Acute glucocorticoid treatment increases urinary biotin excretion and serum biotin

Sara C. Rathman, Brandon Lewis, and Robert J. McMahon

The Center for Nutritional Science and The Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have demonstrated that glucocorticoids alter biotin metabolism. To extend these studies, the effect of dexamethasone on biotin pools was analyzed in rats consuming a purified diet containing a more physiological level of dietary biotin intake (0.06 mg/kg). Acute (5 h) dexamethasone administration (0.5 mg/kg) elicited elevated urinary glucose output as well as elevated urinary biotin excretion and serum biotin. Renal and hepatic free biotin was also significantly elevated by acute dexamethasone administration. Chow-fed rats treated with an acute administration of dexamethasone demonstrated significantly elevated urinary glucose excretion, urinary biotin excretion, and serum biotin, but no change in tissue associated biotin was detected. Chronic administration of dexamethasone (0.5 mg/kg ip) over 4 days significantly elevated urinary glucose excretion 42% but had no effect on urinary biotin excretion, serum biotin, or hepatic- or renal-associated free biotin. These results demonstrate the existence of potentially novel regulatory pathways for total biotin pools and the possibility that experimental models with high initial biotin status may mask potentially important regulatory mechanisms.

dexamethasone; vitamin; AIN76a; rodent


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCOCORTICOIDS are important endogenous regulators of fat, carbohydrate, and protein metabolism. Previous studies have demonstrated in vivo and in vitro that glucocorticoids are involved in carbohydrate metabolism through stimulation of gluconeogenesis (7-10, 12, 13, 16, 20, 36, 39). Additionally, the synthetic glucocorticoid dexamethasone stimulates hepatic triglyceride synthesis and secretion in perfused rat livers, as well as hepatic glucose output in both rodents and humans (5, 40). At doses similar to those used for treatment in premature infants (6, 11, 25, 35, 38), dexamethasone induces protein catabolism in piglets (43), demonstrating a role for glucocorticoids in nitrogen metabolism.

Although the effects of dexamethasone on fatty acid, carbohydrate, and protein metabolism have received significant study, little is known about its effects on the coenzymes required for these processes. In the present investigation, the effect(s) of dexamethasone on free and protein bound biotin pools was analyzed. As a coenzyme for the carboxylases of acetyl-CoA, pyruvate, methylcrotonyl-CoA, and propionyl-CoA, biotin serves a critical role in carbohydrate, fatty acid, and amino acid metabolism. Compounds that alter the metabolism of these macronutrients might therefore reasonably be hypothesized to affect biotin metabolism. Indeed, such a hypothesis has received supporting evidence from a biotin tracer study, where chronic dexamethasone treatment increased the urinary concentration of a biotin catabolite, bisnorbiotin (42). This suggests that dexamethasone stimulated biotin degradation, probably through a beta -oxidation process similar to that of fatty acids (17, 21, 22, 45). These observations suggested the potential for changes among levels of free and protein-bound biotin pools. In the present study, we analyzed the acute and chronic effects of dexamethasone on the total concentration of free biotin in various compartments, as well as the relative abundance of biotin bound to biotin dependent enzymes. These data suggest a potentially distinct glucocorticoid-dependent regulation of free biotin and have implications for the experimental modeling of biotin nutriture in rodent models.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Male Sprague-Dawley rats were purchased from Harlan Laboratories (Indianapolis, IN). Rodent chow was obtained from Harlan Teklad (Rodent Diet no. 8604). Purified biotin-free rodent diet based on a modified AIN76a formulation was purchased from Research Diets (New Brunswick, NJ). The composition of the diet is spray-dried egg white (20% wt/wt), corn starch (15% wt/wt), sucrose (50% wt/wt), cellulose (5% wt/wt), corn oil (5% wt/wt), AIN76a mineral and vitamin mix (without biotin), and choline bitartrate (0.2% wt/wt). D-Biotin, protease inhibitor cocktail, dexamethasone, and o-phenylenediamine dihydrochloride were purchased from Sigma (St. Louis, MO); Neutralite avidin-horseradish peroxidase conjugate was purchased from Pierce Chemical (Birmingham, AL); 96-well microtiter plates (Nunc Maxisorp) and bovine serum albumin (BSA) were purchased from Fisher Scientific (Pittsburgh, PA); and ECL-Plus reagent was purchased from Amersham-Pharmacia (Piscataway, NJ). Biotinylated BSA was synthesized by mixing 50 ml of 10 mg/ml BSA in ice-cold 0.1 mol/l NaHCO3 (pH 7.5) with 5 ml of N-hydroxysuccinimide ester of biotin [12 mg/ml in dimethyl sulfoxide (DMSO)] overnight at 4°C. The mixture was dialyzed for 48 h with gentle stirring at 4°C.

Animals and diet preparation. In this series of experiments, we used either standard rodent chow (Harlan Teklad) containing ~0.22 mg biotin/kg diet (as reported by manufacturer) or a purified diet commonly used for the analysis of dietary components formulated by the American Institute of Nutrition (AIN76a) (3). Although similar to diets used in other studies on biotin nutriture, this purified diet differs significantly from those used earlier in terms of carbohydrate source and amount, fatty acid composition, and some vitamins and minerals (19, 24, 33, 37). This diet has been modified to include spray-dried egg white as its sole protein source. The avidin protein in the egg white in the purified diet binds ~1.44 mg biotin/kg of purified diet, inhibiting biotin absorption (18). The level of dietary biotin designated in these studies represents biotin in excess of the binding capacity of the dietary egg white avidin. All rats used in these experiments were 75-100 g initial weight and were housed individually in hanging wire-bottom cages in an environmentally controlled room with constant temperature (22°C) and a 12:12-h light-dark cycle. All procedures were approved by the University of Florida Animal Care and Use Committee.

Chronic dexamethasone experiment. In this experiment, a dose of 0.5 mg dexamethasone/kg body wt was chosen to mimic a therapeutic level used for treatment of various inflammatory or chronic diseases (34). All rats were acclimated to rodent chow and given free access to water for 7 days before the experimental period. After the acclimation period, rats were randomly assigned to a treatment group (n = 4) that received an intraperitoneal injection of dexamethasone [0.5 mg/kg body wt in 50% DMSO] each day for 4 days or to a control group (n = 4) that received injections of vehicle only. Nineteen hours after the last injection, rats were placed in metabolic chambers for 5 h to allow the discrete collection of urine. Twenty-four hours after the last injection, rats were anesthetized under halothane vapor and killed by exsanguination. Urine, serum, liver, and kidney were collected and prepared as described in Sample preparation.

Acute dexamethasone experiments. These experiments were performed to determine the acute effects of dexamethasone treatment on biotin pools in chow-fed rats. After a 7-day acclimation period when 12 rats consumed rodent chow, they were randomly assigned to a treatment group (n = 6) or a control group (n = 6). Rats in the treatment group were injected intraperitoneally with 0.5 mg/kg dexamethasone or an equal volume of vehicle only and were immediately placed in metabolic chambers for 5 h for urine collection. Rats were anesthetized as described above and killed 5 h after injection by exsanguination. In a second experiment, rats were acclimated to a purified AIN76a diet containing 0.06 mg biotin/kg diet. The 0.06 mg/kg level of biotin was chosen to more closely model human biotin intake (28, 30, 32). Ten rats were acclimated for 7 days on the purified diet and then randomly assigned to a treatment group (n = 5) that received one intraperitoneal injection of 0.5 mg dexamethason/kg body wt or a control group (n = 5) that received vehicle only. Rats were anesthetized as described in Chronic dexamethasone experiment and killed 5 h after injection by exsanguination.

Sample preparation. Whole blood was withdrawn and allowed to clot for 30 min and then centrifuged at 10,000 g for 10 min to collect serum. Approximately 500 mg of liver and kidney were removed and homogenized in 10 volumes of ice-cold homogenization buffer (300 mmol/l mannitol, 10 mmol/l HEPES, pH 7.2, 1 mmol/l EDTA, and protease inhibitor cocktail). An aliquot of the homogenate was retained at stored at -80°C. The remainder of the homogenate was centrifuged at 200,000 g for 30 min at 4°C, and the supernatant (soluble fraction) was recovered. All samples were immediately frozen in a mixture of dry ice and isopropanol and stored at -20°C until needed.

Synthesis of avidin-AlexaFluor 430. NeutrAvidin, an isoelectrically neutral and deglycosylated form of avidin, was conjugated to the succinimidyl ester form of AlexaFluor 430 (Molecular Probes, Eugene, OR). NeutrAvidin (10 mg/ml in 50 mmol/l sodium bicarbonate, pH 8.3) was mixed 5:1 (vol/vol) with a solution of AlexaFluor 430 succinimidyl ester (10 mg/ml in DMSO). The mixture was slowly mixed on a vortexer for 1 h at room temperature. Unconjugated dye was removed by size exclusion chromatography over a DG-10 column (Bio-Rad, Hercules, CA) equilibrated in phosphate-buffered saline (20 mmol/l sodium phosphate, pH 7.2, 150 mmol/l NaCl). Equal fractions (1 ml) were collected, and the fractions that contained the peak with the highest absorbance at 280 nm were combined. Sodium azide (0.02% wt/vol) was added for preservation, and the conjugate was stored at 4°C protected from light.

Competitive binding assay of biotin. For the measurement of serum and tissue free biotin, samples were ultrafiltered using a 5,000-Da NMWCO filter (Millipore, Bedford, MA). The measurement of biotin in urine, serum, liver, and kidney was performed with a coupled high-performance liquid chromatography-competitive binding assay as previously described with minor modifications (29). The reversed-phase column was a Sphereclone 250 × 4.6 mm (Phenomenex, Torrance, CA). Biotin containing chromatography fractions was heated at 65°C and dried under a stream of nitrogen before assay.

Detection and quantification of biotinylated proteins. The protein concentration of liver and kidney homogenates was determined as previously described. (26) Equal amounts (0.1 mg) of liver and kidney homogenate were resolved by 10% SDS-PAGE overnight at 47 V. We have found that maximal resolution of methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase is achieved when the separating gel buffer is adjusted to pH 8 (data not shown). The gel was electroblotted to polyvinylidene difluoride (Immobilon-P, Millipore, Bedford, MA) for 2 h at 12 V as previously described (27). The blot was then washed with two changes of methanol and allowed to air dry. For the detection of pyruvate carboxylase, propionyl-CoA carboxylase, and methylcrotonyl-CoA carboxylase, the blot was incubated in TBS-T [20 mM Tris base, pH 7.4, 150 mM NaCl, 0.05% (vol/vol) Tween-20, 0.5% nonfat dry milk] containing avidin-AlexaFluor 430 conjugate at a 1:750 dilution for 45 min at room temperature on an orbital shaker. Biotinylated proteins on both blots were then detected and emitted fluorescence quantified on a Storm fluorescent optical scanner as described by the manufacturer (Amersham-Pharmacia). Several control experiments were performed to ensure that measurement was in the linear range of detection (Fig. 1).


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Fig. 1.   Specificity and linearity of biotinylated polypeptide detection on the Storm840 fluorescent scanner. PC, pyruvate carboxylase; MCC, methylcrotonyl-CoA carboxylase; PCC, propionyl-CoA carboxylase. A: an equal amount of rat liver homogenate (0.1 mg) was resolved by SDS-PAGE and blotted to polyvinylidene difluoride in duplicate, as specifically described in MATERIALS AND METHODS. The lanes were then probed with avidin-AlexaFluor 430 in the absence (-) or presence (+) of 1 µmol/l biotin. B: various amounts of rat liver homogenate (0.01-0.1 mg) were resolved as in A. The blot was probed with avidin-AlexaFluor 430. C: quantification of emitted fluorescence of the blot in B.

Urine glucose and creatinine measurements. In the acute experiments, dexamethasone-treated rats excreted at least twice as much urine as the control group; this difference and the variability in urine collection during the short time period prompted the standardization of urinary biotin excretion on the basis of excreted creatinine. Dexamethasone administration has been reported to have no significant effect on urinary creatinine excretion (43, 44). Creatinine was measured using a commercially available picric acid-based kit (Sigma no. 555-A). Urine glucose was measured as a marker of the efficacy of the dexamethasone treatment because acute glucosuria occurs after dexamethasone loading (44). Glucose was measured using an Infinity Glucose Reagent kit (Sigma).

Statistical analysis. Results are expressed as means ± SE. The significance of differences (P < 0.05) was tested by Student's t-test. In some cases, the data were log transformed to achieve acceptable homogeneity of variance. Because of the variance in the tissue-associated free biotin, differences were detected by the Mann-Whitney nonparametric t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Acute dexamethasone experiments. In rats consuming the modified AIN76a purified diet containing 0.06 mg biotin/kg, dexamethasone significantly elevated urine glucose 167% (P < 0.05; 1,023.6 ± 94 vs. 382.6 ± 51.3 pmol glucose/mol creatinine) (Fig. 2A). Urinary biotin excretion in dexamethasone-treated rats was 263% higher than in the control group (P < 0.05; 28.7 ± 5.5 vs. 7.9 ± 2.6 pmol/nmol creatinine) (Fig. 2B). Serum biotin in the dexamethasone-treated rats was 311% times higher than the control group (P < 0.05; 10.7 ± 3.3 and 2.6 ± 0.5, nmol/l) (Fig. 2C). There was a significant 75% increase in the concentration of free biotin in the kidney of dexamethasone-treated rats (P < 0.05; 17.5 ± 4.4 vs. 10.0 ± 0.5 pmol/g tissue) and a 106% increase in the concentration of free biotin in the liver (P < 0.05; 12.4 ± 4.9 vs. 6.0 ± 0.9 pmol/g tissue) (Fig. 2D). Acute dexamethasone treatment had no significant effect on the relative abundance of biotinylated pyruvate, methylcrotonyl-CoA, or propionyl-CoA carboxylase polypeptides (Fig. 2E).


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Fig. 2.   Effect of acute dexamethasone administration on purified diet containing 0.06 mg biotin/kg. Rats fed a purified diet were administered a single dose of dexamethasone (0.5 mg/kg body wt ip) or vehicle alone. Rats were then placed in metabolic cages while urine was collected. Five hours after the last injection, rats were killed before serum, liver, and kidney were obtained. Free biotin concentration was measured as described in MATERIALS AND METHODS. The relative abundance of carboxylases was assessed by avidin blotting, as described in MATERIALS AND METHODS. *Statistically significant differences between dexamethasone-treated and control rats. A: urine glucose; B: urine biotin; C: serum biotin; D: free biotin; E: carboxylase.

In rats fed commercially available rodent chow, the baseline urinary biotin excretion (41.5 ± 5.8 pmol/nmol creatinine) and serum biotin concentration (19.4 ± 0.5 nmol/l) were significantly elevated compared with rats fed the purified diet containing 0.06 mg biotin/kg (P < 0.05). An acute dose of dexamethasone to chow-fed rats significantly increased the urine glucose concentration 160% (P < 0.05; 601.6 ± 59.5 vs. 375.8 ± 24.4 pmol glucose/mol creatinine) (Fig. 3A). Urinary biotin excretion in the dexamethasone-treated rats was elevated 101% compared with the control group (P < 0.05; 83.4 ± 11.2 vs. 41.5 ± 5.8 pmol biotin/nmol creatinine) (Fig. 3B). Similarly, serum biotin in dexamethasone-treated rats was 65% higher than control rats (P < 0.05; 32.2 ± 3.6 vs. 19.4 ± 0.5 nmol/l) (Fig. 3C). No significant differences in free biotin concentration were observed in the liver or kidney (Fig. 3D). As assessed by avidin blotting, dexamethasone had no significant effect on the relative abundance of the biotinylated forms of the pyruvate or methylcrotonyl- or propionyl-CoA carboxylase polypeptides (Fig. 3E).


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Fig. 3.   Effect of acute dexamethasone administration on chow-fed rats. Rats fed commercial rat chow were administered a single dose of dexamethasone (0.5 mg/kg body wt ip) or vehicle alone. Rats were then placed in metabolic cages while urine was collected. Five hours after the last injection, rats were killed before serum, liver, and kidney were obtained. Free biotin concentration was measured as described in MATERIALS AND METHODS. The relative abundance of carboxylases was assessed by avidin blotting as described in MATERIALS AND METHODS. *Statistically significant differences between dexamethasone-treated rats and control rats. See Fig. 2 legend for definitions of A-E.

Chronic dexamethasone experiment. Daily administration of dexamethasone for 4 days to chow-fed rats significantly elevated urine glucose concentration 42% compared with control animals (P < 0.05; 245.2 ± 13.5 vs. 171.5 ± 10.9 pmol glucose/mol creatinine) (Fig. 4A). In contrast, this treatment had no significant effect on the rate of urinary biotin excretion or serum biotin or the concentration of free biotin in the liver or kidney (Fig. 4, B, C, and D).


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Fig. 4.   Effect of chronic dexamethasone administration on chow-fed rats. Rats fed commercial rat chow were administered dexamethasone (0.5 mg/kg body wt ip) or vehicle alone for 4 days. Nineteen hours after the last injection, urine was collected in metabolic cages. Rats were then killed before serum, liver, and kidney were obtained and analyzed for free biotin concentration. *Statistically significant differences between dexamethasone-treated rats and control rats. See Fig. 2 legend for definitions of A-D.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we have further characterized previous observations of glucocorticoid-induced changes in biotin metabolism. Using a radiotracer technique, Mock et al. (42) previously described increased catabolism of biotin to bisnorbiotin under the influence of a chronic course of dexamethasone administration. This report extends that work by analyzing chronic and acute doses of dexamethasone both in chow-fed rats and in rats fed a more physiological level of dietary biotin. Because glucocorticoids are important regulatory signals that affect carbohydrate, protein, and fatty acid metabolism, we hypothesized that there might also be concurrent regulation of the total intact biotin pools as a required cofactor for enzymes involved in the metabolism of those macronutrients. We present here for the first time definitive evidence that the total pools of the intact vitamin are altered in response to acute, but not chronic, dexamethasone administration.

In an effort to examine the suitability of commercially available rat chow for nutritional biotin studies, we analyzed acid extracted diet by HPLC-competitive binding assay and confirmed that this rodent chow contains ~0.22 mg/kg. At the rate at which rats consumed the diet, this intake represents ~2.3 mg biotin/day for a 70-kg individual, accounting for the urine and serum biotin values well above those previously reported for humans (30-32). We therefore chose a purified diet based on the AIN76a formulation containing a level of biotin (0.06 mg/kg) that more closely models human intake, although it was admittedly still high compared with the sparse literature on biotin intake estimates (4, 14, 15, 23). Unfortunately, experimental constraints prevent the reproducible formulation and mixing of an egg white diet with much less than this amount of biotin. Urine, serum, and tissue biotin levels were substantially different in rats consuming these two diets. For example, the concentration of biotin in urine, serum, liver, and kidney in rats consuming the purified diet was dramatically lower compared with the same samples from rats consuming normal rodent chow. This suggests that the experiments carried out on chow-fed rats more accurately represents pharmacologically biotin-supplemented animals. It is also interesting to note the rate (8 days) at which biotin status is reset when animals are switched from rodent chow to the purified diet containing the roughly physiological level of biotin, in agreement with another report of a surprisingly rapid loss of biotin (41). In this particular model of biotin status, an acute dose of dexamethasone had a pronounced effect on biotin pools; urinary glucose excretion was elevated ~267%, and changes in tissue-associated free biotin concentration were likewise substantially elevated. Despite the rather large fluctuations in the free biotin pools, we found no difference in the relative abundance of protein-bound biotin, as evidenced by a lack of alterations in abundance of pyruvate, and propionyl-CoA, and methylcrotonyl-CoA carboxylase, three carboxylases expressed at high levels in rat liver.

To assess whether such glucocorticoid-dependent changes could be observed in the biotin-supplemented state, the effect of an acute dexamethasone dose on biotin pools in chow-fed rats was analyzed. As in the physiological model of biotin status, urinary biotin excretion and serum biotin were significantly elevated, although the magnitude of the glucocorticoid effect was attenuated. Consistent with this attenuation, no effect of dexamethasone was observed on the tissue-associated free biotin pool in chow fed rats.

Glucocorticoid therapy often addresses a chronic condition, requiring repeated administrations of dexamethasone. We therefore assessed the effect of chronic glucocorticoid treatment on biotin metabolism. As expected, dexamethasone significantly elevated urine glucose levels, in agreement with earlier reports demonstrating a glucocorticoid-induced stimulation of gluconeogenesis (1, 2, 9). In contrast to the acute experiments, chronic dexamethasone treatment had no such effect on urinary biotin excretion. This lack of effect was mirrored in the measurement of free biotin in the serum, liver, and kidney, which also exhibited no change, suggesting that the effect of glucocorticoids on biotin metabolism is relatively transient.

Because the technique used in these experiments to measure biotin is chemically specific, the results indicate an increase in the intact form of the vitamin, rather than a biotin catabolite or unrelated compound. The source of the additional biotin found in the serum and tissues remains to be described. Because the protein-bound fraction of biotin (carboxylase associated) was not altered by dexamethasone, it appears unlikely that this pool could account for the observed increases. Furthermore, the previous study suggests that there is a modest acceleration of biotin catabolism to bisnorbiotin induced by glucocorticoid, thereby also unsupportive of decreased biotin catabolism as the potential source of the observed increases. Given increases in biotin concentration in one compartment, there might be corresponding decreases in others. The rats in these experiments failed to demonstrate a decrease in either renal or hepatic biotin, suggesting that there may be a pool of free biotin in some other tissue currently not well described. Future experiments will focus on describing possible sources of free biotin that are regulated by glucocorticoids.

Overall, these data demonstrate that significant glucocorticoid regulation of biotin pools in the urine, circulation, and tissues occurs at both supplemented and physiologically relevant biotin intakes. The biological rationale for observed changes in the free biotin pools without concomitant changes in carboxylase expression suggests potentially novel regulatory pathways in glucocorticoid signaling. These data also suggest that the supplemented biotin status induced by chow feeding may mask some important physiological effects of biotin metabolism, in part because specific biotin pools may already be near their maximum capacity. We suggest that further effort into the establishment of physiological models of biotin status be pursued to increase the relevance of studies on biotin nutriture.


    ACKNOWLEDGEMENTS

This research was supported by the Florida Agricultural Experiment Station and approved for publication as Journal Series No. R-08301.


    FOOTNOTES

Address for reprint requests and other correspondence: R. J. McMahon, Box 110370, FSHN Bldg, Univ. of Florida, Gainesville, FL 32611-0370 (E-mail: mcmahon{at}ufl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00357.2001

Received 6 August 2001; accepted in final form 25 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 282(3):E643-E649
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