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The Journal of Nutrition Vol. 127 No. 11 November 1997, pp. 2173-2178
Copyright ©1997 by the American Society for Nutritional Sciences

Ascorbic Acid Deficiency Decreases the Renal Level of Kidney Fatty Acid-Binding Protein by Lowering the alpha 2u-Globulin Gene Expression in Liver in Scurvy-Prone ODS Rats1,2

Saiko Ikeda, Masaharu Takasu, Tsukasa Matsuda*, Atsushi Kakinuma, and Fumihiko Horio3

Laboratory of Nutritional Biochemistry and * Laboratory of Molecular Bioregulation, Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

The evidence for the role of ascorbic acid in gene expression or protein synthesis in vivo is limited. To investigate this role of ascorbic acid, we surveyed proteins whose tissue levels are changed by ascorbic acid deficiency by using ODS rats with a hereditary defect in ascorbic acid biosynthesis. Male ODS rats (7 wk old, body weight ~130 g) were fed a basal diet containing ascorbic acid (300 mg/kg diet) or an ascorbic acid-free diet for 14 d. Ascorbic acid deficiency decreased a renal protein with an apparent molecular mass of 17 kDa. The amino-terminal amino acid sequence of 16 residues of this 17-kDa protein was identical to a kidney fatty acid-binding protein known to be generated by proteolytic degradation of alpha 2u-globulin, a major urinary protein of adult male rats. alpha 2u-Globulin is synthesized in liver, secreted into blood and excreted into urine, but partially reabsorbed by renal proximal tubules. It exists in kidney in a proteolytically modified form. Ascorbic acid deficiency lowered the renal level of kidney fatty acid-binding protein to 53% (P < 0.05) and lowered the serum level of alpha 2u-globulin to 52% (P < 0.05) of the level of the control group, but did not affect the amount of alpha 2u-globulin excreted into urine. The hepatic level of alpha 2u-globulin mRNA of the ascorbic acid-deficient rats was significantly lower (30%) than that of the control rats. These results suggest that in male ODS rats, ascorbic acid deficiency decreases the renal level of kidney fatty acid-binding protein by lowering alpha 2u-globulin gene expression in liver.

KEY WORDS: ascorbic acid · kidney fatty acid-binding protein · alpha 2u-globulin · ODS rats

INTRODUCTION

Ascorbic acid is an essential nutrient for humans and certain other mammals that cannot synthesize the vitamin. Ascorbic acid is a cofactor in some enzymatic hydroxylations, including prolyl and lysyl hydroxylation of collagen (Padh 1991), and is known as a potent antioxidant. Recently, it has also been demonstrated that ascorbic acid has a role in regulating gene expression. Studies on cultured cells have revealed that ascorbic acid upregulates the expression of some genes, including type I collagen gene (Kurata et al. 1993), type X collagen gene (Sullivan et al. 1994) and acetylcholine receptor gene (Liu et al. 1993). However, the evidence for the effect of ascorbic acid on in vivo gene expression is limited. In experiments using the ODS rat (genotype, od/od), which is a mutant Wistar rat with a hereditary defect in ascorbic acid biosynthesis, we recently reported that ascorbic acid deficiency lowered serum apolipoprotein A-I concentration through lowering its mRNA level in liver (Ikeda et al. 1996). Expression of several genes may be modulated in vivo by ascorbic acid.

Although guinea pigs have been used as an experimental animal for investigating the physiologic role of ascorbic acid, the effect of ascorbic acid on in vivo gene expression has rarely been reported in this species. Because the structures of various genes and the regulation of their expression have been more widely investigated in rats, the ODS rat is a useful model for investigating the role of ascorbic acid in in vivo gene expression. The ODS rat lacks L-gulono-gamma -lactone oxidase (EC 1.1.3.8), which catalyzes the terminal step of ascorbic acid biosynthesis as a result of a single base mutation in the gene of this enzyme (Kawai et al. 1992). In this study, we analyzed several tissues of ODS rats to identify proteins whose tissue levels are influenced by ascorbic acid deficiency.

We found that ascorbic acid deficiency lowered the renal level of a 17-kDa protein identified as kidney fatty acid-binding protein (FABP).4 The kidney FABP is a proteolytic fragment of alpha 2u-globulin that is synthesized in liver and secreted into urine. To determine how ascorbic acid deficiency causes this phenomenon, we examined the effects of ascorbic acid deficiency on the serum level of alpha 2u-globulin and the hepatic level of alpha 2u-globulin mRNA.

MATERIALS AND METHODS

Animals and diets. Male ODS (od/od) rats, ~6 wk of age, were purchased from Japan Clea (Tokyo, Japan). Male and female Wistar rats, ~8 wk of age, were purchased from Japan SLC (Shizuoka, Japan). They were housed in individual wire screen-bottomed cages in the animal colony of Nagoya University and maintained at 24°C with a 12-h light cycle (lights on from 0800 to 2000 h). Rats were allowed free access to water and a purified diet. The composition of the basal diet is shown in Table 1. The addition of 300 mg ascorbic acid per kilogram diet is enough to achieve maximum growth and prevent the development of scurvy in ODS rats (Horio et al. 1985). ODS rats were fed the basal diet for 7 d before the start of the experiment. In this experiment, rats were killed by decapitation between 1000 and 1100 h, and all procedures were performed in accordance with the Animal Experimentation Guides of Nagoya University.

Table 1. Composition of the basal diet

[View Table]

Experimental procedures. During the experimental period, male ODS rats were fed a basal diet containing 300 mg of ascorbic acid/kg diet (control group) or a diet without ascorbic acid (ascorbic acid-deficient group). Three to four rats from each group were killed on the morning of d 3, 10 and 14 after the start of the experiment. Because the food intake of ascorbic acid-deficient rats began to decrease slightly on d 12, rats in the control group were pair-fed the amount consumed by rats in the ascorbic acid-deficient group from d 12 to 14. Male and female Wistar rats were fed an ascorbic acid-free diet for 7 d.

Blood was collected, from both ODS and Wistar rats, and serum was prepared by centrifugation at 1500 × g for 10 min. Kidneys were homogenized with PBS and centrifuged at 4°C at 12,000 × g for 15 min. Then, the supernatant was centrifuged at 4°C at 100,000 × g for 60 min; the resultant cytosolic fraction was used for identification of the 17-kDa protein or detection of kidney FABP by Western blot analysis. Liver, kidney, adrenal gland, jejunum, spleen, testis, heart, lung and brain were removed from male ODS rats, frozen immediately in liquid nitrogen and stored at -80°C until use for the extraction of RNA and the determination of ascorbic acid concentration. Urine was collected for 24 h from d 13 to 14.

Determination of ascorbic acid concentration. Liver and kidney were homogenized in ice-cold 50 g/L metaphosphoric acid and centrifuged at 1600 × g for 10 min. The ascorbic acid concentration in the supernatant was measured by a modification of the dinitrophenylhydrazine method (Roe and Kuether 1943), in which the oxidation of ascorbic acid was accomplished with 2,6-dichlorophenol indophenol.

Purification of renal 17-kDa protein and determination of its amino-terminal amino acid sequence. The cytosolic fraction of kidney was dialyzed against 0.02 mol/L Tris-HCl (pH 8.0). The dialysate, containing ~4.5 mg of protein, was applied to an HPLC ion-exchange column (TSK gel DEAE-5PW, 7.5 mm × 7.5 cm, TOSO, Tokyo, Japan). Proteins were eluted with a linear NaCl gradient from 0 to 0.5 mol/L in 0.02 mol/L Tris-HCl (pH 8.0) for 30 min at a flow rate of 1.0 mL/min, and monitored by absorption at 280 nm. Proteins in each fraction of HPLC were then separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon transfer membrane, Nihon Millipore, Tokyo, Japan) with a semi-dry electrophoretic transfer cell (Nippon Bio-Rad Laboratories, Tokyo, Japan) according to the manufacture's instructions. Automated Edman amino-terminal amino acid sequence determinations of the 17-kDa protein on PVDF membrane were performed with a gas-phase peptide sequencer (470/120/900, Applied Biosystems, Perkin-Elmer Japan, Chiba, Japan).

Antibody preparation. The total urinary proteins were dialyzed against 0.02 mol/L Tris-HCl (pH 8.0) and applied onto the HPLC ion-exchange column described above. The proteins separated by HPLC were further purified by Sephadex G-75 gel chromatography (Pharmacia Biotech, Tokyo, Japan); the fractions containing a single protein identified as alpha 2u-globulin were freeze-dried. alpha 2u-Globulin (~0.5 mg) dissociated in 0.75 mL of PBS was mixed with an equal volume of Freund's complete adjuvant and emulsified. The emulsion was injected intradermally into a female New Zealand White rabbit (Japan Clea). Three weeks after the first injection, the rabbit received the second intradermal injection of alpha 2u-globulin emulsified with Freund's incomplete adjuvant. Blood was drawn from the rabbit 7 d after the second injection, and the antiserum was used for determination of the levels of kidney FABP and alpha 2u-globulin. In immunoblot analysis of serum, a single band of alpha 2u-globulin (19 kDa) was detected using this antiserum. In the same analysis of renal cytosol, a single band of kidney FABP (17 kDa) and a single band of alpha 2u-globulin (19 kDa) were detected.

Immunoblot analysis. The renal cytosolic fraction (equivalent to 6.25 µg of kidney), serum (1 µL) and urine were subjected to SDS-PAGE on 12.5 g/L acrylamide-gel, and the proteins in the gel were transferred onto PVDF membranes. The filters were blocked for 1.5 h with PBS containing 30 g/L skim-milk powder, and incubated for 1.5 h with a rabbit anti-rat alpha 2u-globulin antiserum that had been diluted 1:500 with PBS containing 0.5 g/L Tween-20 and 10 g/L skim-milk powder. The filter was then washed twice with PBS containing 0.5 g/L Tween-20 and incubated for 1.5 h with 125I-labeled donkey anti-rabbit immunoglobulin G antibody (Amersham, Tokyo, Japan). The filter was washed twice with PBS and autoradiographed; the radioactivities on the bands were quantified with a Bioimage Analyzer System (BAS 2000II, Fuji Photographic Film, Kanagawa, Japan). The amount of alpha 2u-globulin excreted into urine from d 13 to 14 was expressed per 100 grams body weight.

Northern blot analysis. Total RNA was extracted from liver and other tissues by the method of Chomczynski and Sacchi (1987) and subjected to Northern blot analysis. The extracted RNA (20 µg) was separated by electrophoresis on 10 g/L agarose gel containing 66 g/L formaldehyde, 40 mmol/L 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0), 10 mmol/L sodium acetate and 1 mmol/L EDTA. RNA was denatured by heating at 55°C for 15 min in MOPS buffer containing 500 g/L formamide and 66 g/L formaldehyde. The electrophoresis buffer was 40 mmol/L MOPS (pH 7.0) containing 10 mmol/L sodium acetate and 1 mmol/L EDTA. After electrophoresis, RNA was transferred directly onto a nitrocellulose membrane (Hybond N+, Amersham) in 10 × SSC (1 × SSC is 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.0). The membrane was then baked at 80°C for 2 h.

cDNA probes were labeled with [32P]dCTP using a labeling system kit (Megaprime, Amersham). Hybridization with the probe (0.9 MBq/L of 32P-labeled cDNA) was performed overnight at 42°C in a solution containing 500 g/L formamide, 5 × SSC, 5 × Denhardt's solution, 10 g/L SDS, 50 mmol/L sodium phosphate (pH 6.5) and 0.5 g/L denatured salmon sperm DNA. The membrane was washed twice with 2 × SSC containing 1 g/L SDS at room temperature for 15 min, and twice with 0.1 × SSC containing 1 g/L SDS at 55°C for 15 min. The washed membrane was subjected to autoradiography. For quantifying the hepatic levels of alpha 2u-globulin mRNA and apolipoprotein E mRNA, RNA slot blot analysis was performed using 5 µg of total RNA. The baking of the membrane and the hybridization with 32P-labeled cDNA were identical to the process used in Northern blot analysis. The radioactivity on each slot was quantified as described above.

cDNA clones. The cDNA clone for rat alpha 2u-globulin was synthesized using the polymerase chain reaction (PCR). The upstream and downstream primers for alpha 2u-globulin were 5'-CCGGATCCATGGAAGAAG-3' (nucleotide -11 to 7) and 5'-ATGCTCGAGTCCTGGTGA-3' (inverse complement of nucleotide 527-544), respectively (Unterman et al. 1981). One microgram of poly(A)+ RNA from male rat liver was reversely transcribed into cDNA by incubating with 0.25 µg of the downstream primer, 20 mmol/L MgCl2, 200 U M-MLV reverse transcriptase (SUPERSCRIPT II, GIBCO BRL, Life Technologies, Tokyo, Japan), 0.5 mmol/L dNTP, 0.01 mmol/L dithiothreitol, 0.1 mol/L Tris-HCl (pH 8.3) in a final volume of 20 µL at 42°C for 2 h. The two primers (each 0.1 µmol) were added to a standard PCR mixture (final volume, 100 µL) containing the synthesized cDNA as a template. The major reaction product (550 bp) was isolated and its DNA sequence was determined by the dideoxy chain termination method (Sanger et al. 1977).

cDNA clones for mouse beta -actin (Tokunaga et al. 1986) and rat apolipoprotein E (McLean et al. 1983) were kindly provided by K. Hitomi of Nagoya University and J. M. Taylor of Gladstone Foundation Laboratories (San Francisco), respectively.

Statistical analysis. Values in the text are means ± SEM. The number of rats in each group is shown in each legend. When variances of each group were equal, mean values obtained for the control and the ascorbic acid-deficient rats were compared using Student's t test. When variances of each group were unequal, significance of differences was determined using Welch's test (Aspin 1949, Trickett et al. 1956). Differences with a P-value < 0.05 were considered significant.

RESULTS

Effects of ascorbic acid deficiency on body weight gain and tissue ascorbic acid concentration. The initial and the final body weights of the control and the ascorbic acid-deficient rats and their renal and hepatic ascorbic acid concentrations on d 14 are shown in Table 2. The final body weights did not differ in the two groups; throughout the course of experiment, no signs of scurvy were observed in any rats of the ascorbic acid-deficient group. On d 14, the renal and hepatic concentrations of ascorbic acid in the deficient rats were significantly lower than those in the control rats.

Table 2. Initial and final body weights, and renal and hepatic ascorbic acid concentrations on d 14 of the control and the ascorbic acid-deficient rats1

[View Table]

Identification of 17-kDa protein as kidney fatty acid-binding protein. SDS-PAGE analyses were performed on the cytosolic fractions from several tissues, such as liver, kidney, adrenal glands, spleen and brain, of the control and the ascorbic acid-deficient rats. Ascorbic acid deficiency did not affect the levels of any proteins stained with Coomassie Brilliant Blue in these tissues except for kidney (data not shown). In the cytosolic fraction from kidney of the ascorbic acid-deficient rats, the level of a protein with an apparent molecular mass of 17 kDa was markedly lower than that in the control rats (Fig. 1).
Fig. 1. SDS-PAGE analysis of the renal cytosolic fractions of the control and ascorbic acid-deficient rats on d 14. Results from two rats of each group are shown. The renal cytosolic fractions (20 µg of protein) of the control and the ascorbic acid-deficient rats were subjected to SDS-PAGE, and proteins were stained with Coomassie Brilliant Blue.
[View Larger Version of this Image (78K GIF file)]

To identify the 17-kDa protein, the cytosolic fraction from kidney of the control rats was purified on an HPLC ion-exchange column. The elution pattern monitored at 280 nm and the results of SDS-PAGE analyses of the fractions 14-16 containing the 17-kDa protein are shown in Figure 2A and 2B, respectively. The 17-kDa protein in fraction 14 separated by SDS-PAGE was electroblotted onto a PVDF membrane for amino-terminal amino acid sequence analysis. The sequence of the sixteen amino-terminal amino acid residues determined for this protein is shown in Figure 3. The protein data base (GENETYX) indicated that this sequence was identical with the amino terminus of kidney FABP.

Fig. 2. Ion-exchange HPLC separation of renal cytosolic proteins of a male rat. A) The elution profile of the renal cytosolic proteins monitored by the absorbance at 280 nm. The renal cytosolic fraction was dialyzed against 0.02 mol/L Tris-HCl (pH 8.0). The dialysate, containing ~4.5 mg of protein, was applied to an HPLC ion-exchange column as described in Materials and Methods. A linear line indicates the concentration of NaCl in eluent. The eluted solution was collected by 1-mL aliquots, and the fractions 14-16 were used for SDS-PAGE analyses. The horizontal bar in the graph indicates the fractions 14-16. B) Results of SDS-PAGE analysis of fractions 14-16. The arrow indicates the 17-kDa protein.
[View Larger Version of this Image (40K GIF file)]

Fig. 3. Amino-terminal amino acid sequence of the 17-kDa protein. The sequences of kidney fatty acid-binding protein (FABP) and alpha 2u-globulin proposed by Kimura et al. (1989) are presented for reference.
[View Larger Version of this Image (9K GIF file)]

Effect of ascorbic acid deficiency on the renal level of kidney fatty acid-binding protein. alpha 2u-Globulin, the precursor protein of kidney FABP, is synthesized in the liver by adult male rats but not by female rats (Ekstrom et al. 1984, Roy et al. 1983, Vandoren et al. 1983). The results of immunoblot analyses of renal kidney FABP, urinary alpha 2u-globulin and serum alpha 2u-globulin in a male rat and a female rat using anti-alpha 2u-globulin antiserum are shown in Figure 4A. In female rats, neither kidney FABP nor alpha 2u-globulin was detected in kidney, urine and serum. The apparent molecular mass of alpha 2u-globulin in urine and serum of male rats (19 kDa) was higher than that of kidney FABP in renal cytosol (17 kDa). The immunoblot analysis of kidney FABP in renal cytosol of the control rats and the ascorbic acid-deficient rats is shown in Figure 4B, and the measured level of this protein in each group is shown in Figure 4C. The apparent level of kidney FABP in the ascorbic acid-deficient rats was reduced to 53% of that in the control rats.
Fig. 4. Effect of ascorbic acid deficiency on the renal level of kidney fatty acid-binding protein (FABP) in control and ascorbic acid-deficient rats. A) Immunoblot analysis of renal cytosol, urine and serum of a male (M) and a female (F) Wistar rat. Urine was collected for 24 h. B) Immunoblot analysis of renal cytosol of three rats in the control group and the ascorbic acid-deficient group. Immunoblot analysis was performed as described in Materials and Methods by using a rabbit anti-rat alpha 2u-globulin antiserum. C) The renal level of kidney FABP calculated from the results of immunoblot analysis. Values are means ± SEM (n = 3) and are presented as a percentage of the control value. *Significantly different (P < 0.05) than the control group by Student' t test.
[View Larger Version of this Image (32K GIF file)]

Effect of ascorbic acid deficiency on the serum level of alpha 2u-globulin and the amount of alpha 2u-globulin in urine. The serum level of alpha 2u-globulin and the amount of alpha 2u-globulin in urine of both groups are shown in Figure 5. There were no significant differences between the two groups in the serum level of alpha 2u-globulin on d 3 and 10; on d 14, however, the level of alpha 2u-globulin of the ascorbic acid-deficient rats was reduced to 52% of that of the control rats. In contrast, the excreted amount of urinary alpha 2u-globulin on d 13-14 was not affected by ascorbic acid deficiency.
Fig. 5. Effects of ascorbic acid deficiency on the serum level of alpha 2u-globulin and the amount of alpha 2u-globulin excreted into urine in control and ascorbic acid-deficient rats. Values are means ± SEM, n = 3 (d 3 and 10) or n = 4 (d 14) and are presented as a percentage of the control value. Urine was collected from d 13 to 14 from four rats of each group. *Significantly different (P < 0.05) than the control group by Student' t test.
[View Larger Version of this Image (30K GIF file)]

Effect of ascorbic acid deficiency on the hepatic level of alpha 2u-globulin mRNA. The Northern blot analysis of RNA from various tissues of the control rats is shown in Figure 6A. Although beta -actin mRNA was detected in all tissues studied, alpha 2u-globulin mRNA was detected only in liver. On d 3 and 10, there were no differences in the hepatic level of alpha 2u-globulin mRNA between the control and the ascorbic acid-deficient rats (Fig. 6B). On d 14, the level was significantly lower in the ascorbic acid-deficient rats (70% of the control rats). On the other hand, ascorbic acid deficiency did not affect the hepatic level of apolipoprotein E mRNA (Fig. 6C).
Fig. 6. Effect of ascorbic acid deficiency on the hepatic level of alpha 2u-globulin mRNA in control and ascorbic acid-deficient rats. A) Northern blot analysis of total RNA from liver and other tissues of a male rat. Total RNA (20 µg) isolated from various tissues as separated on 10 g/L agarose gel containing 66 g/L formaldehyde. RNA in the gel was transferred to a Hybond N+ membrane and hybridized with 32P-labeled alpha 2u-globulin cDNA or 32P-labeled beta -actin cDNA. B) The hepatic level of alpha 2u-globulin mRNA in the control group and the ascorbic acid-deficient group. For quantifying the level, slot blot analyses were performed using 5 µg of total RNA. Values are means ± SEM, n = 3 (d 3 and 10) or n = 4 (d 14) and are presented as a percentage of the respective control group. *Significantly different (P < 0.05) than the control group by Student' t test. C) The hepatic level of apolipoprotein E mRNA in the control and the ascorbic acid-deficient groups. For quantifying the level, slot blot analyses were performed using 5 µg of total RNA. Values are means ± SEM, n = 3 (d 3 and 10) or n = 4 (d 14) and are presented as a percentage of the respective control group.
[View Larger Version of this Image (35K GIF file)]

DISCUSSION

In this study, severe reduction of food intake and signs of scurvy, such as hemorrhages around the eyes and nose, were not observed in the ascorbic acid-deficient rats fed an ascorbic acid-free diet for 14 d. Because a severe reduction of food intake seems to affect protein syntheses in several tissues, we chose 14 d of ascorbic acid deficiency. The renal and hepatic concentrations of ascorbic acid in the ascorbic acid-deficient rats on d 14 were significantly lower than those in the control rats. Ascorbic acid deficiency lowered the level of 17-kDa protein in kidney; that protein was regarded as a kidney FABP, a proteolytically modified form of alpha 2u-globulin (Kimura et al. 1989 and 1991), by its amino-terminal amino acid sequence.

alpha 2u-Globulin is a member of the alpha 2u-globulin superfamily, which consists of a dozen proteins with molecular masses around 20 kDa (Pevsner et al. 1988). Each member of this superfamily, such as serum retinol-binding protein and apolipoprotein D, is thought to be a transporter of hydrophobic molecules. The ligand for alpha 2u-globulin has not yet been identified. alpha 2u-Globulin (18.7 kDa) is synthesized mainly in liver in male rats, secreted into blood and rapidly excreted into urine (Neuhaus 1986 and 1992). However, female rats scarcely synthesize this protein either in liver or in other tissues (Neuhaus 1986 and 1992). In this study too, alpha 2u-globulin was not detected immunochemically in serum and urine in a female rat (Fig. 3A). In male rats, alpha 2u-globulin is synthesized in liver and approximately half of the amount is excreted into urine. The other half is reabsorbed into the proximal tubules of nephrons, processed by proteases associated with the brush border membrane, and eventually localized as kidney FABP in endosomes and lysosomes of epithelia of the proximal tubules (Kimura et al. 1991, Neuhaus 1986 and 1992).

FABP is thought to play a role in intracellular transport and metabolism of fatty acids (Veerkamp et al. 1991 and 1992). There are five FABP subtypes, i.e., liver FABP, intestinal FABP, heart FABP, adipocyte FABP and myelin FABP. However, kidney FABP differs from these subtypes in structure, although kidney FABP can efficiently bind to oleic acid in vitro (Lam et al. 1988).

We found that 14 d of ascorbic acid deficiency significantly lowered both the serum concentration of alpha 2u-globulin and the renal level of kidney FABP, although it did not affect the amount of alpha 2u-globulin excreted into urine from d 13 to 14 (Figs. 4C, 5). Ascorbic acid deficiency also lowered the hepatic level of alpha 2u-globulin mRNA (Fig. 6B). From these data, we speculate that ascorbic acid deficiency lowers the biosynthesis of alpha 2u-globulin in liver through lowering the hepatic level of its mRNA, subsequently causing the low level of kidney FABP in renal cytosol.

Because the amount of alpha 2u-globulin excreted into urine from d 13 to 14 was not decreased by ascorbic acid deficiency, it cannot be excluded that ascorbic acid deficiency suppressed the reabsorption of alpha 2u-globulin in the proximal tubules of nephrons or the proteolytic processing of this protein. On d 14, the serum concentrations of creatinine and uric acid of the ascorbic acid-deficient rats were not different than those of the control rats (data not shown). By histologic examinations of kidney, no abnormality was observed in nephrons of the ascorbic acid-deficient rats (data not shown). These results suggest that ascorbic acid deficiency does not impair the reabsorption of alpha 2u-globulin in proximal tubules of nephrons. Ascorbic acid deficiency decreased the kidney FABP (17 kDa) but did not increase the alpha 2u-globulin (19 kDa) in kidney (Fig. 4B). Because kidney FABP is synthesized from alpha 2u-globulin by proteolytic processing, these results also suggest that ascorbic acid deficiency does not suppress the proteolytic processing of alpha 2u-globulin.

This investigation has proved that ascorbic acid deficiency lowers the level of hepatic alpha 2u-globulin mRNA. The mechanism of action of ascorbic acid in maintaining the hepatic level of alpha 2u-globulin mRNA remains unclear. The hormonal regulation of alpha 2u-globulin biosynthesis and its gene expression have been widely investigated; androgens (Kurtz et al. 1976, Song et al. 1991), glucocorticoids (Addison and Kurtz 1989, Chan et al. 1991) and growth hormone (Lynch et al. 1982) are required for alpha 2u-globulin synthesis, whereas estrogens strongly suppress its synthesis (Kurtz et al. 1976, Van Dijck and Verhoeven 1992). Although data were not shown, we measured the serum concentrations of testosterone (androgen) and corticosterone (glucocorticoid) because these hormones are secreted into blood from testes and adrenal glands, respectively. The serum concentration of testosterone was not different in the ascorbic acid-deficient rats than in control rats. The serum concentration of corticosterone was generally higher in the ascorbic acid-deficient rats. Therefore it is not likely that ascorbic acid deficiency lowers the hepatic level of alpha 2u-globulin mRNA by lowering the serum level of testosterone or corticosterone. Further studies are required to determine how ascorbic acid maintains the hepatic level of alpha 2u-globulin mRNA.

Although a potent affinity of kidney FABP for oleic acid was clearly demonstrated in vitro (Lam et al. 1988), the role and the function of this protein in intracellular transport and metabolism of fatty acids remain to be elucidated. The processing of alpha 2u-globulin into kidney FABP by proteases associated with the brush border membrane during its reabsorption is quite unique. Further studies are required to determine whether ascorbic acid deficiency causes the abnormality of fatty acid metabolism in kidney of male rats by decreasing the amount of kidney FABP.

In this study, by analyzing the cytosolic fractions from several tissues of male ODS rats, we have found for the first time a decrease of kidney FABP due to ascorbic acid deficiency. There were no changes detected in the levels of cytosolic fractions of other tissues induced by ascorbic acid deficiency. Ascorbic acid deficiency may cause the decrease in kidney FABP by lowering the hepatic mRNA level of its precursor protein, alpha 2u-globulin.

FOOTNOTES

1   Supported in part by Grant-in-Aid for Scientific Research 09660134 from the Ministry of Education, Science and Culture, Japan, and a grant from the Elizabeth Arnold Fuji Foundation, Japan.
2   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3   To whom correspondence and reprint requests should be addressed.
4   Abbreviations used: FABP, fatty acid-binding protein; MOPS, 3-(N-morpholino)propanesulfonic acid; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride.

Manuscript received 9 May 1997. Initial reviews completed 13 June 1997. Revision accepted 7 August 1997.

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0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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