Enzymic analysis of rat liver carbohydrate: Glucose and oligoglucoside contents as additional indices in metabolic studies

ANALYTICAL

BIOCHEMISTRY

Enzymic

18, 107-117 (1967)

Analysis

of Rat

Liver

Carbohydrate:

Glucose and Oligoglucoside Contents as Additional Indices in Metabolic Studies’

JOHN Lkision

A. JOHNSON

AND

RAMON

M. FUSARO

of Dermatology, University of Minnesota Minneapolis, Minnesota 66466

Hospitals,

Received May 18, 1966

We recently described a method for the direct enzymic (DE)2 assay of glycogen in liver homogenate (1). The unique features of this technique are: (1) Glycogen content is determined without prior isolation and purification of the carbohydrate. (2) Oligoglucosides (OG) are detected as glycogen. (3) Tissue glucose content is determined simultaneously. In order to reconcile the difference in glycogen yield between the enzymic technique and that based on ethanol precipitation of glycogen from KOH digests (KOH glycogen), it was necessary to develop an independent assay for OG. The determination of glucose, OG, and glycogen contents of liver homogenates obtained under a variety of conditions led to the conclusion that the relative quantities of these fractions provide useful indices for the investigation of in uivo and in vitro carbohydrate metabolism of liver. In particular, the practical problem of obtaining glycogen values which accurately reflect the carbohydrate content of liver in the intact animal can be examined in detail. An obvious source of error in the determination of liver glycogen content is the nonuniform distribution of carbohydrate within the organ. Many investigators, rather than employ the cumbersome technique of processing the entire organ, analyze a relatively large piece of each test specimen, assuming that sampling error will be minimized by examining a large number of specimens in each experimental group. A more reliable alternative is the application of a technique (2) which converts an intact rat liver to a homogeneous frozen powder, small portions of which can be analyzed for average carbohydrate content of the specimen. A second type of error in carbohydrate analysis results from unavoid1This investigation *The abbreviations direct enaymic.

was supported in part by USPHS research grant AM-06841. employed in this report are: OG, oligoglucoside(s) : DE, 107

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able enzymic breakdown of glycogen during preparation of test samples. The observation (1) that glucose content of excised rat liver increases steadily whereas OG levels exhibit no consistent post-mortem increase suggests that rapid deactivation of tissue enzymes after death of the donor will minimize OG production, and that glucose content of a test homogenate provides a reliable index of the care exercised in processing the specimen. The technique of sacrificing a rat quickly, excising the liver and converting it to a frozen powder, and heat-deactivating weighed samples of powder before homogenization yields homogenates with consistently low glucose contents of 0.2 to 0.3%. However, even these levels are considerably higher than those of blood (0.1%) or of skin and muscle (0.02 to 0.05% ; unpublished data, this laboratory). Since the quantity of OG in liver preparations may be of general interest in future carbohydrate studies, the reliability of the technique employed for their assay was investigated. It was concluded that the fractionation procedure described in this report yields good reproducibility between replicates processed simultaneously, and that some variation occurs between repeat OG assays. The purpose of this communication is to describe a reproducible technique for isolating an OG fraction from a rat liver homogenate, and to cite examples in which the measurement of glucose and OG levels provides useful information concerning in vivo and in vitro carbohydrate metabolism of rat liver. EXPERIMENTAL

Preparation of Test Specimens

Except as otherwise noted, animals were sacrificed by stunning and decapitation. The intact liver was quickly excised, frozen between blocks of dry ice, and pulverized in a dry-ice cooled mill (2). Weighed portions of frozen liver powder were immersed in hot water, heated, and homogenized (I). Carbohydrate

Analysis

The procedures for DE assay of test solutions and enzymic determination of KOH glycogen were described earlier (1). The technique for isolation of KOH glycogen was modified to the extent of evaporating homogenate aliquots to dryness before addition of KOH. Although DE assay was reported in detail earlier, a brief description of the method is pertinent. Aliquots of liver homogenates or of OG fractions are incubated with Diazyme (Miles Laboratories, Inc.) and deproteinized with ZnSO, and Ba(OH)Z; tissue glucose controls are prepared by deproteinization of aliquots of the original test mixtures. The resulting test solutions are

ENZYMIC

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CARBOHYDRATE

AKALYSIS

109

subjected to glucose oxidase assay with Glucostat Regular (Worthington Biochemical Corp.) reconstituted with tris buffer (3). Total glucose equivalents (Diazyme-treated solutions) are corrected for tissue glucose, to yield glucose equivalents due to glycogen or to OG. Oligoglwoside

Assay

The fractionation technique employed in the present investigation is a modification of the original method (1). Since OG yield is highly sensitive to the isolation conditions employed, the technique now in use will be described in detail. Aliquots (1 ml) of homogenate containing 20 to 40 mg tissue are placed in polypropylene tubes (Nalgene No. 3110, 16 X 100 mm), and 1.3 ml 95% ethanol is added to each. The mixtures are heated to boiling, cooled at least 45 min in ice water, and centrifuged 30 min at -10” in an International Model HR-1 centrifuge at 10,000 rpm (angle head No. 858, approximately 13,OOOg). The supernatant fluids are decanted into labeled tubes, and the residues are each dispersed in 1 ml distilled water and treated with ethanol as in the first step. The second supernatant fluids are combined with the corresponding first extracts, and these solutions are evaporated to dryness in streams of warm air. The residues so obtained are dissolved in appropriate volumes of distilled water and subjected to direct enzymic assay, to yield glucose and OG contents. The insoluble tissue residues obtained after decanting the second aqueous ethanol extracts are analyzed for DE glycogen. RESULTS Reproducibility

of Oligoglucoside

Assay

Table 1 lists the results of subjecting groups of six replicate aliquots to repeat fractionations. DE assays of OG fractions, insoluble tissue residues (residue glycogen), and of whole homogenates (DE glycogen) were performed. For a given fractionation, the reproducibility for replicate aliquots is good, whereas minor variations in technique cause some variation between repeat fractionations. Increasing the ionic strength of aqueous mixtures by addition of LiCl before ethanol precipitation (expt. A2) did not lower OG values, as would be expected if the conditions for “glycogen” precipitation had been improved. The results of experiment B indicate that OG yield is not greatly affected by decreasing the tissue concentration of homogenate from 50 to 25 mg/ml. Direct enzymic assay of whole homogenates detects OG as glycogen; therefore the close agreement between DE glycogen and total nonglucose carbohydrate (OG plus residue glycogen) indicates that the fractionation procedure does not cause significant error in the estimation of the carbohydrate fractions.

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JOHNSON

Reproducibility Expt.

Group

A

1 2 3 1 2

B

AND

TABLE 1 of Oligoglucoside Oligoglucoside

GlUCOS.2

0.23 0.23 0.23 0.21 0.22

(0.01) (0.01) (0.01) (0) (0.01)

FUSARO

0.26 0.27 0.30 0.44 0.47

Assay”

Residue

(0) (0.01) (0.01) (0.01) (0.01)

7.79 7.81 7.66 5.04 5.10

glyeogen

Total6

(0.03) (0.06) (0.11) (0.05) (0.09)

DE glycogen

8.05 8.08 7.96 5.48 5.57

8.11 5.39 5.53

0 Groups of six replicate ahquots of rat liver homogenates were subjected to ethanol fractionation as described in the text, with the following deviations from the normal procedure: Expt. A2, addition of LiCl to aqueous mixtures; expt. B, 50 mg tissue/ml homogenate (group 1) and 25 mg/ml (group 2). Direct enzymic assay was performed in triplicate on each test homogenate (DE glycogen). Results are expressed as gm glucose equivalents per 100 gm wet weight of tissue, with S.D. in parentheses. b OG plus residue glycogen.

Oligoglucoside

Production

in Rat

Liver

Homogenate

Eight grams of frozen rat liver was immersed in 10 ml ice-cold water in the tube of a friction-type tissue grinder. Homogenization was accomplished in a cold room at -ll’, by rotating the pestle manually at a rate

sufficient

homogenate deactivated,

to prevent

Carbohydrate so.

1 2 3 4 5 6 7 8 9 10

the mixture

from

freezing.

Aliquots

of the

were incubated at 37’ for varying periods of time, heat diluted to 20 to 30 mg/ml tissue content, and analyzed for

Th?

0 0 3 6 9 12 15 18 21 24

KOH glycogen

7.55 5.57 3.29 3.05 2.69 2.76 2.61 2.60 2.21 2.37

TABLE 2 Content of Incubated Rat Liver Homogenate” DE glyygen

7.77 7.14 6.92 6.87 6.89 6.83 6.66 6.48 6.64 6.70

Glugc~~e

0.22 0.61 0.82 0.84 0.88 0.92 0.94 1.00 1.05 1.10

Oligqgluyde

0.59 1.28 3.59 3.52 3.52 3.72 3.94 4.01 4.21 4.18

Residue glyggen

7.04 5.61 3.40 3.32 3.29 3.20 2.77 2.76 2.43 2.50

Total AS-B

7.99 7.75 7.74 7.71 7.77 7.75 7.60 7.48 7.69 7.80

carbohydrate B+C+D

7.85 7.50 7.81 7.68 7.69 7.84 7.65 7.77 7.69 7.78

0 Specimens 2-10 were aliquots of a viable rat liver homogenate prepared with diztilkd water, with external cooling of the tissue grinder. Specimen 1 was heat-deactivated before homogenization. Numbers 2-10 were incubated at 37” for the times specified in the table, heat-deactivated, and subjected to carbohydrate analysis as described in the text. Results are expressed as gm glucose equivalents per 100 gm tissue. b Minutes at 37”.

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111

ANALYSIS

carbohydrate. A control homogenate was prepared from another portion of the liver specimen by heat deactivation before grinding. External cooling does not completely inhibit glycogen breakdown, as evidenced by the higher glucose and OG contents, and the corresponding lower glycogen level, of specimen 2 compared to those of specimen 1 (Table 2). Incubation at 37” caused a steady rise in glucose content, a rapid increase in OG, and a corresponding abrupt fall in ethanol-precipitable “glycogen” (KOH glycogen, residue glycogen). DE glycogen decreased slowly, accompanied by a corresponding rise in glucose content. Total carbohydrate, calculated as DE glycogen plus glucose or as the sum of carbohydrate fractions, remained constant during incubation. Effect

of Mode

of Sacrifice

on Rat Liver

Carbohydrate

The effects on liver carbohydrate of sacrifice by stunning and decapitation, anesthesia and desapitation, and immersion in liquid nitrogen were investigated. In a preliminary experiment, six young adult male rats were divided into three groups and decapitated under the following conditions: stunned; 3 to 5 min after intraperitoneal injection of sodium pentobarbital, 60 mg/kg body weight; mild diethyl ether anesthesia. Carbohydrate assays were performed on homogenates prepared from heat-deactivated specimens. The effects on liver glycogen were in qualit,ative agreement with the observation of Burton et al. (4) that glycogen decrease in perfused rat livers obtained under light ether anesthesia was less than that in livers obtained from pentobarbital-anesthetized rats. In order to verify that the decreased liver OG content of anesthetized donors was not a chance observation, an additional experiment employing three stunned rats and three animaIs anesthetized with sodium pentobarbital was performed. Anesthesia caused significant changes in liver glucose (increased) , OG (decreased) and glycogen (decreased). StatieTABLE 3 Effect of Pentoharbital

Anesthesia on Rat Liver Carbohydrat,eo

k Stunned Carbohydrate

Direct enzymic glycogen Glucose Oligoglucosides

Range

B. Pentobarbital Mean”

Range

Meant

Significanw

6.17-7.76

6.92 (0.75)

5.30-6.30

5.66 (0.48)

p < 0 02

0.21-0.31 0.13-0.90

0.25 (0.04) 0.41(0.291

0.28-0.41 0.05-0.11

0.32 (0.05) 0.10(0.03)

p
a Two groups of rats (five animals in each group) were sacrificed hv decapitat,ion after: (A) stunning; (B) pentobarbital anesthesia. Results are expressed as gm glumtie equivalents per 100 gm tissue. b SD. in parentheses. c Values obtained by application of the t test.

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tioal analysis (t test) of the data obtained from the five experiments (Table 3) indicates that the effect of pentobarbital on rat liver carbohydrate was highly significant, for glucose (increased, p < 0.01) and strongly significant, for glycogen and OG (decreased, p < 0.02). It, is worth noting that the relatively high mean OG content of livers of stunned animals (0.41%) is due to a single anomalous value, 0.90%. The mean of the remaining data of this group is 0.28%, S.D. 0.11% ; and the corresponding t test statistic is p < 0.01. Sacrifice by immersion in liquid nitrogen yielded a specimen with high glycogen content (6.46%)) increased glucose (0.37%)) and approximately “normal” OG (0.20%). Reliability

‘of Analytical

Techniques

To evaluate the reliability of the methods employed in this investigation when performed on a routine basis homogenates prepared from the livers of 12 weanling female rats were analyzed twice by each technique. It, should be noted that a single aliquot of each homogenate was employed in a given assay, and each repeat analysis involved repetition

Reproducibility

TABLE 4 of Routine Carbohydrate Assay

Analysis

Direct enzymic Oligoglucoside

KOH glycogen Enzymic Anthrone

Carbohydrate

Glucose Glycogen Glucose OG Residueglycogen Glycogen Glycogen

Range

1

Assays” Assay

M.%Wl

Range

2 Mean

6s

0.21-0.27 3.12-7.09 0.21-0.26 0.11-0.18 2.88-7.31

0.24 4.92 0.25 0.15 4.85

0.22-0.26 2.80-7.19 0.21-0.27 0.11-0.24 3.04-7.30

0.24 4.84 0.25 0.15 4.84

0 0.08 0 0.01 0.01

2.42-6.93 2.52-7.29

4.31 4.61

2.46-6.78 2.55-6.98

4.26 4.51

0.05 0.11

= Twelve rat liver homogenates were subjected to two independent assays by each of the analytical procedures employed in this investigation. Results are in gm glucose equivalents per 100 gm tissue. b Mean difference, calculated by dividing the algebraic sum of differences of paired observations by the number of paired observations (twelve).

of the entire analytical procedure on an additional aliquot of each homogenate. Solutions prepared from KOH glycogen samples were analyzed enzymically and by the anthrone method (5). Table 4 lists the range, sample mean, and mean difference between repeat assays for each analytical technique. Good reproducibility was obtained for repeat assays of single test aliquots with every analysis performed.

ENZYMIC

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CARBOHYDRATE

ANALYSIS

113

DISCUSSIOIL Oligoglwode

Assay

Precipitation techniques for isolation of glycogen usually employ relatively low centrifugation forces, and slight mechanical loss of carbohydrate upon decantation of supernatant fluid can be tolerated. However, such losses in OG isolation represent increases in carbohydrate content of the aqueous ethanol extracts and therefore cause nonreproducible elevation of OG values. Therefore, high centrifugation force and heat coagulation after ethanol addition are considered essential for reproducible isolation of OG from liver homogenate. It must be emphasized that the fractionation conditions employed for OG assay were originally chosen in order to reconcile DE glycogen values with those obtained for KOH glycogen. The OG fractions defined by these conditions are therefore arbitrary, and one must not attach undue significance to their absolute values. One can, however, examine quantitative differences in OG contents of test specimens when the assays are performed under rigorously defined fractionation conditions. Oligoglucoside

Production

in Rat

Liver

Homogenate

The study of in vitro carbohydrate metabolism of liver slices and homogenates requires that endogenous glycogen disappearing during the course of an experiment be accounted for. Since analytical procedures based on ethanol precipitation of glycogen do not quantitatively detect OG, experiments performed under conditions allowing OG formation cannot be adequately studied by these techniques. Therefore, past attempts to equate glycogen disappearance with increases in glucose and lactate have failed. The data presented in Table 2 illustrate the large errors inherent in attempting to follow glycogen disappearance in liver homogenate by techniques based on prior isolation of glycogen. For example, failure to account for a large portion of the carbohydrate as glucose or KOH glycogen would lead to the conclusion that glucose was rapidly metabolized, whereas the constant level of total carbohydrate indicates that no glucose disappeared during incubation. Since DE assay of homogenate det’ects OG as glycogen and requires simultaneous determination of glucose content, this technique provides a convenient means of monitoring carbohydrate content of incubated test mixtures. Effect of Mode of Sacrifice on Rat Liver

Carbohydrate

Since liver carbohydrate of the intact animal is released as glucose under conditions of stress, it is likely that any technique employed to

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obtain a specimen will result in loss of glycogen. The massive bleeding following decapitation ensures rapid cessation of circulatory response of the donor; however, the trauma induced by this mode of sacrifice may cause such intense stimulation of glycogenolysis that, even though the effect operated for only a short time interval, significant loss of glycogen could occur. The effect of decapitation under anesthesia was therefore investigated, in anticipitation that the general glycogen-lowering effect of anesthetics might be offset by reduced stress and concomitant reduction of glycogenolysis at sacrifice. The desired improvement in glycogen yield was not realized; glycogen levels of livers obtained from anesthetized animals were lower than those observed in livers of stunned donors. It was surprising to observe that anesthesia caused a rise in liver glucose content and a decrease in OG levels. These results suggest that glucose and OG levels provide additional indices for the evaluation of the effects of pharmacological agents on in viva liver carbohydrate balance. Evaluation of the effects of pharmacological agents on liver carbohydrate is not within the scope of our investigations; however, our techniques provide useful tools for such studies. Obvious lines of investigation are to examine alterations of liver and blood OG, rate of production of OG in liver preparations, and discharge of OG by perfused organs. Our primary objective in evaluating modes of sacrifice was to develop a technique with which one can routinely collect intact livers from small animals with minimal post-mortem glycogenolysis. The requirement of uniform samples whose carbohydrate contents represent average levels of the entire organs, precludes the use of in situ freezing techniques which, although they may reduce glycogenolysis considerably, do not permit convenient removal of the entire organ. Our present technique of stunning, decapitation, and immediate removal and freezing of the liver provides a means of collecting specimens with a relatively constant, minimal amount of glycogen breakdown. Presence of OG in the Intact

Animal

The question of the physiological presence or absence of OG in animal liver is pertinent to this investigation only to the extent that their presence in test homogenates may reflect a shortcoming in the technique employed in obtaining and processing specimens. Although careful adherence to the procedure described in this report enables one to obtain homogenates with consistently low OG content, every specimen examined to date contained detectable quantities of these compounds. Since OG content is calculated as the difference between total glucose equivalents and glucose content of test solutions, it is tempting to state that values as low as 0.08% based on tissue wet weight are within the limits of error

ENZYMIC

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CARBOHYDRATE

ANALYSIS

115

of the analytical technique. However, multiple analyses of liver homogenates with low OG content have without exception yielded total glucose equivalents greater than glucose content. It therefore must be concluded that the OG levels cited in this report are genuine, and that our data do not allow a definite decision to be made concerning the presence of OG in the liver of the intact animal. SUMMARY

A technique for the enzymic determination of oligoglucosides in liver homogenate has been described and evaluated. Examples were cited to illustrate the utility of determining glucose and oligoglucoside contents in the investigation of in vivo and in vitro liver carbohydrate metabolism. The reliability of routine application of the analytical methods employed in this investigation was evaluated by performing repeat assays of several homogenates. Although numerous liver homogenates with high glycogen and low oligoglucoside contents have been analyzed, detectable quantities of oligoglucosides were present in each specimen. ADDENDUM

While this report was in preparation, Mordoh, Krisman, and Leloir (6) described a technique in which liver homogenate is prepared in 3% HgCl, to prevent the action of a-amylase. In Table II of their report, they present data which indicate that OG content of the livers of fasted rats remained constant for up to 5.5 hr after refeeding with sucrose, whereas glycogen content increased from 0.0‘2 to 2.3%. We are not prepared to comment on this observation without additional knowIedge concerning the analytical techniques employed. However, the data cited for OG content (1.3 pmoles/gm liver, based on a maltose standard; equivalent to 0.05%) are considerably lower than the smallest value obtained by us (0.08%) for heat-deactivated rat liver homogenate. It therefore seemed worth while for us to repeat the work of these authors. It should be noted that the procedure employed by Mordoh et al. for isolating and assaying OG differs from that described in this report in several ways: (1) Oligoglucosides are isolated from the supernatant fluid obtained after tissue debris is sedimented by centrifuging liver homogenate at low speed. (2) Glyeogen is precipitated by addition of 2 vol 95% ethanol, rather than 1.3 vol. (3). The aqueous ethanol extract is passed through a mixed-bed resin (Amberlite MB-3) before OG assay. (4). An indirect assay is employed in which reducing groups are reduced to alcohol functions with NaBH, to prevent glucose interference, and the resulting mixture is analyzed with phenol sulfuric acid against a maltose standard.

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The above-mentioned investigators perhaps encountered difficulties in performing glucose and OG assays in the presence of mercuric ion. (A control experiment performed by us indicated that MB-3 does not completely remove the ion, as evidenced by formation of a black precipitate upon subsequent treatment with H,S.) We quickly learned that mercuric ion, at a concentration corresponding to the assay dilution described in this report, does not inhibit the enzyme (Diazyme) employed by us for glycogen or OG hydrolysis, but does effect complete inhibition of the glucose oxidase system. However, tris-glucose oxidase is not affected by the presence of mercuric ion .3 Since this reagent is employed in DE assay of whole homogenate and of OG fractions, we are able to perform these analyses in the presence of HgCL. A preliminary experiment established that heat deactivation (A) and homogenization in 3% HgCI, (B) yield essentially equivalent rat liver carbohydrate fractions. The corresponding values (%) for DE glycogen, glucose, and OG are, respectively: (A) 7.97, 0.26, 0.10; (B) 7.72, 0.27, 0.07. In comparing carbohydrate distribution of the two homogenates, results are considered equivalent within experimental error. The reader will note that the OG content obtained by us for HgCl, homogenate was higher than that reported by Mordoh et al. We therefore performed an experiment in which the technique employed by these authors was compared with our procedure involving fractionation of whole homogenate. Accordingly, a rat liver homogenate was prepared in 3% aqueous HgCl,. A portion of this test mixture was centrifuged at 2000 rpm for 5 min; and the resulting supernatant fluid was fractionated for OG, employing 1.3 vol 95% ethanol. Simultaneously, an aliquot of whole homogenate was subjected to the same OG fractionation procedure. The corresponding OG and glucose values for supernatant fluid and whole homogenate were, respectively: 0.041, 0.17; 0.085, 0.26. It therefore appears that the low OG values reported by Mordoh et al. are more apparent than real. Passage of OG fractions through Amberlite MB-3 resulted in a loss of enzymic glucose (0.24 vs. 0.17%), and no change in OG content (0.08 vs. 0.09%). The loss of enzymic glucose may be due to the effect of the strongly basic anion exchanger present in the mixed-bed resin. The interested reader is referred to our earlier publication (7) for the effect of alkali-catalyzed isomerization of glucose, and its effect on the enzymic detection of glucose. ‘This result is not surprising in view of a current report by Hanlon et al. CAnal. Biochem. 16, 225 (1966)l in which they state that tris buffer strongly binds divalent mercury ion.

ENZYMIC

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117

ACKNOWLEDGMENT We are indebted to Miss Carolyn Guse for expert technical assistance. We also wish to thank Dr. L. A. Underkofler, Miles Laboratories, Inc., for providing generous amounts of Diazyme. REFERENCES 1. 2. 3. 4. 5.

JOHNSON, J. A., AND FUSAEO, R. M., Anal. Biochem. 15, 140 (1966). SMITH, Q. T., AND JOHNSON, J. A., J. Invest. Dermatol. 45, 303 (1965). DAHLQVIST, A., Anal. Biochem. 7, 18 (1964). BURTON, S. D., ST. GEORO~, S., AND ISHIDA, T., J. AppE. Physiol. 15, 128 (1960). CARROLL, N. V., LONGLEY, R. W., AND RCYE,J. H., J. Bd. Chem. 220, 633 (1956).

Por Addendum 6. MORWH, J., KRISMAP~, C. R., AND LELOIR, L. F., Arch. Biochem. 113, 265 (1966). 7. JOHNSON, J. A., AND FUSARO, R. M., And. Biochem. 13, 412 (1965).

Biophys.