blood metabolites after intestinal absorption of amino acids in locusts

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Nov 18, 1971 - In a previous paper we have shown that L-glutamate passes from the gut lumen to the haemocoel of the locust slowly compared to L-alanine ...
J. Exp. Biol. (1972). S*, 795-8o8 With 8 text-figures in Great Britain

795

BLOOD METABOLITES AFTER INTESTINAL ABSORPTION OF AMINO ACIDS IN LOCUSTS BY L. L. MURDOCK AND B. KOIDL Fachbereich Biologie, Universitdt Konstanz, 775 Konstanz, West Germany (Received 18 November 1971) INTRODUCTION

In a previous paper we have shown that L-glutamate passes from the gut lumen to the haemocoel of the locust slowly compared to L-alanine and glycine (Murdock & Koidl, 1972). Furthermore, it was found that L-glutamate undergoes extensive metabolism, probably in the gut wall. Limited permeability and metabolism would be expected to severely restrict the amount of L-glutamate entering the blood from the diet. It has been claimed that the neuromuscular synapses of locusts have no structural or enzymic barriers between them and the blood, and that there is little 'free' L-glutamate in the blood of these animals (Usherwood & Machili, 1968). If this is so, then it is possible to view the gut wall as a sort of first line of synaptic defense, because the limited permeability and destruction of the amino acid in the alimentary tract wall would prevent or reduce increases in blood L-glutamate, thus helping avoid interference with synaptic transmission. To complement and extend these observations, it was of interest to know the nature of metabolites of L-glutamate and other amino acids that appear in the blood of locusts during absorption. The present paper examines this, and considers the metabolism of these substances during and after absorption. Because considerable destruction of L-glutamate appears to be taking place during absorption, it was of especial interest to discover the nature of its metabolites produced by the gut wall. The gastric caeca were chosen for study because they have been shown to be the major tissue site of amino acid absorption in locusts (Treherne, 1959). The fact that the caeca are richly tracheated may be an indication of their oxidative capacity. METHODS

Absorption without perfusion

The absorption of an amino acid in locusts was studied by examining blood for it and its metabolites 1 h after infusion of the liC-labelled substance into the alimentary tract. Radioisotopes, gut cannulation, and injection were as described earlier (Murdock & Koidl, 1972). To collect blood, a shallow cut in the cervical membrane was made, and the blood was allowed to drip into a calibrated centrifuge tube held in ice. Two volumes of ethanol were added, the contents mixed well, and the tube allowed to stand 30 min in ice. After centrifugation at 5000 x g for 10 min, the supernatant was withdrawn and used for chromatography immediately. The concentration of the radioactive amino acid injected into the gut lumen was 50 mM, containing 2-5 or

796

L. L. MURDOCK AND B. KOIDL

5*0 /tCi of " C made up in the 200 mM-NaCl saUne described earlier. This amount o j | radioactivity allowed the separation and identification of metabolites using only 5 fi\ or1 the extract, thus avoiding additional purification steps. In vitro metabolism experiments Metabolism of the amino acids by isolated gastric caeca was studied as follows. Gastric caeca were dissected out of locusts that had been deprived of food during the previous 24-36 h. Usually the anterior-directed caeca from three animals were removed, taking care that they did not contact the digestive fluid leaking from cuts in the abdominal wall. After being cut off, each caecum was pulled over the surface of filter paper to remove adhering fat and Malpighian tubules. It was then slit lengthwise, and washed in a stream of ice-cold saline. Individual caeca were held on a watch glass on crushed ice until the required number had been accumulated. They were then blotted to remove excess saline, and placed on the depression of a spot plate in which 1 or 3/tCi of the 14C-precursor had been dried. After 150 fi\ of oxygen-saturated saline had been added, the incubation system was stirred well with the tip of a glass melting-point tube, and the depression covered with a microscope slide coverslip. At intervals of 20-30 min the coverslip was removed, and the system reflushed with oxygen. This was repeated at intervals until the required incubation time had been reached (1 or 3 h). The caeca were then lifted out, washed for a few seconds in saline, homogenized in ethanol/H2O (4/1) using an all-glass homogenizer. The homogenate was transferred to a centrifuge tube and spun down as above. The supernatant was subjected to chromatography at once. In some experiments the bath fluid was also collected at the end of the incubation period and analysed by chromatography. Thin-layer chromatography Thin-layer chromatography was carried out with prepared cellulose plates (Merck Alufolie). In all experiments the appropriate amino acid standards were placed directly on the extract spot. After spraying with ninhydrin, the very dark spots due to the standards could easily be recognized over the background of amino acids naturally occurring in the extract. This obviated the need to correct RF values due to the presence of salts and interfering substances. Further identification of metabolites employed a preliminary separation on paper chromatograms, elution of radioactive spots with ethanol/HaO (4/1), followed by reconcentration and re-chromatography on at least four other thin-layer systems. Solvent systems used were: 1, n-butanol/acetic/HaO(4/i/5); 2, phenol/H2O/ formic acid (75 g/25 ml/i ml); 3, 96% ethanol/25% ammonia (7/3); 4, methanol/ pyridine/acetic acid/H2O (80/4/1/20); 5, methanol/H2O/pyridine (20/5/1). Enzyme determinations Considering the marked metabolic activity of the gut wall met with in this and the previous study (Murdock & Koidl, 1972), it was of interest to discover more about the enzyme activities of the alimentary tract tissues, particularly of the gastric caeca and ventriculus. To enable comparisons to be made with other tissues, Malpighian tubules and fat body were also examined. Tissues were dissected out of 3-week-old adult Locusta rmgratoria that had been deprived of food during the previous 24 h..

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Fig. i. Blood metabolites i h after infusion of L-glutamate-U-"C into the alimentary tract of a female L. migratoria. The major area of radioactivity corresponds with the position of glutamine. A, Shows the distribution of radioactivity on the chromatogram. B, shows the position of ninhydrin-positive spots (both standards and naturally occurring substances). Smaller amounts of radioactivity are found in the position of alanine. Note the absence of radioactivity in glutamate. Abbreviations: glutamate - Glu; alanine-Ala; glutamine - Glu(NH,); proline - Pro; serine - Ser; glycine - Gly.

After mincing with scissors, the tissues were homogenized in ioo mM-Na, K phosphate buffer pH 7-2 by 4 x 20 sec sonication (Branson Instruments, Sonifier). Enzyme activities were determined in the supernatant after centrifugation at 144,000 # for 30 min. Phosphorylase (EC. 2.4.1.1) and glyceraldehyde phosphate dehydrogenase (EC. 1.2.1.12) were assayed as described in Bass et al. (1969). Citrate synthetase (EC. 4.1.3.7), malate dehydrogenase (EC. 1.1.1.37), 3-hydroxyacyl-CoA dehydrogenase (EC. 1.1.1.35), and glutamate dehydrogenase (EC. 1.4.1.2) were measured according to Brdiczka et al. (1968) and aspartate aminotransferase (EC. 2.6.1.1), alanine aminotransferase (EC. 2.6.1.2), and [NADP] malate dehydrogenase [decarboxylating] (EC. 1.1.1.38) after Biicher et al. (1964). Assay of phosphoenolpyruvate carboxykinase (EC. 4.1.1.32) was as described by Nolte et al. (1971). RESULTS

Blood metabolites after absorption

After infusion of L-glutamate-U-14C into Schistocerca or Locusta adults of either sex, by far the greatest amount of blood radioactivity was recovered in glutamine (Fig. 1). In four or five experiments radioactive L-glutamate could not be seen in two-dimensional chromatograms; in one case radioactive glutamate was found, but the quantity of radioactivity in this form was only a few per cent, at most, of that found in glutamine. Considering that the blood bathed the alimentary tract which contained 50 mM L-glutamate, this appears to be a striking demonstration of the efficiency with which a build-up of L-glutamate in the blood can be prevented. The second most abundant radioactive substance recovered from the blood was alanine. Usually it was present in much smaller quantity than radioactive glutamine (Figs. 1 and 2). Additional minor metabolites were seen, but not identified.

L. L . MURDOCK AND B. KoiDL

ooo F

Ala

GIu Glu(NH2)

S

14

Fig. 2. Blood metabolites i h after infusion of L-glutamate-U- C into the alimentary tract of male L. mxgratoria. One-dimensional separation using system 4 (see text). The major radioactive peak is concurrent with glutamine. The other significant peak indicates the presence of alanine. Note that there is no radioactivity apparent in glutamate. Abbreviations: glutamate — Glu; alanine - Ala; glutamine - Glu (NH,).

9 Glu

Gly + Scr

System 3 — A B Fig. 3. Blood metabolites 1 h after infusion of L-alanine-i-"C into the alimentary tract of a female L. migratoria. A, Shows the distribution of radioactivity on the chromatogram. B, Shows the position of ninhydrin-positive spots (both standards and naturally occurring substances). The densest spot of radioactivity corresponds in position to the alanine standard. Considerable amounts of radioactivity are also discernible in glutamine. There also appears to be some radioactivity in glycine/serine. Abbreviations: glutamate - Glu; alanine-Ala; glutamine - Glu (NH,); serine - Ser; glycine - Gly; proline - Pro.

When the experiments were repeated with L-alanine i-14C as substrate, the major radioactive substance recovered from the blood was unchanged alanine (Fig. 3). The other considerable metabolite was glutamine. The presence of glutamine derived from alanine shows that the glutamate carbon skeleton can be built up from that of alanine, but again that it is glutamine rather than glutamate that appears in the blood. Low levels of radioactivity were sometimes observed in the area of glycine/serine. After gut infusion of glycine-i-MC, the only radioactive substance found in the blood was unchanged glycine (Fig. 4). It cannot be ruled out that some serine was

Blood metabolites

799 Pro-O

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A B Fig. 4. Blood metabolites 1 h after infusion of glycine-i-"C into the alimentary tract lumen of a female L. imgratoria. A, Shows the distribution of radioactivity on the chromatogram. B, Shows the position of ninhydrin-positive spots (both standards and naturally occurring substances). Only one radioactive spot is seen, which corresponds in position with the location of glycine. Because of the overlap of the glycine spot with serine, it is not possible to determine whether small amounts of radioactivity are present in the latter compound. Abbreviations: glycine - Gly; serine - Ser; glutamine - Glu (NH»); proline - Pro.

produced from glycine, because the chromatographic systems employed separate the two amino acids poorly. Nevertheless, it is clear that the great majority of the recovered radioactivity is present in unchanged glycine. It would thus appear that glycine passes into the blood in large measure unchanged. This observation, together with those for L-alanine, provides a good perspective for appreciating the effectiveness with which the build-up of L-glutamate in the blood is prevented. To investigate the specificity of the mechanism preventing entry and build-up of L-glutamate in the blood, experiments were carried out in which D-glutamate-i-14C was infused. With both species of locusts the major radioactive substance found in blood was unchanged glutamate (Fig. 5). It is reasonable to assume that this is the D-form, because the L-enantiomorph, as has been shown, is apparently not absorbed unchanged. This would suggest that the mechanism that so effectively screens out L-glutamate from the blood is incapable of acting against the D-isomer. The only metabolite of D-glutamate was glutamine. Whether this represents D-glutamine, or a fraction of the D-glutamate that has been converted to the L-form and thence to Lglutamine, cannot be answered with certainty. It is of interest, however, to note that glutamine synthetase from peas, pigeon liver, sheep brain (Levintow & Meister, 1953) and Prodema larvae (Levenbook & Kuhn, 1962) can synthesize D-glutamine from D-glutamate. The most prominent metabolite of L-glutamate in gastric caeca was found to be glutamine (Fig. 6). The second most abundant product was alanine. Smaller quantities of radioactivity were present in other metabolites, but these were not identified. The metabolic products extracted from the tissue were also recovered from the bathing fluid. This indicates that they were produced in the tissue and released into the medium. To confirm the identity of alanine and glutamine as metabolites of L-glutamate, portions of an extract made from gastric caeca incubated with L-glutamate-U-1*C were subjected to a preliminary separation on paper chromatograms. The radioactive bands were located by scanning, eluted off, and subjected to re-chromatography

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System 2 • A B Fig. 5. Blood metabolites i h after infusing D-glutamate-i- u C into the alimentary tract of female L. imgratoria. A, Shows the distribution of radioactivity on the chromatogram. B, Shows the position of ninhydrin-positive spots (both standards and naturally occurring substances). The major peak of radioactivity corresponds in position with D-glutamate. The presence of significant quantities of radioactivity in glutamine shows this compound to be a metabolite of D-glutamate. Whether this represents D - or L-glutamine is not clear. Note that the direction of the two chromatographic systems are reversed compared to the earlier figures. Abbreviations: D-glutamate - D-Glu; glutamine - Glu (NH,); alanine - Ala; proline - Pro.

Table i. Major metabolites of h-glutamate-U-llC in Schistocerca gregariagastric caeca. Re-chromatography of individual peaks together with standards* Rr value Solvent systemf

1

2

3

4

L-Glutamate standard radioactive peak i L-Glutamine standard radioactive peak 2 L-Alanine standard radioactive peak 3

0-18 0-19

0-36 O-37 0-54 °-54

028 028

039 0-40 o-a8 oa8 0-54 o-53

o-is 0-15 O'35 0-25

052

os 1

0-47 0-46 0-64 0-64

5 0-37 0-36 0-32 0-32 o-SS

o-ss

14

• An extract made from gastric caeca incubated with L-glutamate-U- C was streaked on the base of Whatman No. 3 MM paper, radioactive peaks separated with system 3, each individual peak was located by scanning, eluted with ethanol/H!O (4/1), and re-chromatographed on each of the T L C systems together with the standard amino acid. t See text for composition of solvent systems.

(Table 1). The correspondence of radioactivity with the RF of standards confirms the identity of alanine and glutamine. The presence of glutamine was also supported by the observation that incubation of portions of the extract with glutaminase caused the glutamine peak to disappear. Also, radioactivity appeared in glutamine when gastric caeca were incubated with L-glutamate-i-14C, as would be expected. When glycine-i-14C was incubated with caeca, the only radioactive peak recovered corresponded to the unchanged starting material. This provides further evidence for thinking that the metabolism of glycine during absorption is minimal.

Blood metabolites

801

Glu(NH2) ^-Ala

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Fig. 6. Metabolites of L - G 1 U - U - " C after 3 h incubation with ScMstocerca gastric caeca. A, Shows the distribution of radioactivity on the chromatogram. B, Shows the position of ninhydrin-positive spots (both standards and and naturally occurring substances). The main metabolites are glutamine and alanine. Smaller amounts of radioactivity are seen at other positions, but the compounds responsible for them were not identified. Abbreviations: glutamate - Glu; alanine - Ala; glutamine - Glu (NH,); glycine - Gly; serine - Ser.

Fate of L-glutamate in blood

The gut infusion/haemocoel perfusion experiments (Murdock & Koidl, 1972) established that when L-glutamate-i-uC is infused into the locust gut lumen, only about one-fifth of the perfusate radioactivity is in a stable, non-volatile form. In view of the extensive metabolism which this amino acid undergoes, and the apparent coupling of released residual-radioactivity with that in "C/bicarbonate, it appeared probable that the residual-radioactivity actually represents a metabolic product, possibly glutamine. Glutamine is indeed the main product of L-glutamate metabolism in the major tissue-site of absorption, and the blood of animals that have been absorbing L-glutamate contain glutamine and not L-glutamate. Nevertheless, it is possible to imagine that some unchanged L-glutamate is released from the alimentary tract, but that the blood, acting perhaps in conjunction with other tissues, is capable of metabolizing it to glutamine sufficiently fast to prevent a L-glutamate build-up.

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L. L . MURDOCK AND B. KOIDL

F

Glu(NHj)

Glu

S

Fig. 7. Blood metabolites in blood of Scfustocerca after glow infusion of L-glutamate-i-14C into the haemocoel during the preceding hour (for details see text). Note the absence of radioactivity in L-glutamate. The radioactivity is recovered in glutamine. Chromatographic system 2. Abbreviations: glutamate - Glu; glutamine - Glu (NH,).

To evaluate this possibility, experiments were designed to closely simulate the release of small amounts of L-glutamate into the blood from the alimentary tract. The rationale of the experiments was as follows. If after slow infusion of small amounts of the amino acid into the blood, the L-glutamate is recovered unchanged, then it would be clear that the blood and associated tissues do not have the capacity to rapidly convert L-glutamate to L-glutamine; therefore the glutamine found in blood from cannulated gut-infused locusts must have been synthesized in the gut wall and released. If, however, glutamine is recovered as the exclusive product, it will be clear that blood and/or tissues do have the capacity of rapidly forming glutamine from glutamate. In this case it will not be possible to decide whether L-glutamate is released from the alimentary tract, but evidence for a possible complementary mechanism of reducing the level of blood L-glutamate will be obtained. The experiments were carried out as follows. A locust was cannulated and gut-infused with 100 /il of saline containing 50 mM non-radioactive L-glutamate. A second syringe, containing 10/il of saline in which 37-5 /ig L-glutamate and o-5-1-0/iCi of L-glutamate-i-MC had been dissolved, was inserted through an intersegmental membrane of the abdomen, and waxed in place. Beginning at the moment of injection of fluid into the gut, 1 fi\ of fluid from the second syringe was injected into the haemocoel. Every 6 min thereafter an additional 1 /A was injected, until after 54 min, the entire 10 /A had been administered. One hour after the gut infusion and 6 min after the last haemocoel injection, a sample of blood was taken from the cervical membrane and examined by chromatography. A typical result is shown in Fig. 7. No radioactivity was recovered in unchanged glutamate. The major product was glutamine. A second metabolite, in much smaller

Blood metabolites

803

Table 2 Fat body Phosphorylase Glyceraldehydephosphate dehydrogenase Citrate synthetase Malate dehydrogenase Aspartate aminotransferase Glutamate dehydrogenase /?-Hydroxyacyl-CoA dehydrogenase Phosphoenolpyruvate carboxykinase Alanine aminotransferase Malate dehydrogenase (decarboiylating)

Malpighian tubule*

Gastric caeca

Ventriculu*

3 760

870

1

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13

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766 49

70

65

4770 229S

2840 1280 263 S3O

526

920

1250

915

264

396

210 40

216 11

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186

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Activity of key metabolic enzymes in fat body, Malpighian tubules, gastric caeca, and midgut ventriculus of Locusta mgratoria. Activities given as /imoles x g"1 wet weight x h"1. • Values were measured after freezing the enzyme preparation overnight at — 30 °C.

quantity, was sometimes seen, but not identified. These experiments suggest that should L-glutamate enter the haemocoel from the gut or from other tissues, it will rapidly be converted to glutamine. Enzymic patterns in gastric caeca and other tissues

The activities of several key enzymes of major metabolic systems in gastric caeca and other tissues are presented in Table 2. A few general remarks will be given here; consideration of the role of certain enzymes in the metabolism of L-glutamate and L-alanine will be postponed until the Discussion. It is interesting that although the activity of the glutamate-oxaloacetate transaminase is higher than the glutamate-pyruvate transaminase in all four tissues examined (see also Kilby & Neville, 1957), L-aspartate does not appear as a significant metabolic product of either L-glutamate or L-alanine, both of which undoubtedly lead to the biosynthesis of MC-oxaloacetate, which could be transaminated to L-aspartate. 3-Hydroxyacyl-CoA dehydrogenase, an enzyme of fatty acid oxidation, is somewhat more active in fat body and in Malpighian tubules than in gut tissues. Phosphorylase, representing glycogenolytic capacity, is relatively low in the tissues of the gut. Glyceraldehyde phosphate dehydrogenase, a key glycolytic enzyme, is present at similar activity levels in all four tissues. The relatively low value of the ratio 'malic' enzyme/malic dehydrogenase in gut tissues (1/13 and 1/16) compared to Malpighian tubules (1/81) may reflect a relatively greater metabolic flow from malate to pyruvate in the gut wall. Very interesting is the finding of phosphoenolpyruvate carboxykinase in all tissues examined. The enzyme is particularly prominent in the caeca. It catalyses one of the initial steps of gluconeogenesis, so the caecum may be regarded as a possible site for this process. It is clear from this survey that the caeca have a high and characteristic metabolic capacity, rivalling the fat body in many respects. DISCUSSION

It would appear that rises in the blood concentration of L-glutamate in locusts are prevented. As has been previously shown (Murdock & Koidl, 1972), metabolism and limited permeability in the gut wall would severely restrict the entry of dietary

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L. L. MURDOCK AND B. K O I D L

L-glutamate into the blood. Post-absorption levels of L-glutamate, as shown here, usually so low as not to be detectable by the methods employed. By contrast, glycine" and alanine pass largely intact into the blood and are recovered. Even more striking is it that D-glutamate does enter the blood and can be recovered. Auclair (1959) also noted that feeding large quantities of L-glutamate to cockroaches did not result in an increased blood level of this amino acid, whereas feeding amino acids such as alanine, serine, and phenylalanine was followed by marked rises in their blood concentration. It also seems clear that if L-glutamate should enter the blood, either from the diet or from the tissues, it would be rapidly converted to glutamine. The site of glutamine synthesis is unknown. The L-glutamate may be taken up into certain tissues and undergo synthesis and release back into the blood as glutamine. Levenbook & Kuhn (1962) observed that the glutamine synthetase activity of Prodenia blood is very low, or absent. It is of interest to consider the metabolic pathways followed by luminal L-alanine and L-glutamate. It is not yet possible to designate all tissue sites of this metabolism, but it may be supposed that the gastric caeca are one significant site. In the case of L-alanine the major radioactive metabolites were XiCO2 (Murdock & Koidl, 1972) and glutamine. A probable major metabolic pathway begins with the transamination of alanine (Fig. 8). Transaminases capable of biosynthesizing pyruvate from alanine using L-glutamate or L-aspartate as co-substrates are widely distributed in locust tissues, including the gut wall (Kilby & Neville, 1957; see also Table 2). The next step would involve the action of the pyruvate dehydrogenase complex, forming acetyl-CoA and U CO 2 . The acetyl-CoA would then condense with oxaloacetate, a reaction catalysed by citrate synthetase; this enzyme is present in the locust gastric caeca (Table 2), where its activity is comparable with that of the other tissues, including fat body. The citrate, carrying a portion of the original carbon skeleton of alanine, would then cycle to the level of a-ketoglutarate, which itself would act as a co-substrate for the transamination of another molecule of alanine. This pathway accounts for the M CO 2 produced from alanine, but does not explain the appearance of WC in glutamine (formed via a-ketoglutarate and glutamate). The experiments were carried out with L-alanine-1-MC. In the pathway just considered, the No. 1 carbon of alanine is lost during the action of pyruvate dehydrogenase, so radioactivity would not appear in subsequent products of the citric acid cycle. To account for the appearance of radioactivity in glutamine it is necessary to find an alternative route whereby the entire carbon skeleton of alanine can enter the citric acid cycle. Such reactions can theoretically be catalysed by the malic enzyme, condensing COa and pyruvate to form malate. Malic enzyme is present in the locust tissues examined (Table 2), and has also been found in Schistocerca fat body (Walker & Bailey, 1969). It is thought, however, that this enzyme functions mainly in the reverse direction (Mahler & Cordes, 1966; Walker & Bailey, 1969). A more likely route involves the action of pyruvate carboxylase (Utter & Keech, 1963), which uses metabolic CO2 and pyruvate to form oxaloacetate. It might be thought that entrance of the UC via this route would likewise not lead to the appearance of 14C in glutamine: After condensation the pyruvate- i-14C appears in oxaloacetate-i-14C and, after condensation to form citrate and cycling to the level of oxalosuccinate, the labelled carbon is lost again as 14COa during a-ketoglutarate synthesis. However, equilibration of the oxalo-

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Blood metabolites OPOJHJ

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