89
Clinica Chimica Acta, 187 (1990) 89-94 Elsevier
CCA 04659
A new sensitive microassay for the measurement of erythrocyte glycogen James Farquharson,
E. Cherry Jamieson, Gordon B. MacPhee and Robert W. Logan
Department of Biochemistry, Royal Hospital for Sick Children and Queen Mother’s Hospital, Yorkhill, Glasgow, Scotland (UK) (Received
10 June 1989; revision
12 October
Key words: Glycogen;
1989; accepted
Erythrocyte;
16 October
1989)
Microassay
Summary Conventional ethanol precipitation of sub-microgram amounts of glycogen leads to low yields ( < 50%). Quantitative recoveries of 90% were attained, however, when the isolation temperature was raised to 50°C and ethanol was replaced by the less polar propan-2-01. This improvement enabled development of an erythrocyte assay for glycogen which was both sensitive (0.1 pg glycogen) and required only 1 ml of whole blood. 26 paediatric specimens were analysed and a reference range of values from undetected to 78 pg glycogen/g haemoglobin (Hb) was obtained.
Introduction Although the concentration of glycogen in normal erythrocytes is very low active glycogen metabolism is undoubtedly present [l]. It is believed that glycogen does not accumulate intracellularly, except in disease states, because the relative rates of activity of the erythrocyte phosphorylase and debrancher enzymes are substantially greater than that of glycogen synthetase and the brancher system. To overcome the problems associated with the analysis of micro amounts of glycogen the concentrating of cell extracts by dialysis, lyophilisation or ethanol precipitation was proposed [2]. Early attempts utilising ethanol demonstrated that glycogen was not completely precipitated without the addition of ethanol-soluble salts such as sodium sulphate [3] and lithium chloride [4]. Using such salts Huijing [5] was, however, unable to precipitate more than 50% of the micro quantities of glycogen from tissue digests.
Correspondence and requests for reprints to: Dr. R.W. Logan, Department of Biochemistry, Royal Hospital for Sick Children and Queen Mother’s Hospital, Yorkhill, Glasgow G3 8SJ. Scotland, UK.
0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
90
To overcome the difficulties we present here a sensitive microassay which incorporates a new alcohol precipitation step and leads to quantitative recoveries of sub microgram amounts of glycogen. Materials Instrumentation All absorbances were measured using a Model 25 spectrophotometer Instruments, Fullerton, CA, USA). Ultrasonication was performed FS200 Ultrasonicator (Decon Ultrasonics Ltd., Hove, Sussex, UK).
(Beckman in a Decon
Chemicals and enzymes Glycogen (Oyster extract G-8751) and amyloglucosidase (from Aspergillus niger; amylo-a-1,6-a-1,4 glucosidase, EC 3.2.1.3, A-3514) were both obtained from the Sigma Chemical Co., Poole, Dorset, UK). Glucose was measured by the Gluco-quant system (Cat. No. 263826) from Boehringer Mannheim GmBH, Mannheim, FRG and consisted of hexokinase (HK, from yeast; ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1) and glucosed-phosphate dehydrogenase (G6P-D, from yeast; D-glucose-6-phosphate : NADP l-oxidoreductase, EC 1.1.1.49). In addition, phosphate buffer (70 mmol/l), pH 7.7, Mgzf (4 mmol/l), NADP (1.3 mmol/l) and ATP (1.3 mmol/l) were also supplied. Ethanol and propan-2-01 were of Spectrosol grade and together with all other chemicals of Analar grade were purchased from BDH Chemicals Ltd, Poole, UK. Methods In the determination of reference paediatric erythrocyte glycogen concentration no bloods were included from patients receiving glucose infusions, glucocorticoid therapy or suspected of having any form of glycogen storage disease (GSD). As soon as possible after venepuncture 1 ml of lithium-heparinised blood was centrifuged (1100 X g, 5 min) at 4°C. The erythrocytes were washed twice with sodium chloride (0.15 mol/l) and the haemoglobin content was determined. 200 ~1 of the washed cells was mixed thoroughly with 600 ~1 ice-cold perchloric acid (0.6 mol/l) and after centrifugation (900 X g, 10 min) at 4°C 400 ~1 of the clear supernatant was transferred to a 1.5-ml Sarstedt reaction tube and 500 ~1 of propan-2-01 was added slowly with vortex mixing. At the same time standards over the range O-10 pg glycogen were prepared by appropriate dilution of a freshly prepared working solution (50 mg/l) in perchloric acid (0.6 mol/l). The volume of each standard was made up to 400 ~1 with the perchloric acid and 500 ~1 of propan-2-01 was again slowly added with mixing. All the tubes were incubated at 50°C for one hour to precipitate the glycogen and subsequently centrifuged (1500 x g, 10 min) at room temperature, The supernatants were discarded and the tubes inverted and allowed to dry. The glycogen precipitates were dissolved by the addition with ultrasonication (20 s) of 200 ~1 sodium acetate buffer (0.1 mol/l), pH 4.8 to the test and 100 ~1 buffer to each standard tube. 100 ~1 of the test solution
was transferred to a separate tube to act as a blank, 5 ~1 of amyloglucosidase reagent (4 U) added to the test and each standard and 5 ~1 distilled water added to the blank. The specimens were mixed, incubated for 30 min at 37°C and centrifuged (900 x g, 5 min) at room temperature. 100 ~1 of supernatant was added in turn to 400 ~1 of the Gluco-quant phosphate buffer (70 mmol/l), pH 7.7 and initial absorbance was measured at 340 nm against a distilled water reference. 2 ~1 of the combined HK (2 0.2 U): G6P-D(> 0.36 U) reagent was added and the final absorbance was measured after 3 min reaction at room temperature. Results and discussion Figure 1 shows typical glucose and glycogen standard curves illustrating an overall recovery of glycogen at all concentrations of about 80%, comprising 90% glycogen precipitation by propan-2-01 and 90% hydrolysis by amyloglucosidase to glucose. The sensitivity of the method was 0.1 pg glycogen (95% confidence). Allowing for the initial specimen dilution in the glycogen extraction this represented an assay detection limit of 1.5 pg glycogen/ml erythrocytes (= 5 pg glycogen/g Hb). Linearity pertains to at least 30 pg glycogen (off-scale on Fig. 1 at an absorbance of 1.647) and the paediatric range of 26 results was distributed logarithmically from less than 5 to 78 pg glycogen/g Hb (geometric mean = 12 and 95% confidence limits 1.7-84.4). This range compared favourably with that found by other authors [6]. The distribution of results seen in Fig. 2 illustrates the reason for logarithmic transformation prior to statistical analysis. Values determined in 3 renal transplant patients receiving prednisolone were 89, 130 and 174 pg/g Hb. In one patient receiving intravenous dextrose erythrocyte glycogen was 462 pg/g Hb. In the course of our investigations we encountered the previously discussed and recently confirmed [7] difficulties in isolating micro quantities of glycogen using
Concentration
@g/assay)
Fig. 1. Standard curves for glucose (o), non precipitated glycogen (*) glywgen ( f 1.
and propan-2-ol-precipitated
92
0’
4
25 Precipitation temperature
37 (OC)
50
Fig. 2. Dist~buti~n of normal erythrocyte glycogen concentrations. Fig. 3. 2 pg glycogen reacted in each assay. Ethanol (.), propan-2-01 precipitation (*)
conventional ethanol precipitation (Fig. 3). Knowing that thermal agitation had some positive effect in achieving more rapid glycogen precipitation [4], we decided to increase the temperature of the procedure to 50°C sufficient to increase precipitation without detriment to the glycogen structure (Fig, 3). In spite of knowing that Steinitz [3] reported that raising the ethanol concentration was ineffective due to the fact that increasing the overall volume nullified any gains from the decreased glycogen solubility we sought to achieve the latter by employing propan-2-01 without increasing the volume used. The combination of higher temperature together with the use of a less polar alcohol resulted in what we believe is the first successful quantitative recovery of micro amounts of glycogen using a simple alcohol precipitation procedure. Although the assay of glycogen in erythrocytes is generally confined to those patients with possible debrancher or phosphorylase deficiency GSD (types III or VI), where the high concentrations present are readily detected without precipitation of the glycogen, we nevertheless consider that our sensitive microassay which permits accurate deter~nation of glycogen using small blood volumes is clearly advantageous. In addition, the quantitative recovery of sub-microgram amounts of glycogen enabled the authors to isolate sufficient normal human erythrocyte glycogen to assess its behaviour in a preliminary investigation of GSD type III, which is hindered by the con~~tant hydrolysis of limit dextrin substrates by lysosomal a-glucosidase [8,9]. The limit dextrins currently employed are derived from glycogen precursors of non human origin and in some cases from non mammalian sources and may not behave like the natural substrate. This advance in cellular glycogen meas~ement may in futumreduce the requirement for tissue biopsy specimens as part of the investigation of glycogen storage disease.
93
Acknowledgements We are grateful to all our clinical colleagues whose cooperation rendered this study possible. We would also like to express our thanks to Mr. A. Irwin and Miss J. Hyslop of the Department of Medical Illustration for preparation of the figures and to Mrs. B. Nisbet for the typing of the manuscript. References 1 Moses SW, Bashan N, Gutman A. Glycogen metabolism in the normal red blood cell. Blood 1972;40(6):836-843. 2 Sidbury JB, Cornblath M, Fisher J, House E. Glycogen in erythrocytes of patients with glycogen storage disease. Paediatrics 1961;27:103-111. 3 Steinitz K. Laboratory diagnosis of glycogen diseases. In: Sobota H, Stewart CP, (editors). Advances in Clinical Chemistry, Volume 9. Academic: New York, 1967;251-266. 4 Roe JH, Dailey RE. Determination of glycogen with the anthrone reagent. Anal Biochem 1966;15:245-250. 5 Huijing F. Rapid enzymic method for glycogen estimation in very small tissue samples. Clin Chim Acta 1970;30:567-572. 6 Collins W, Brown I, Taylor WH. Interference in the measurement of red cell glycogen by glycogen from white cells. Clin Chim Acta 1987;162:207-213. 7 Brodal BP, Gehrken BB. Enzymatic microanalysis of glycogen. Stand J Clin Lab Invest 1986;46:193-195. 8 Gutman A, Barash V. Incorporation of [i4C] glucose into a-1,4 bonds of glycogen by leukocytes and fibroblasts of patients with type III glycogen storage disease. Pediatr Res 1985;19:28-32. 9 Van Diggelen OP, Janse HC, Smit GPA. Debranching enzyme in fibroblasts, amniotic fluid cells and chorionic villi: pre- and postnatal diagnosis of glycogenosis type III. Clin Chim Acta 1985;149:129-134.
Clinica Chimica Acta, 187 (1990) 89-94 Elsevier
CCA 04659
A new sensitive microassay for the measurement of erythrocyte glycogen James Farquharson,
E. Cherry Jamieson, Gordon B. MacPhee and Robert W. Logan
Department of Biochemistry, Royal Hospital for Sick Children and Queen Mother’s Hospital, Yorkhill, Glasgow, Scotland (UK) (Received
10 June 1989; revision
12 October
Key words: Glycogen;
1989; accepted
Erythrocyte;
16 October
1989)
Microassay
Summary Conventional ethanol precipitation of sub-microgram amounts of glycogen leads to low yields ( < 50%). Quantitative recoveries of 90% were attained, however, when the isolation temperature was raised to 50°C and ethanol was replaced by the less polar propan-2-01. This improvement enabled development of an erythrocyte assay for glycogen which was both sensitive (0.1 pg glycogen) and required only 1 ml of whole blood. 26 paediatric specimens were analysed and a reference range of values from undetected to 78 pg glycogen/g haemoglobin (Hb) was obtained.
Introduction Although the concentration of glycogen in normal erythrocytes is very low active glycogen metabolism is undoubtedly present [l]. It is believed that glycogen does not accumulate intracellularly, except in disease states, because the relative rates of activity of the erythrocyte phosphorylase and debrancher enzymes are substantially greater than that of glycogen synthetase and the brancher system. To overcome the problems associated with the analysis of micro amounts of glycogen the concentrating of cell extracts by dialysis, lyophilisation or ethanol precipitation was proposed [2]. Early attempts utilising ethanol demonstrated that glycogen was not completely precipitated without the addition of ethanol-soluble salts such as sodium sulphate [3] and lithium chloride [4]. Using such salts Huijing [5] was, however, unable to precipitate more than 50% of the micro quantities of glycogen from tissue digests.
Correspondence and requests for reprints to: Dr. R.W. Logan, Department of Biochemistry, Royal Hospital for Sick Children and Queen Mother’s Hospital, Yorkhill, Glasgow G3 8SJ. Scotland, UK.
0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
90
To overcome the difficulties we present here a sensitive microassay which incorporates a new alcohol precipitation step and leads to quantitative recoveries of sub microgram amounts of glycogen. Materials Instrumentation All absorbances were measured using a Model 25 spectrophotometer Instruments, Fullerton, CA, USA). Ultrasonication was performed FS200 Ultrasonicator (Decon Ultrasonics Ltd., Hove, Sussex, UK).
(Beckman in a Decon
Chemicals and enzymes Glycogen (Oyster extract G-8751) and amyloglucosidase (from Aspergillus niger; amylo-a-1,6-a-1,4 glucosidase, EC 3.2.1.3, A-3514) were both obtained from the Sigma Chemical Co., Poole, Dorset, UK). Glucose was measured by the Gluco-quant system (Cat. No. 263826) from Boehringer Mannheim GmBH, Mannheim, FRG and consisted of hexokinase (HK, from yeast; ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1) and glucosed-phosphate dehydrogenase (G6P-D, from yeast; D-glucose-6-phosphate : NADP l-oxidoreductase, EC 1.1.1.49). In addition, phosphate buffer (70 mmol/l), pH 7.7, Mgzf (4 mmol/l), NADP (1.3 mmol/l) and ATP (1.3 mmol/l) were also supplied. Ethanol and propan-2-01 were of Spectrosol grade and together with all other chemicals of Analar grade were purchased from BDH Chemicals Ltd, Poole, UK. Methods In the determination of reference paediatric erythrocyte glycogen concentration no bloods were included from patients receiving glucose infusions, glucocorticoid therapy or suspected of having any form of glycogen storage disease (GSD). As soon as possible after venepuncture 1 ml of lithium-heparinised blood was centrifuged (1100 X g, 5 min) at 4°C. The erythrocytes were washed twice with sodium chloride (0.15 mol/l) and the haemoglobin content was determined. 200 ~1 of the washed cells was mixed thoroughly with 600 ~1 ice-cold perchloric acid (0.6 mol/l) and after centrifugation (900 X g, 10 min) at 4°C 400 ~1 of the clear supernatant was transferred to a 1.5-ml Sarstedt reaction tube and 500 ~1 of propan-2-01 was added slowly with vortex mixing. At the same time standards over the range O-10 pg glycogen were prepared by appropriate dilution of a freshly prepared working solution (50 mg/l) in perchloric acid (0.6 mol/l). The volume of each standard was made up to 400 ~1 with the perchloric acid and 500 ~1 of propan-2-01 was again slowly added with mixing. All the tubes were incubated at 50°C for one hour to precipitate the glycogen and subsequently centrifuged (1500 x g, 10 min) at room temperature, The supernatants were discarded and the tubes inverted and allowed to dry. The glycogen precipitates were dissolved by the addition with ultrasonication (20 s) of 200 ~1 sodium acetate buffer (0.1 mol/l), pH 4.8 to the test and 100 ~1 buffer to each standard tube. 100 ~1 of the test solution
was transferred to a separate tube to act as a blank, 5 ~1 of amyloglucosidase reagent (4 U) added to the test and each standard and 5 ~1 distilled water added to the blank. The specimens were mixed, incubated for 30 min at 37°C and centrifuged (900 x g, 5 min) at room temperature. 100 ~1 of supernatant was added in turn to 400 ~1 of the Gluco-quant phosphate buffer (70 mmol/l), pH 7.7 and initial absorbance was measured at 340 nm against a distilled water reference. 2 ~1 of the combined HK (2 0.2 U): G6P-D(> 0.36 U) reagent was added and the final absorbance was measured after 3 min reaction at room temperature. Results and discussion Figure 1 shows typical glucose and glycogen standard curves illustrating an overall recovery of glycogen at all concentrations of about 80%, comprising 90% glycogen precipitation by propan-2-01 and 90% hydrolysis by amyloglucosidase to glucose. The sensitivity of the method was 0.1 pg glycogen (95% confidence). Allowing for the initial specimen dilution in the glycogen extraction this represented an assay detection limit of 1.5 pg glycogen/ml erythrocytes (= 5 pg glycogen/g Hb). Linearity pertains to at least 30 pg glycogen (off-scale on Fig. 1 at an absorbance of 1.647) and the paediatric range of 26 results was distributed logarithmically from less than 5 to 78 pg glycogen/g Hb (geometric mean = 12 and 95% confidence limits 1.7-84.4). This range compared favourably with that found by other authors [6]. The distribution of results seen in Fig. 2 illustrates the reason for logarithmic transformation prior to statistical analysis. Values determined in 3 renal transplant patients receiving prednisolone were 89, 130 and 174 pg/g Hb. In one patient receiving intravenous dextrose erythrocyte glycogen was 462 pg/g Hb. In the course of our investigations we encountered the previously discussed and recently confirmed [7] difficulties in isolating micro quantities of glycogen using
Concentration
@g/assay)
Fig. 1. Standard curves for glucose (o), non precipitated glycogen (*) glywgen ( f 1.
and propan-2-ol-precipitated
92
0’
4
25 Precipitation temperature
37 (OC)
50
Fig. 2. Dist~buti~n of normal erythrocyte glycogen concentrations. Fig. 3. 2 pg glycogen reacted in each assay. Ethanol (.), propan-2-01 precipitation (*)
conventional ethanol precipitation (Fig. 3). Knowing that thermal agitation had some positive effect in achieving more rapid glycogen precipitation [4], we decided to increase the temperature of the procedure to 50°C sufficient to increase precipitation without detriment to the glycogen structure (Fig, 3). In spite of knowing that Steinitz [3] reported that raising the ethanol concentration was ineffective due to the fact that increasing the overall volume nullified any gains from the decreased glycogen solubility we sought to achieve the latter by employing propan-2-01 without increasing the volume used. The combination of higher temperature together with the use of a less polar alcohol resulted in what we believe is the first successful quantitative recovery of micro amounts of glycogen using a simple alcohol precipitation procedure. Although the assay of glycogen in erythrocytes is generally confined to those patients with possible debrancher or phosphorylase deficiency GSD (types III or VI), where the high concentrations present are readily detected without precipitation of the glycogen, we nevertheless consider that our sensitive microassay which permits accurate deter~nation of glycogen using small blood volumes is clearly advantageous. In addition, the quantitative recovery of sub-microgram amounts of glycogen enabled the authors to isolate sufficient normal human erythrocyte glycogen to assess its behaviour in a preliminary investigation of GSD type III, which is hindered by the con~~tant hydrolysis of limit dextrin substrates by lysosomal a-glucosidase [8,9]. The limit dextrins currently employed are derived from glycogen precursors of non human origin and in some cases from non mammalian sources and may not behave like the natural substrate. This advance in cellular glycogen meas~ement may in futumreduce the requirement for tissue biopsy specimens as part of the investigation of glycogen storage disease.
93
Acknowledgements We are grateful to all our clinical colleagues whose cooperation rendered this study possible. We would also like to express our thanks to Mr. A. Irwin and Miss J. Hyslop of the Department of Medical Illustration for preparation of the figures and to Mrs. B. Nisbet for the typing of the manuscript. References 1 Moses SW, Bashan N, Gutman A. Glycogen metabolism in the normal red blood cell. Blood 1972;40(6):836-843. 2 Sidbury JB, Cornblath M, Fisher J, House E. Glycogen in erythrocytes of patients with glycogen storage disease. Paediatrics 1961;27:103-111. 3 Steinitz K. Laboratory diagnosis of glycogen diseases. In: Sobota H, Stewart CP, (editors). Advances in Clinical Chemistry, Volume 9. Academic: New York, 1967;251-266. 4 Roe JH, Dailey RE. Determination of glycogen with the anthrone reagent. Anal Biochem 1966;15:245-250. 5 Huijing F. Rapid enzymic method for glycogen estimation in very small tissue samples. Clin Chim Acta 1970;30:567-572. 6 Collins W, Brown I, Taylor WH. Interference in the measurement of red cell glycogen by glycogen from white cells. Clin Chim Acta 1987;162:207-213. 7 Brodal BP, Gehrken BB. Enzymatic microanalysis of glycogen. Stand J Clin Lab Invest 1986;46:193-195. 8 Gutman A, Barash V. Incorporation of [i4C] glucose into a-1,4 bonds of glycogen by leukocytes and fibroblasts of patients with type III glycogen storage disease. Pediatr Res 1985;19:28-32. 9 Van Diggelen OP, Janse HC, Smit GPA. Debranching enzyme in fibroblasts, amniotic fluid cells and chorionic villi: pre- and postnatal diagnosis of glycogenosis type III. Clin Chim Acta 1985;149:129-134.
