats. Nutr. Rep. Int. 6: 107-110. 7. Fehling, C., Jagerstad, M., Lindstrand, K. & Elmquist, D. (1976) Reduction of folate levels in the rat: difference in depletion between the central and the peripheral nervous system. Z. Ernaehrungswiss. 15: 1-8. 8. Akesson, B., Fehling, C., Jagerstad, M. & Stenram, U. (1982) Effect of experimental folate deficiency on lipid metabolism in liver and brain. Br. J. Nutr. 47: 505-520. 9. Hakim, A. M., Arrieta, M. J., Cooper, B. A. & Pappius, H. M. (1984) Effect of folate deficiency on local cerebral glucose utilization in the rat. J. Neurochem. 42: 1582-1587. 10. Whitehead, V. M. (1986) Pharmacokinetics and physiological disposition of folate and its derivatives. In: Folates and Pterins (Blakley, R. L. & Whitehead, V. M., eds.), vol. 3, pp. 177-205. John Wiley and Sons, New York, NY. 11. Weir, D. G., McGing, P. G. & Scott, J. M. (1985) Folate metabolism, the enterohepatic tirculation and alcohol. Biochem. Pharmacol. 34: 1-7. 12. Kamen, B. A. & Capdevila, A. (1986) Receptor-mediated folate accumulation is regulated by the cellular folate content. Proc. Nati. Acad. Sci. U.S.A. 83: 5983-5987. 13. Henderson, G. B., Tsuji, J. M. & Kumar, H. P. (1988) Mediated uptake of folate by a high-affinity binding protein in sublines
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of L1210 cells adapted to nanomolar concentrations of folate. J. Membr. Biol. 101: 247-258. Chanarin, L, Mollin, D. L. & Anderson, B. B. (1958) The clearance from the plasma of folie acid injected intravenously in normal subjects and patients with megaloblastic anaemia. Br. I. Haematol. 4: 435-446. McMartin, K. E., Virayotha, V. &. Tephly, T. R. (1981) Highpressure liquid chromatography separation and determination of rat liver folates. Arch. Biochem. Biophys. 209: 127-136. McMartin, K. E. (1984) Increased urinary folate excretion and decreased plasma folate levels in the rat after acute ethanol treatment. Alcohol. Clin. Exp. Res. 8: 172-178. McMartin, K. E., Collins, T. D., Eisenga, B. H. & Bhandari, S. D. (1990) Increased folate uptake prevents dietary development of folate deficiency in the rat brain. FASEB J. 4: A673 (abs.). Yamada, S., Wilson, I. S. & Lieber, C. S. (1985) The effects of ethanol and diet on hepatic and serum yglutamyltranspeptidase activities in rats. J. Nutr. 115: 1285-1290. Thenen, S. W. (1978) Blood and liver folacin activity, formiminoglutamic acid excretion, growth and hematology in guinea pigs fed a folacin-deficient diet with and without sulfonamides. J. Nutr. 108: 836-842. Home, D. W. & Patterson, D. (1988) Lactobacillus casei microbiological assay of folie acid derivatives in 96-well microtiter plates. Clin. Chem. 34: 2357-2359. Eisenga, B. H., Collins, T. D. & McMartin, K. E. (1989) Effects of acute ethanol on urinary excretion of 5methyltetrahydrofolic acid and folate derivatives in the rat. J. Nutr. 119: 1498-1505. Zamierowski, M. M. & Wagner, C. (1977) Effect of folacin deficiency on folacin-binding proteins in the rat. J, Nutr. 107: 1937-1945. Hörne,D. H., Briggs, W. T. & Wagner, C. (1979) Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol. Arch. Biochem. Biophys. 196: 557-565. Ward, G. J. & Nixon, P. F. (1990) Modulation of pteroylpolyglutamate concentration and length in response to altered folate nutrition in a comprehensive range of rat tissues, f. Nutr. 120: 476-484. Spector, R. (1989) Micronutrient homeostasis in mammalian brain and cerebrospinal fluid. J. Neurochem. 53: 1667-1674. Botez, M. I. & Bachevalier, J. (1981) The blood-brain barrier and folate deficiency. Am. J. Clin. Nutr. 34: 1725-1730. Young, S. N. & Ghadirian, A. M. (1989) Folie acid and psychopathology. Prog. Neuro-Psychopharmacol. & Biol. Psy chiatry 13: 841-863. Rothberg, K. G., Ying, Y., Kolhouse, J. F., Kamen, B. A. & Anderson, R.G.W. (1990) The glycophospholipid-linked folate receptor internalizes folate without entering the clathrincoated pit endocytic pathway. J. Cell Biol. 110: 637-649. Spector, R. & Lorenzo, A. V. (1975) Folate transport by the choroid plexus in vitro. Science (Washington, DC) 187: 540-542. Selhub, }. & Franklin, W. A. (1984) The folate-binding protein of rat kidney. Purification, properties, and cellular distribu tion. J. Biol. Chem. 259: 6601-6606. DaCosta, M. & Rothenberg, S. P. (1988) Characterization of the folate-binding proteins associated with the plasma mem brane of rat liver. Biochim. Biophys. Acta 939: 533-541. McGuire, J. J. & Berlino, I. R. (1981) Enzymatic synthesis and function of folylpolyglutamates. Mol. Cell. Biochem. 38: 19-48. Eisenga, B. H., Collins, T. D. & McMartin, K. E. (1988) Differ ential effects of acute ethanol on urinary excretion of folate derivatives in the rat. J. Pharmacol. Exp. Ther. 248: 916-922.
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liver was [3H]PteGlu and the rest of the label occurred as H4PteGlu derivatives only. The present study was designed to assess the effects of folate deficiency on the in vivo incorporation of folate, i.e., on the end result of the processes of uptake and retention in the cell. Conclusions cannot be made in terms of specific cellular metabolic changes due to folate deficiency. However, we can conclude that folate deficiency alters the tissue incorporation of 3H-label from folie acid, with different effects in different tissues.
IN FOLATE DEFICIENCY
