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Estimation of glycogen in small amounts of tissue
ANALYTICAL
BIOCHEMISTRY
Estimation
of
11,
2!%-265
Glycogen EMILE
From
the Florida
State
(1965)
in Small VAN
Amounts
October
Tissue
HANDEL
Roard of Health. Entolnological Vera Beach, Flokln Received
of
Reseaxh
Center,
1, 1964
Most investigators favor, for isolation of total glyeogen from animal tissues, the modification of Good et al. (1) of Pfluger’s method (2), and assay the precipitate with the anthrone reagent (3). This combination of procedures yielded low and unreproducible results when applied to 0.1 mg of glycogen, to individual mosquitoes, or to 10 mg of liver. Therefore, each step of these procedures was reinvestigated. By taking into consideration factors affecting the precipit.ability of aqueous glycogen solutions by ethanol, quantitative isolation of as little as 1 pg of glycogen from animal tissue was achieved. REACTION
WITH
ANTHRONE
As the estimation of glycogen depended on anthrone, this reaction will be described first. The procedure of Seifter et al. (3) was used, modified t,o avoid heat of mixing and to keep the maximum color stable for a considerable time. While stirring and cooling, 760 ml sulfuric acid (d = 1.84) is carefully poured into 300 ml water. A solution of 0.15% ant’hrone in this diluted acid is prepared on the day of USC. The reaction is carried out in 16 X 100 mm culture tubes. If the glycogen aliquot is present as an aqueous solution or dispersion, it should be concentrat,ed at 110°C until about 0.05 ml remains* ; if the aliquot is available as a precipitate, it should be dispersed in 0.05 ml water. Three ml reagents arc added and the tubes are heated for 20 min at 9O”,l chilled in ice water, and measured at 620 mp. The optical density reaches a maximum in 15 min and remains constant for about 10 min. (When the tubes are heated in boiling water the maximum color is reached in 5 min and remains constant for not more than 2 min.) 1 The 16 x 100 mm 80 mm holes, mounted and at 9O’C.
tubes were heated in aluminum blocks, on Thermolyne (type 2200) hot plates. 2.56
provided with 17 x maintained at 110”
257 PRECIPIT-ITION
OF GLTCOGI’,X
WITH
ETHANOI,
Glycogen? is extremely insoluble in 50 or 6670 ethanol. This was determined by placing portions of 10 mg glycogcn in a glass-stoppered cent,rifuge tube, shaking for 0.5 hour with 10 ml diluted ethanol (40-8070), centrifuging for 20 min at 2000 rpm, and reacting the supernatant with anthrone (Table la). However, when glycogen was dissolved in 5 ml water and 5 ml ethanol was added, far more appeared in the supernatant than would be expected from it,s solubility. When the precipitate was redissolved in 5 ml water and reprecipitated with 5 ml ethanol, 33% was lost from 10 mg and 98% from 1 mg glycogen aft,er 3 precipitations (Table lb). TABLE 1 EFFECT OF ETH.ANOL ON GLYCOGEK PRECIPITATION Solubility .f($ mg glycogeu in 10 ml diluted ethanol Recoveredin Ethanol roncn., supernatant. u/o mg 40 41 -2’2 43 44 45 50 66 80
10 6 2 0.4 0.3 0.12 0.01 0.005 0.000
(b)
Precipitability of 5 ml aqueous glycogen by 5 ml ethanol __-Glycogen Fracticm 10 mg 1 mg First sup. Second sup. Third sup. Left in ppt,.
0.2 mK 0.3 ‘2 s
0.65 mg 0.28 0.05
6.5
0.02
-
Several workers have suggested that electrolytes accelerate the precipitation. In the Pfluger procedure, KOH would provide such an electrolyte. In 1929, Osterberg used a 1% sodium sulfate solution to coprecipitate with glycogen on addition of ethanol and showed that 20 pg glycogen, added to protein, could be quantitatively recovered (4). As Osterberg did not discuss recoveries without addition of a salt, experiments were carried out to illustrate the effect of Na,SO, and some other electrolytes (trichloroacetic acid, KOH, and LiBr). To 50 pg glycogen (0.05 ml 0.1% solution) were added 0.4 ml water and 0.05 ml of an electrolyte solution. Ethanol (1 ml) was added and ‘The pure glycogen used was either rabbit liver or oyster glycogen (Nutritional Biochemical Co.). According to the manufacturer, both were prepared by boiling the raw material with KOH and repeated precipitation with alcohol. No difference was found between the two types.
258
EMILE
VAN
HANDEL
glycogen was determined in the centrifugate (first precipitate). In a concomitant experiment the centrifugate was redissolved in 0.5 ml water and reprecipitated with 1 ml ethanol (second precipitate). Of these 4 electrolytes only NaSO, precipitates on addition of ethanol. Alcoholsoluble electrolytes are ineffective, as prohibitive losses occur even in the first precipitation (Table 2a). Obviously, quantitative precipitation of micro amounts of glycogen does not depend on electrolytes per se, but on those electrolytes which coprecipitate with glycogen. TABLE EFFECT
OF ELECTROLYTES
ON
(4
Glycogen (0.05 ml), PIi?
~~,~~ly
m
2
RECOVERY OF GLYCOQEN Recovered in first ppt., PI3
Recovered in second ppt., cg
10 30 16 23 25 49
5 10 8 5 15 49
50 50 50 50 50 50
(Water) 100% TCA 50% LiBr 60% KOHo 5% NazfO Satd. Na2S04
Glycogen (0.05 ml), Pg
Electrolyte (0.05 ml)
0.4 ml water
50 50 50 50
(Water) 5% NapSO 10% NazSOc Satd. Na&OI
5 15 30 50
Beef liver powder, w
(b)
(4 Glyoogen, !a
0.05 ml satd. NarS01
10
-
-
10 10 10
100 -
+
10
100
+
Recovery
in second
ppt.,
pg
0.4 ml ‘30% KOHa
15 49 50 50
Recovery in second ppt., !a
14 57 28 48 147
Vol.
ethanol
1.2 1.2 2 2 2
a COS-free.
In the Pfluger method glycogen is always precipitated from strongly alkaline solutions. Therefore the simultaneous effect of KOH and Na,S04 was determined. To 50 pg glycogen and 0.05 ml Na,SO, was added 0.4 ml water or 0.4 ml 30% KOH. Ethanol (1 ml) was added, and the centrifugate was dissolved in 1 ml water and reprecipitated with 1 ml ethanol. The results are presented in Table 2b.
MICRO
ASSAY
OF
GLYCOGEN
259
The effect of KOH and Na,SO, is strongly additive, but as purification of glycogen in biological material often requires repeated precipitation, which rapidly exhausts the KOH, it is recommended that saturated Na,SO, solution be used as the coprecipitant: when 50 pg glycogen in 0.5 ml water was precipitated with 1 ml ethanol in the presence of 0.05 ml saturated Na,SO,, 47 pg could be recovered after 5 such precipitations. In biological products losses were equally severe in the absence of Na2S04. Beef liver powder (Nut,ritional Biochemical Co.) was heated at 100” for 10 min with 0.5 ml 30% KOH (CO,-free) and brought to a boil after addition of 0.6 to 1 ml ethanol. The centrifugate was dispersed in 0.5 ml water and again brought to a boil with 0.6 to 1 ml ethanol (Table 2~). This is the procedure of Good et al. (1). Heating after addition of ethanol to flocculate glycogen is unnecessary in the presence of Na,SO, and was no help in cases in which no salt was added. NaZSOI is more efficient as a coprecipitant than other salts: it is very soluble in water (~30%), but only slightly soluble in 66% ethanol (0.18%) ; it crystallizes readily on addition of ethanol and does not interfere with the anthrone reaction. EFFECT
OF CARBONATE
Practical KOH solutions contain varying amounts of carbonate, which could also provide a vehicle for precipitat,ion of glycogen on addition of ethanol. Therefore carbonate-free KOH solutions were used throughout (prepared from saturated KOH by addition of 10 vol ethanol, decantation of the supernatant, addition of some water, and evaporation of alcohol). The solution should remain miscible with 10 vol ethanol. Carbonate may fulfil the same role as sodium sulfate when it crystallizes on addition of ethanol, but more often an excess carbonate concentrates in the bottom of the tube as a liquid drop. When the supernatants are decanted and the tubes are allowed to drain in an inverted position, that drop will be lost, carrying along a portion of the glycogen. This loss occurs even in the presence of well crystallized Na,SO,. When KOH is heated for a prolonged time in open tubes, it absorbs increasing amounts of CO,, mixed with increasing amounts of silicates from t.he glass walls. Unawareness of these facts may lead to the false conclusion that pure glycogen or glycogen in tissue is not stable toward prolonged boiling with concentrated alkali. On prolonged boiling the apparent glycogen content of beef liver powder fell from 0.47% (15 min, 30% KOH) to 0.25% (6 hr, 30% KOH) and from 0.48% (15 min, 60% KOH) to 0.12% (6 hr, 60% KOH). The effect of carbonate can be produced instantaneously by adding K&O, to carbonate-free KOH. To 250 pg glycogen (0.05 ml) and 0.05 ml saturated Na,SO, were added portions of 0.4 ml 60% KOH containing 0, 1, 2, and 5% saturated K,CO, solution. Glycogen was precipitated
260
EMILE
VAIi
HANDEL
with 1 ml ethanol, the centrifugate was decanted, and the tubes were allowed to drain in inverted position. The recovery of glycogen in the precipitate was 250, 160, 110, and 75 pg, respectively. The error can be remedied by dilution of an aliquot wit,h sufficient. water to prevent formation of a subnatant on addition of ethanol, or, more practically, by removal of the supernatant with a syringe, carefully avoiding removal of any subnatant. SOLUBILITY
OF
GLYCOGEN
IN
WATER
AFTER
TREATMENT
WITH
KOH
When liver is treated with KOH, the digest is turbid, but, can be clarified by centrifugation. The question has arisen whether this precipitate contains glycogen. Seifter et al. argue that the washed material behaves like glycogen toward anthrone and toward iodine, They caution that centrifugation before aliquots are taken causes error (3). Carroll et al. claimed that it is not glycogen because it is insoluble in KOH and water, though it responded to anthrone, and after hydrolysis, to alkaline copper solution (5). In tissue of low glycogen content the proportion of waterinsoluble anthrone-positive material may be very high and we have therefore investigated under what conditions this insoluble mat,erial occurs in alkaline digests. Chicken liver was treated with 60 and 30% KOH containing 5% saturated NaSO,. The tubes were kept at 100°C for 15 min, 1 hr, 4 hr, 8 hr, and 20 hr. At each time interval the tubes were rapidly cooled, and shaken vigorously prior to removing an aliquot. Glycogen was precipitated with 2 vol ethanol, dispersed in water, and reprecipitated. Supernatants were removed with a syringe. The precipitates were dispersed in water and the anthrone-reactive material measured before and after centrifugation. The results show that, when chicken liver was heated from 15 min to 20 hr with KOH, the glycogen content did not. change; but, when from the material precipitated with ethanol only the mater-soluble fraction was reacted with anthrone, the estimate was erratic and irreproducible (Table 3). We have further determined that, in livers of fed and fasted rats, in beef liver and muscle, and in mosquitoes, the amount of anthrone-positive, alcohol-precipitable material after 24-hr treatment with KOH was 9%105% of that found after 15 min, but only if in the 15-min samples the glycogen dispersions were not clarified by centrifugation. After 24 hr, centrifugation made no difference. When an aqueous solution of 25 Kg glycogen was heated with 30% KOH from 3-15 min, then precipitated twice with ethanol, IO-15 pg had become insoluble in water. When heating with KOH was continued, it
MICRO
ASSAY
OF
261
GLTCOGEX
again became water-soluble. This was not proportional t’o the amount of glycogen present, as out of 100 pg glycogen not more than lo-15 pg became insoluble between 3 and 15 min.
EFFECT
OF
PROLONGED
HE.ATING
UPON
GLYCOGEN Glycogen,
30%
chicken
KOII Watersoluble
Total
mirl
2 i0
1.90
2.60
hr hr hr hr
2.70 ‘1.75 “.iO 2.65
1.35 0.95 2.45 2.60
2.55 2.45 2.60 2.iO
EVIDENCE
FOR
IN
liver 60%
Total
Timr
15 1 4 8 20
mg/gm
(CONCENTRATION
LIVER
__. IiOH watersoluble
1.60 1 .:x3 1.55 2.30 2.iO
AT LEBST TWO WATER-INSOLUBLE GLTCOGES COMPLEXES
In the preceding section it was shown that, during alkaline digestion, the water-insoluble material first increases, then decreases. This suggests the occurrence or the formation of at least two water-insoluble products, proof of which is given in the following experiment: cheken liver was heated at 100°C for 15 min wit,h 30% KOH. The digest was cooled, centrifuged, and separated into an alkali-soluble supernatant and an alkali-insoluble precipitate. Aliquots of the alkali-soluble supernatant were precipitated twice with ethanol or they were heated again for 4 and 8 hr and then precipitated twice with ethanol. This precipitate was separated int,o a water-soluble and a water-insoluble fraction (Table 4al. The alkali-insoluble precipitate was washed wit,h water and heated with fresh 30% KOH. At intervals the digest was centrifuged, the supernatant precipitated with ethanol, and the precipitate reacted with anthrone, to determine the amount solubilizcd by prolonged boiling with KOH (Table 4b). Table 4a shows that, on addition of ethanol to the clarified alkalisoluble fraction of liver digest’, new water-insoluble products are formed. They reappeared in the water-soluble fraction after prolonged heating of the alkali-soluble digest. The fraction originally insoluble in 30% KOH was also gradually solubilized after prolonged heat,ing with KOH. Therefore, it seems a safe assumption to calculate as glycogen all anthronepositive material precipitable with ethanol after digestion with alkali. This includes material insoluble in water. The chitin skeleton of insects is insoluble in KOH. After digestion and
EMILE
VAN
HANDEL
TABLE GLYCOQEN
WATER-INSOLUBLE
4 COMPLEXES
IN LIVER
Alkali-soluble chicken liver digest (supernatant after 16min treatment with 30% KOH) Heated with KOH. then pptd. with ethanol
Glycogen,
0 hr 4 hr 8 hr Alkali-insoluble after
liver Water-soluble
3.0 3.0 3.0
1.8 2.6 2.9
(b)
lbmin
m&pm
Total
chicken liver treatment
digest (centrifugate with KOH) Glycogen,
Heated with KOH. then pptd. with ethanol
mg/gm
liver
Solubilized
O-2 hr 24 4-8 8-24
0.12 0.21 0.12 0.06 Total solubilised: 0.51 Still
24 hr
insoluble
0.09 Total: 0.60
with ethanol, the precipitate is dispersed in water and an aliquot is removed with a syringe, avoiding the transfer of gross particles. In mosquitoes, contrary to liver or beef muscle, we found that the supernatants are quite clear and the anthrone-sensitive material is the same after centrifugation.
precipitation
SENSITIVITY
OF
THE
GLYCOGEN
DETERMINATION
Liver of a rat, starved for 5 days, was heated with 30% KOH for 15 min. Portions of 5 mg (0.4 ml) of the digest were pipetted into centrifuge tubes; 0.05 ml Na,SO,, glycogen solution, and 1 ml ethanol were added. The centrifugate was dispersed in 0.5 ml water and reprecipitated with 1 ml ethanol. With 2 mg liver, 0.2 ml aqueous phase was precipitated with 0.4 ml ethanol and groups of 4 precipitates were pooled in 2 ml anthrone in order to obtain sufficient material to give an optica density reading. Mosquitoes, starved to death, were analyzed individually. Their average dry weight was 0.7 mg (Table 5).
MICRO
ASSAY
OF
263
tiLTCOGEN
TABLE 5 SENSITIVITY
OF THE
Substrate
Rat liver (5X repeated) 5w
GLYCOGEN Glycogen
added,
-
5 5
1:
2
-
2
DETERMINATION
r
2
100
Rlosquito (8 X repeated) 1 each 1 each
pg
Glycogen recovered -f S.E., fig
3.3
+
0.2
9.0
f
0.4
13.0
k 0.5
1.2
+ 0.1
3.6 66
4 10
14
k
0.2
t1
f0.4 & 0.4
Microgram quantities could be quantitatively isolated from tissue of very low glycogen content, but the analytical application is limited by the sensitivity of the anthrone reaction. DISCUSSIO?; Three sources of error were recognized in Pfluger’s and Good’s methods, all leading to losses of glycogen: (a) discrepancy between solubility of glycogen in dilute ethanol and its precipitability from aqueous solutions by ethanol (Table 1) ; (b) presence of carbonate in aqueous concentrated KOH; (c) formation of a water-insoluble glycogen complex (Tables 3 and 4). With the proposed modifications the method is suitable for biological material containing only a few micrograms of glycogen (Table 5). Quantitative precipitation of glycogen by ethanol was achieved by addition of sodium sulfate to the aqueous solution. Electrolytes soluble in ethanol were not effective (Table 2). Addition of ethanol to carbonatecontaining KOH may cause a concentration of noncrystalline, liquid carbonate and unprecipitated glycogen as a liquid subnatant that would be lost on drainage of the supernatant. The use of carbonate-free KOH or removal of the supernatant with a syringe prevented this loss. Glycogen precipitated with ethanol from alkaline liver digests does not redissolve entirely in water. Calculation of the water-soluble fraction only, as true glycogen, leads to unreproducible and low results. At least 20 hr boiling with concentrated KOH was required to render all glycogen soluble in water after precipitation with ethanol (Table 3). Carroll et al. observed that, when liver was boiled for a prolonged time with 30% KOH, the anthrone-positive material precipitable with ethanol continuously diminished, whereas pure glycogen did not change in this way upon boiling. They concluded that this was due to decom-
264
lSMILE
VAN
HANDEL
position of a nonglycogen complex into fragments not precipitable by ethanol (5). The presence of such a nonglycogen complex would invalidate any glycogcn determination. However, WC believe this observation to be in error, as these authors calculated glycogen as the water-soluble anthrone-positive fraction of the precipitate; they were unaware of the water-insoluble complexes formed during digestion and precipitation with ethanol (Tables 3 and 4). It has long been thought that glycogen in tissues exism in at least two forms: a “free” form, extractable with trichloroacet.ic acid, and a “bound” form extractable with hot KOH. Roe et al. showed that any such differentiation is artifactual and indicative of incomplete extraction in the first place. When tissue was dispersed in TCA at 45,000 rpm in a tissue homogenizer in the presence of homogenizing glass beads, no residual glycogen could be extracted with boiling KOH (6). Furthermore, as seen in Table 2a, TCA does not bring about quantitative precipitation of glycogen on addition of ethanol. In mosquitoes, approximately 65% of t,he glycogen could be extracted with hot water, t,he remainder with hot KOH. When alkaline mosquito digests were treated with hot ethanol, the precipitated glycogen redissolved completely in water. This contrasts with the glycogen of alkaline liver digests. Following convention, we defined as glycogen all anthrone-sensitive material with an absorpt,ion peak at 620 m,u precipitable with ethanol after alkaline digestion. This definition requires some qualification. Glycogen is a polydisperse material, inhomogeneously distributed within cells and suspected to exist in different forms. The molecular weight of glycogen is not known with certainty, as ext,raction with either acid or alkali causes structural changes, apparent by a gradual decline of molecular weight (7). Yet, in our experience, heating at 100°C with 60% KOH for 24 hr did not diminish the total amount of anthrone-positive mat,erial precipitable by ethanol from liver, mosquitoes, and pure glycogen. Overestimat,ion, especially in tissue of low glycogen content, could bc clue to the presence of polysaccharides metabolically different from glycogen or from other anthrone-positive products precipitable under the same conditions. Little or none of such inert material was present in the biological material used to illustrate the recovery of added glycogen in Table 5: in a fed rat the apparent glycogen content of liver was 670, whereas in a rat fasted for 5 days it was 0.06%; similarly, in mosquitoes glycogen fell from 0.3 mg in fed specimens to 0.002 mg in mosquitoes starved to death. Either case demonstrates that 99F of the material calculated as glycogen could be metabolized.
SUMMARY
Some physical properties of glycogen have been investigated and applied to micro isolation and assay in biological material containing only a few micrograms of glycogen. Losses of glycogen in the ethanol precipitation step were found to be preventable by the use of a coprecipitant, of which Na,SO, was the most satisfactory. Evidence for the existence or formation of at least two water-insoluble glycogen complexes in liver was presented. Treatment at 100°C with concentrated KOH for 20 hr rendered all glycogen soluble in water. ACKNOWLEDGMENTS This work was supported bp a grant (AI 05054) from the Sational Health. The analyses mere performed by J. E. Guirn.
Institutes
of
REFEREKCEB A., KHAMER, H., AND Smocrr, M.. J. Riol. Chem. 100, 485 (1933). E., Arch. Ges. Phyxiol. 129, 362 (1909). S., D.ATTON, S., XOVIC. B., .4~n MCNTWYLER, E., Arch. Biochem. Biophys. 25, 191 (1950). 4. OSTERBERG, il. E.. J. Riol. Chem. 85, 97 (1929). 5. CARROLL, N. v,, LOSGLEY. R. ITT., ASD ROE, J. H.. J. Biol. C&m. 220, 583 (1956). 6. ROE, J. H., B.~II,EY, J. M., GRAY, R. R., .~SD RORISSON, J. N., J. Riol. Chem. 23& 1244 (1961). 7. STETTEN, M. R., KATZFX, H. M., AND STETTEX. D., JR.. J. Biol. Chem. 232, 475 (1958) 1. GOOD,
3. PFLUGER, 3. SEIFTER,
C.
BIOCHEMISTRY
Estimation
of
11,
2!%-265
Glycogen EMILE
From
the Florida
State
(1965)
in Small VAN
Amounts
October
Tissue
HANDEL
Roard of Health. Entolnological Vera Beach, Flokln Received
of
Reseaxh
Center,
1, 1964
Most investigators favor, for isolation of total glyeogen from animal tissues, the modification of Good et al. (1) of Pfluger’s method (2), and assay the precipitate with the anthrone reagent (3). This combination of procedures yielded low and unreproducible results when applied to 0.1 mg of glycogen, to individual mosquitoes, or to 10 mg of liver. Therefore, each step of these procedures was reinvestigated. By taking into consideration factors affecting the precipit.ability of aqueous glycogen solutions by ethanol, quantitative isolation of as little as 1 pg of glycogen from animal tissue was achieved. REACTION
WITH
ANTHRONE
As the estimation of glycogen depended on anthrone, this reaction will be described first. The procedure of Seifter et al. (3) was used, modified t,o avoid heat of mixing and to keep the maximum color stable for a considerable time. While stirring and cooling, 760 ml sulfuric acid (d = 1.84) is carefully poured into 300 ml water. A solution of 0.15% ant’hrone in this diluted acid is prepared on the day of USC. The reaction is carried out in 16 X 100 mm culture tubes. If the glycogen aliquot is present as an aqueous solution or dispersion, it should be concentrat,ed at 110°C until about 0.05 ml remains* ; if the aliquot is available as a precipitate, it should be dispersed in 0.05 ml water. Three ml reagents arc added and the tubes are heated for 20 min at 9O”,l chilled in ice water, and measured at 620 mp. The optical density reaches a maximum in 15 min and remains constant for about 10 min. (When the tubes are heated in boiling water the maximum color is reached in 5 min and remains constant for not more than 2 min.) 1 The 16 x 100 mm 80 mm holes, mounted and at 9O’C.
tubes were heated in aluminum blocks, on Thermolyne (type 2200) hot plates. 2.56
provided with 17 x maintained at 110”
257 PRECIPIT-ITION
OF GLTCOGI’,X
WITH
ETHANOI,
Glycogen? is extremely insoluble in 50 or 6670 ethanol. This was determined by placing portions of 10 mg glycogcn in a glass-stoppered cent,rifuge tube, shaking for 0.5 hour with 10 ml diluted ethanol (40-8070), centrifuging for 20 min at 2000 rpm, and reacting the supernatant with anthrone (Table la). However, when glycogen was dissolved in 5 ml water and 5 ml ethanol was added, far more appeared in the supernatant than would be expected from it,s solubility. When the precipitate was redissolved in 5 ml water and reprecipitated with 5 ml ethanol, 33% was lost from 10 mg and 98% from 1 mg glycogen aft,er 3 precipitations (Table lb). TABLE 1 EFFECT OF ETH.ANOL ON GLYCOGEK PRECIPITATION Solubility .f($ mg glycogeu in 10 ml diluted ethanol Recoveredin Ethanol roncn., supernatant. u/o mg 40 41 -2’2 43 44 45 50 66 80
10 6 2 0.4 0.3 0.12 0.01 0.005 0.000
(b)
Precipitability of 5 ml aqueous glycogen by 5 ml ethanol __-Glycogen Fracticm 10 mg 1 mg First sup. Second sup. Third sup. Left in ppt,.
0.2 mK 0.3 ‘2 s
0.65 mg 0.28 0.05
6.5
0.02
-
Several workers have suggested that electrolytes accelerate the precipitation. In the Pfluger procedure, KOH would provide such an electrolyte. In 1929, Osterberg used a 1% sodium sulfate solution to coprecipitate with glycogen on addition of ethanol and showed that 20 pg glycogen, added to protein, could be quantitatively recovered (4). As Osterberg did not discuss recoveries without addition of a salt, experiments were carried out to illustrate the effect of Na,SO, and some other electrolytes (trichloroacetic acid, KOH, and LiBr). To 50 pg glycogen (0.05 ml 0.1% solution) were added 0.4 ml water and 0.05 ml of an electrolyte solution. Ethanol (1 ml) was added and ‘The pure glycogen used was either rabbit liver or oyster glycogen (Nutritional Biochemical Co.). According to the manufacturer, both were prepared by boiling the raw material with KOH and repeated precipitation with alcohol. No difference was found between the two types.
258
EMILE
VAN
HANDEL
glycogen was determined in the centrifugate (first precipitate). In a concomitant experiment the centrifugate was redissolved in 0.5 ml water and reprecipitated with 1 ml ethanol (second precipitate). Of these 4 electrolytes only NaSO, precipitates on addition of ethanol. Alcoholsoluble electrolytes are ineffective, as prohibitive losses occur even in the first precipitation (Table 2a). Obviously, quantitative precipitation of micro amounts of glycogen does not depend on electrolytes per se, but on those electrolytes which coprecipitate with glycogen. TABLE EFFECT
OF ELECTROLYTES
ON
(4
Glycogen (0.05 ml), PIi?
~~,~~ly
m
2
RECOVERY OF GLYCOQEN Recovered in first ppt., PI3
Recovered in second ppt., cg
10 30 16 23 25 49
5 10 8 5 15 49
50 50 50 50 50 50
(Water) 100% TCA 50% LiBr 60% KOHo 5% NazfO Satd. Na2S04
Glycogen (0.05 ml), Pg
Electrolyte (0.05 ml)
0.4 ml water
50 50 50 50
(Water) 5% NapSO 10% NazSOc Satd. Na&OI
5 15 30 50
Beef liver powder, w
(b)
(4 Glyoogen, !a
0.05 ml satd. NarS01
10
-
-
10 10 10
100 -
+
10
100
+
Recovery
in second
ppt.,
pg
0.4 ml ‘30% KOHa
15 49 50 50
Recovery in second ppt., !a
14 57 28 48 147
Vol.
ethanol
1.2 1.2 2 2 2
a COS-free.
In the Pfluger method glycogen is always precipitated from strongly alkaline solutions. Therefore the simultaneous effect of KOH and Na,S04 was determined. To 50 pg glycogen and 0.05 ml Na,SO, was added 0.4 ml water or 0.4 ml 30% KOH. Ethanol (1 ml) was added, and the centrifugate was dissolved in 1 ml water and reprecipitated with 1 ml ethanol. The results are presented in Table 2b.
MICRO
ASSAY
OF
GLYCOGEN
259
The effect of KOH and Na,SO, is strongly additive, but as purification of glycogen in biological material often requires repeated precipitation, which rapidly exhausts the KOH, it is recommended that saturated Na,SO, solution be used as the coprecipitant: when 50 pg glycogen in 0.5 ml water was precipitated with 1 ml ethanol in the presence of 0.05 ml saturated Na,SO,, 47 pg could be recovered after 5 such precipitations. In biological products losses were equally severe in the absence of Na2S04. Beef liver powder (Nut,ritional Biochemical Co.) was heated at 100” for 10 min with 0.5 ml 30% KOH (CO,-free) and brought to a boil after addition of 0.6 to 1 ml ethanol. The centrifugate was dispersed in 0.5 ml water and again brought to a boil with 0.6 to 1 ml ethanol (Table 2~). This is the procedure of Good et al. (1). Heating after addition of ethanol to flocculate glycogen is unnecessary in the presence of Na,SO, and was no help in cases in which no salt was added. NaZSOI is more efficient as a coprecipitant than other salts: it is very soluble in water (~30%), but only slightly soluble in 66% ethanol (0.18%) ; it crystallizes readily on addition of ethanol and does not interfere with the anthrone reaction. EFFECT
OF CARBONATE
Practical KOH solutions contain varying amounts of carbonate, which could also provide a vehicle for precipitat,ion of glycogen on addition of ethanol. Therefore carbonate-free KOH solutions were used throughout (prepared from saturated KOH by addition of 10 vol ethanol, decantation of the supernatant, addition of some water, and evaporation of alcohol). The solution should remain miscible with 10 vol ethanol. Carbonate may fulfil the same role as sodium sulfate when it crystallizes on addition of ethanol, but more often an excess carbonate concentrates in the bottom of the tube as a liquid drop. When the supernatants are decanted and the tubes are allowed to drain in an inverted position, that drop will be lost, carrying along a portion of the glycogen. This loss occurs even in the presence of well crystallized Na,SO,. When KOH is heated for a prolonged time in open tubes, it absorbs increasing amounts of CO,, mixed with increasing amounts of silicates from t.he glass walls. Unawareness of these facts may lead to the false conclusion that pure glycogen or glycogen in tissue is not stable toward prolonged boiling with concentrated alkali. On prolonged boiling the apparent glycogen content of beef liver powder fell from 0.47% (15 min, 30% KOH) to 0.25% (6 hr, 30% KOH) and from 0.48% (15 min, 60% KOH) to 0.12% (6 hr, 60% KOH). The effect of carbonate can be produced instantaneously by adding K&O, to carbonate-free KOH. To 250 pg glycogen (0.05 ml) and 0.05 ml saturated Na,SO, were added portions of 0.4 ml 60% KOH containing 0, 1, 2, and 5% saturated K,CO, solution. Glycogen was precipitated
260
EMILE
VAIi
HANDEL
with 1 ml ethanol, the centrifugate was decanted, and the tubes were allowed to drain in inverted position. The recovery of glycogen in the precipitate was 250, 160, 110, and 75 pg, respectively. The error can be remedied by dilution of an aliquot wit,h sufficient. water to prevent formation of a subnatant on addition of ethanol, or, more practically, by removal of the supernatant with a syringe, carefully avoiding removal of any subnatant. SOLUBILITY
OF
GLYCOGEN
IN
WATER
AFTER
TREATMENT
WITH
KOH
When liver is treated with KOH, the digest is turbid, but, can be clarified by centrifugation. The question has arisen whether this precipitate contains glycogen. Seifter et al. argue that the washed material behaves like glycogen toward anthrone and toward iodine, They caution that centrifugation before aliquots are taken causes error (3). Carroll et al. claimed that it is not glycogen because it is insoluble in KOH and water, though it responded to anthrone, and after hydrolysis, to alkaline copper solution (5). In tissue of low glycogen content the proportion of waterinsoluble anthrone-positive material may be very high and we have therefore investigated under what conditions this insoluble mat,erial occurs in alkaline digests. Chicken liver was treated with 60 and 30% KOH containing 5% saturated NaSO,. The tubes were kept at 100°C for 15 min, 1 hr, 4 hr, 8 hr, and 20 hr. At each time interval the tubes were rapidly cooled, and shaken vigorously prior to removing an aliquot. Glycogen was precipitated with 2 vol ethanol, dispersed in water, and reprecipitated. Supernatants were removed with a syringe. The precipitates were dispersed in water and the anthrone-reactive material measured before and after centrifugation. The results show that, when chicken liver was heated from 15 min to 20 hr with KOH, the glycogen content did not. change; but, when from the material precipitated with ethanol only the mater-soluble fraction was reacted with anthrone, the estimate was erratic and irreproducible (Table 3). We have further determined that, in livers of fed and fasted rats, in beef liver and muscle, and in mosquitoes, the amount of anthrone-positive, alcohol-precipitable material after 24-hr treatment with KOH was 9%105% of that found after 15 min, but only if in the 15-min samples the glycogen dispersions were not clarified by centrifugation. After 24 hr, centrifugation made no difference. When an aqueous solution of 25 Kg glycogen was heated with 30% KOH from 3-15 min, then precipitated twice with ethanol, IO-15 pg had become insoluble in water. When heating with KOH was continued, it
MICRO
ASSAY
OF
261
GLTCOGEX
again became water-soluble. This was not proportional t’o the amount of glycogen present, as out of 100 pg glycogen not more than lo-15 pg became insoluble between 3 and 15 min.
EFFECT
OF
PROLONGED
HE.ATING
UPON
GLYCOGEN Glycogen,
30%
chicken
KOII Watersoluble
Total
mirl
2 i0
1.90
2.60
hr hr hr hr
2.70 ‘1.75 “.iO 2.65
1.35 0.95 2.45 2.60
2.55 2.45 2.60 2.iO
EVIDENCE
FOR
IN
liver 60%
Total
Timr
15 1 4 8 20
mg/gm
(CONCENTRATION
LIVER
__. IiOH watersoluble
1.60 1 .:x3 1.55 2.30 2.iO
AT LEBST TWO WATER-INSOLUBLE GLTCOGES COMPLEXES
In the preceding section it was shown that, during alkaline digestion, the water-insoluble material first increases, then decreases. This suggests the occurrence or the formation of at least two water-insoluble products, proof of which is given in the following experiment: cheken liver was heated at 100°C for 15 min wit,h 30% KOH. The digest was cooled, centrifuged, and separated into an alkali-soluble supernatant and an alkali-insoluble precipitate. Aliquots of the alkali-soluble supernatant were precipitated twice with ethanol or they were heated again for 4 and 8 hr and then precipitated twice with ethanol. This precipitate was separated int,o a water-soluble and a water-insoluble fraction (Table 4al. The alkali-insoluble precipitate was washed wit,h water and heated with fresh 30% KOH. At intervals the digest was centrifuged, the supernatant precipitated with ethanol, and the precipitate reacted with anthrone, to determine the amount solubilizcd by prolonged boiling with KOH (Table 4b). Table 4a shows that, on addition of ethanol to the clarified alkalisoluble fraction of liver digest’, new water-insoluble products are formed. They reappeared in the water-soluble fraction after prolonged heating of the alkali-soluble digest. The fraction originally insoluble in 30% KOH was also gradually solubilized after prolonged heat,ing with KOH. Therefore, it seems a safe assumption to calculate as glycogen all anthronepositive material precipitable with ethanol after digestion with alkali. This includes material insoluble in water. The chitin skeleton of insects is insoluble in KOH. After digestion and
EMILE
VAN
HANDEL
TABLE GLYCOQEN
WATER-INSOLUBLE
4 COMPLEXES
IN LIVER
Alkali-soluble chicken liver digest (supernatant after 16min treatment with 30% KOH) Heated with KOH. then pptd. with ethanol
Glycogen,
0 hr 4 hr 8 hr Alkali-insoluble after
liver Water-soluble
3.0 3.0 3.0
1.8 2.6 2.9
(b)
lbmin
m&pm
Total
chicken liver treatment
digest (centrifugate with KOH) Glycogen,
Heated with KOH. then pptd. with ethanol
mg/gm
liver
Solubilized
O-2 hr 24 4-8 8-24
0.12 0.21 0.12 0.06 Total solubilised: 0.51 Still
24 hr
insoluble
0.09 Total: 0.60
with ethanol, the precipitate is dispersed in water and an aliquot is removed with a syringe, avoiding the transfer of gross particles. In mosquitoes, contrary to liver or beef muscle, we found that the supernatants are quite clear and the anthrone-sensitive material is the same after centrifugation.
precipitation
SENSITIVITY
OF
THE
GLYCOGEN
DETERMINATION
Liver of a rat, starved for 5 days, was heated with 30% KOH for 15 min. Portions of 5 mg (0.4 ml) of the digest were pipetted into centrifuge tubes; 0.05 ml Na,SO,, glycogen solution, and 1 ml ethanol were added. The centrifugate was dispersed in 0.5 ml water and reprecipitated with 1 ml ethanol. With 2 mg liver, 0.2 ml aqueous phase was precipitated with 0.4 ml ethanol and groups of 4 precipitates were pooled in 2 ml anthrone in order to obtain sufficient material to give an optica density reading. Mosquitoes, starved to death, were analyzed individually. Their average dry weight was 0.7 mg (Table 5).
MICRO
ASSAY
OF
263
tiLTCOGEN
TABLE 5 SENSITIVITY
OF THE
Substrate
Rat liver (5X repeated) 5w
GLYCOGEN Glycogen
added,
-
5 5
1:
2
-
2
DETERMINATION
r
2
100
Rlosquito (8 X repeated) 1 each 1 each
pg
Glycogen recovered -f S.E., fig
3.3
+
0.2
9.0
f
0.4
13.0
k 0.5
1.2
+ 0.1
3.6 66
4 10
14
k
0.2
t1
f0.4 & 0.4
Microgram quantities could be quantitatively isolated from tissue of very low glycogen content, but the analytical application is limited by the sensitivity of the anthrone reaction. DISCUSSIO?; Three sources of error were recognized in Pfluger’s and Good’s methods, all leading to losses of glycogen: (a) discrepancy between solubility of glycogen in dilute ethanol and its precipitability from aqueous solutions by ethanol (Table 1) ; (b) presence of carbonate in aqueous concentrated KOH; (c) formation of a water-insoluble glycogen complex (Tables 3 and 4). With the proposed modifications the method is suitable for biological material containing only a few micrograms of glycogen (Table 5). Quantitative precipitation of glycogen by ethanol was achieved by addition of sodium sulfate to the aqueous solution. Electrolytes soluble in ethanol were not effective (Table 2). Addition of ethanol to carbonatecontaining KOH may cause a concentration of noncrystalline, liquid carbonate and unprecipitated glycogen as a liquid subnatant that would be lost on drainage of the supernatant. The use of carbonate-free KOH or removal of the supernatant with a syringe prevented this loss. Glycogen precipitated with ethanol from alkaline liver digests does not redissolve entirely in water. Calculation of the water-soluble fraction only, as true glycogen, leads to unreproducible and low results. At least 20 hr boiling with concentrated KOH was required to render all glycogen soluble in water after precipitation with ethanol (Table 3). Carroll et al. observed that, when liver was boiled for a prolonged time with 30% KOH, the anthrone-positive material precipitable with ethanol continuously diminished, whereas pure glycogen did not change in this way upon boiling. They concluded that this was due to decom-
264
lSMILE
VAN
HANDEL
position of a nonglycogen complex into fragments not precipitable by ethanol (5). The presence of such a nonglycogen complex would invalidate any glycogcn determination. However, WC believe this observation to be in error, as these authors calculated glycogen as the water-soluble anthrone-positive fraction of the precipitate; they were unaware of the water-insoluble complexes formed during digestion and precipitation with ethanol (Tables 3 and 4). It has long been thought that glycogen in tissues exism in at least two forms: a “free” form, extractable with trichloroacet.ic acid, and a “bound” form extractable with hot KOH. Roe et al. showed that any such differentiation is artifactual and indicative of incomplete extraction in the first place. When tissue was dispersed in TCA at 45,000 rpm in a tissue homogenizer in the presence of homogenizing glass beads, no residual glycogen could be extracted with boiling KOH (6). Furthermore, as seen in Table 2a, TCA does not bring about quantitative precipitation of glycogen on addition of ethanol. In mosquitoes, approximately 65% of t,he glycogen could be extracted with hot water, t,he remainder with hot KOH. When alkaline mosquito digests were treated with hot ethanol, the precipitated glycogen redissolved completely in water. This contrasts with the glycogen of alkaline liver digests. Following convention, we defined as glycogen all anthrone-sensitive material with an absorpt,ion peak at 620 m,u precipitable with ethanol after alkaline digestion. This definition requires some qualification. Glycogen is a polydisperse material, inhomogeneously distributed within cells and suspected to exist in different forms. The molecular weight of glycogen is not known with certainty, as ext,raction with either acid or alkali causes structural changes, apparent by a gradual decline of molecular weight (7). Yet, in our experience, heating at 100°C with 60% KOH for 24 hr did not diminish the total amount of anthrone-positive mat,erial precipitable by ethanol from liver, mosquitoes, and pure glycogen. Overestimat,ion, especially in tissue of low glycogen content, could bc clue to the presence of polysaccharides metabolically different from glycogen or from other anthrone-positive products precipitable under the same conditions. Little or none of such inert material was present in the biological material used to illustrate the recovery of added glycogen in Table 5: in a fed rat the apparent glycogen content of liver was 670, whereas in a rat fasted for 5 days it was 0.06%; similarly, in mosquitoes glycogen fell from 0.3 mg in fed specimens to 0.002 mg in mosquitoes starved to death. Either case demonstrates that 99F of the material calculated as glycogen could be metabolized.
SUMMARY
Some physical properties of glycogen have been investigated and applied to micro isolation and assay in biological material containing only a few micrograms of glycogen. Losses of glycogen in the ethanol precipitation step were found to be preventable by the use of a coprecipitant, of which Na,SO, was the most satisfactory. Evidence for the existence or formation of at least two water-insoluble glycogen complexes in liver was presented. Treatment at 100°C with concentrated KOH for 20 hr rendered all glycogen soluble in water. ACKNOWLEDGMENTS This work was supported bp a grant (AI 05054) from the Sational Health. The analyses mere performed by J. E. Guirn.
Institutes
of
REFEREKCEB A., KHAMER, H., AND Smocrr, M.. J. Riol. Chem. 100, 485 (1933). E., Arch. Ges. Phyxiol. 129, 362 (1909). S., D.ATTON, S., XOVIC. B., .4~n MCNTWYLER, E., Arch. Biochem. Biophys. 25, 191 (1950). 4. OSTERBERG, il. E.. J. Riol. Chem. 85, 97 (1929). 5. CARROLL, N. v,, LOSGLEY. R. ITT., ASD ROE, J. H.. J. Biol. C&m. 220, 583 (1956). 6. ROE, J. H., B.~II,EY, J. M., GRAY, R. R., .~SD RORISSON, J. N., J. Riol. Chem. 23& 1244 (1961). 7. STETTEN, M. R., KATZFX, H. M., AND STETTEX. D., JR.. J. Biol. Chem. 232, 475 (1958) 1. GOOD,
3. PFLUGER, 3. SEIFTER,
C.