Plasma lipases in chronic renalfailure
(1977), with a column (9 mm x 8 mm) containing agarose beads (Sepharose 4B, Pharmacia Fine Chemicals, Uppsala, Sweden) to which heparin (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia) had been covalently bound as described by Iverius (1971). The buffer used throughout for affinity chromatography was sodium barbital (5 mmol/l) at pH 7.4, to which was added NaCl at various concentrations. A 1 : 1 mixture (4 ml) of whole plasma and buffer containing sodium chloride 0.45 mol/l was placed on the column, to which were applied, in succession: (1) 4 ml of buffer containing NaCl (0.3 molh); (2) and (3) two 3 ml portions of buffer containing NaCl (0.72 mol/l); (4) and (5) two 3 ml portions of buffer containing NaCl (1.1 mol/l); (6) 10 ml of buffer containing NaCl (0.15 mol/l). A steady flow of 0.5 ml/min through the column was maintained, and all procedures were performed at 4OC. Each column was used twice. The six fractions were checked for lipolytic activity. Elution profiles were similar in all subject groups, and more than 70% of the total lipase activity eluted was present in fractions (2) and (4). These two fractions had the characteristics of hepatic and lipoprotein lipase respectively (Boberg et al., 1977), and were thus always used to assay these enzymes.
157
Each sample was incubated in quadruplicate (duplicates on each of 2 different days) for 30 min at 28OC with gentle mixing. 14C-labelledfatty acid released during incubation was extracted and counted in a Packard Tri-carb Liquid Scintillation Spectrometer 3255 (Packard Instrument Co. Inc., Downers Grove, Ill., U.S.A.) by the method of Belfrage & Vaughan (1969) as modified by Nilsson-Ehle 8c Schotz (1976). The efficiency of extraction was determined by adding [14C]oleic acid, in place of the labelled triacylglycerol, to a triolein emulsion prepared identically to the enzyme substrate. The amount of fatty acid released was calculated after correcting for incomplete extraction and quenching, and subtracting the activity of substrate incubated alone.
Determination of optimum conditions for enzyme activity In preliminary experiments it was found that each lipase reached its maximum activity 15 min after injection of heparin (Fig. l), and the optimum pH of the incubation medium was 8.6 for both enzymes (Fig. 2). NaCl (1 mol/l, final concentration) abolished lipoprotein lipase activity but reduced hepatic lipase by only 9% in normal and 2 1% in uraemic samples.
Enzyme assay with [“Cltriolein Each working day, fresh substrate emulsion was made up by using a standardized technique. This aqueous emulsion, comprising triolein (Sigma Chemical Co., St Louis, Mo., U.S.A.), 22.5 mmol/l, glycerol tri-[ l-14Cloleate (The Radiochemical Centre, Amersham, Bucks., U.K.), 0.04 pCi/pmol, and soya-bean lecithin (Sigma), 1 g/l (final concentration), was prepared by using the microtip attachment of a Branson Sonifier Cell Disruptor 200 (Branson Sonic Power Co., Danburry, Conn., U.S.A.) for 4 min at setting 3, whilst packed in ice. A stock solution of defatted bovine serum albumin (Sigma), 160 g/l in Tris-chloride (0.8 mol/l) buffer, pH 8.6, was made up each month. The incubation mixture contained 0-1 ml of substrate emulsion, 0-05 ml of stock albumin solution, 0.02 ml of the column fraction being assayed and water to a total volume of 0.2 ml. Thus the final concentration of triolein in the mixture was 11.3 mmol/l. A portion (0.02 ml) of pooled plasma drawn from healthy fasting subjects without prior injection of heparin, and inactivated by heating at 56OC for 1 h, was included, at the expense of water, for the assay of lipoprotein lipase.
I 0
,
1
10
20
30
40
50
60
Time (min)
FIG.1. Mean activity of plasma lipases at different time intervals after administration of heparin (50 i.u./kg body weight): e--., hepatic lipase from healthy subjects; e - - 0 , hepatic lipase from uraemic patients; A-A, lipoprotein lipase from healthy subjects; A---A, lipoprotein iipase from uraemic patients. Enzyme fractions were incubated with 11-3 mmol of triolein at pH 8.6, with 20 pl of added plasma for lipoprotein lipase assay. NEFA, Non-esterifled fatty acids.
G . A. Crawford, E . Savdie and J. H . Stewart
158
7
8
9
10
PH FIG.2. Mean activity of plasma lipases at different p H values: M, hepatic lipase from healthy subjects; 0---., hepatic lipase from uraemic patients; A-4 lipoprotein lipase from healthy subjects; A---A, lipoprotein lipase from uraemic patients. The activity was measured I5 min after intravenous administration of heparin (50 i.u./kg body weight). Enzyme fractions were incubated with 11.3 mmol of trioleidl with 20 pI of added plasma for lipoprotein lipase assay. A substrate blank was subtracted for each p H value.
Poobd, heat-inactivated pre-heparin plasma, from fasting normal subjects, added to the incubation medium was slightly inhibitory for hepatic lipase and generally stimulating for lipoprotein lipase (Fig. 3). However, this stimulation was significantly (P < 0-05)greater for normal lipoprotein lipase than for the enzyme from uraemic
patients, when 20 pl or more of pooled plasma was added. Both hepatic and lipoprotein lipase appeared to conform to Michaelis-Menten enzyme-substrate kinetics, and the substrate concentration used in the definitive assays was shown not to be rate-limiting for either enzyme (Fig. 4). Mean apparent Michaelis constants calculated from Lineweaver-Burk plots were 0.8 ( r = 0.98) and 3-0( r = 0-98) mmol of triolein/l for normal subjects, and 0.3 ( r = 0.77) and 5.0 (r = 0.96) mmol/l in uraemic subjects, for hepatic lipase and lipoprotein lipase respectively. With optimum assay conditions the within-run coefficient of variation was 12% for hepatic lipase and 7% for lipoprotein lipase (four portions assayed in duplicate with different columns). When post-heparin plasma was divided into eight portions, stored frozen and assayed on separate days spread over a 3 month period, activity did not decline significantly despite a between-run coefficient of variation for hepatic lipase of 22% and for lipoprotein lipase of 31%. This rather large variation was attributable to day-to-day differences in the substrate emulsion, which we were unable to eliminate despite rigidly standardized conditions of preparation. The variation was monitored by including a standard plasma source of enzyme activity with each assay, and rejecting results in which the standard’s activity was more than 10% above or below the expected value.
0
-
.-> m y
g
i
.-I
0
,
, 10
,
, 20
,
, 30
Plasma (/.11/0,2rnl of assay medium)
FIG.3. Mean activity of plasma lipases (as a percentage of that activity with no added plasma) with increasing amounts of pooled plasma added to the incubation medium: + 0 . hepatic lipase from healthy subjects; O---O, hepatic lipase from uraemic patients; A-A, lipoprotein lipase from healthy subjects; A---A, lipoprotein lipase from uraemic patients. The activity was measured 15 min aRer intravenous administration of heparin (50 i.u./kg body weight). Enzyme fractions were incubated with 11.3 mmol of triolein/l at pH 8.6.
Plasma lipases in chronic renal failure
159
concentration) heated pooled plasma. Fatty acids released were extracted by the method of Dole (1956) and measured by the spectrophotometric method of Duncombe (1963).
5
-
v
Statistical calculations Results were analysed by conventional parametric statistical methods including linear regression analysis and Student’s t-test (Snedecor & Cochran, 1967).
Triolein (pnol)
FIG.4. Mean activity of plasma lipases with increasing hepatic lipase from substrate concentration: W, hepatic -.lipase , from uraemic healthy subjects; +patients; A-4 lipoprotein lipase from healthy subjects; A--- A, lipoprotein lipase from uraemic patients. Activity was measured 15 min after intravenous administration of heparin (50 i.u./kg body weight). Enzyme fractions were incubated at pH 8.6 with addition of 20 pl of pooled plasma for lipoprotein lipase measurement. K,, Apparent Michaelis constant.
Enzyme assay with Intralipid Fractions from the heparin-Sepharose columns containing enzyme activity were incubated with an unlabelled Intralipid (Vitrum, Stockholm, Sweden) substrate mixture by the method of Boberg & Carlson (1964). The incubation conditions were modiied from those of Boberg & Carlson as follows: (i) incubation at 28OC, (ii) samples taken at 20 and 65 min for fatty acid estimation, (iu) assay of hepatic lipase at pH 9.3, (iv) assay of lipoprotein lipase at pH 8-6 with addition of 10% (final
Results
Results with “Cltriolein substrate Twelve male and 18 female patients with undialysed chronic renal failure, eight male and seven female renal transplant recipients and 17 male and 15 female healthy control subjects were so studied (Table 1). Male and female patients with chronic renal failure had elevated fasting serum triacylglycerol concentrations and expanded plasma volumes, but body weights and serum cholesterol concentrations which were not significantly different from those of control subjects. In addition to slightly elevated serum creatinine concentrations, the male and female transplant recipients had significantly raised serum triacylglycerols, but normal serum cholesterol values, body weights and plasma volumes. Hepatic lipase (Table 3a and Fig. 5). Hepatic lipase activity tended to be higher in males than in females, but significantly so only in controls (P
(1977), with a column (9 mm x 8 mm) containing agarose beads (Sepharose 4B, Pharmacia Fine Chemicals, Uppsala, Sweden) to which heparin (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia) had been covalently bound as described by Iverius (1971). The buffer used throughout for affinity chromatography was sodium barbital (5 mmol/l) at pH 7.4, to which was added NaCl at various concentrations. A 1 : 1 mixture (4 ml) of whole plasma and buffer containing sodium chloride 0.45 mol/l was placed on the column, to which were applied, in succession: (1) 4 ml of buffer containing NaCl (0.3 molh); (2) and (3) two 3 ml portions of buffer containing NaCl (0.72 mol/l); (4) and (5) two 3 ml portions of buffer containing NaCl (1.1 mol/l); (6) 10 ml of buffer containing NaCl (0.15 mol/l). A steady flow of 0.5 ml/min through the column was maintained, and all procedures were performed at 4OC. Each column was used twice. The six fractions were checked for lipolytic activity. Elution profiles were similar in all subject groups, and more than 70% of the total lipase activity eluted was present in fractions (2) and (4). These two fractions had the characteristics of hepatic and lipoprotein lipase respectively (Boberg et al., 1977), and were thus always used to assay these enzymes.
157
Each sample was incubated in quadruplicate (duplicates on each of 2 different days) for 30 min at 28OC with gentle mixing. 14C-labelledfatty acid released during incubation was extracted and counted in a Packard Tri-carb Liquid Scintillation Spectrometer 3255 (Packard Instrument Co. Inc., Downers Grove, Ill., U.S.A.) by the method of Belfrage & Vaughan (1969) as modified by Nilsson-Ehle 8c Schotz (1976). The efficiency of extraction was determined by adding [14C]oleic acid, in place of the labelled triacylglycerol, to a triolein emulsion prepared identically to the enzyme substrate. The amount of fatty acid released was calculated after correcting for incomplete extraction and quenching, and subtracting the activity of substrate incubated alone.
Determination of optimum conditions for enzyme activity In preliminary experiments it was found that each lipase reached its maximum activity 15 min after injection of heparin (Fig. l), and the optimum pH of the incubation medium was 8.6 for both enzymes (Fig. 2). NaCl (1 mol/l, final concentration) abolished lipoprotein lipase activity but reduced hepatic lipase by only 9% in normal and 2 1% in uraemic samples.
Enzyme assay with [“Cltriolein Each working day, fresh substrate emulsion was made up by using a standardized technique. This aqueous emulsion, comprising triolein (Sigma Chemical Co., St Louis, Mo., U.S.A.), 22.5 mmol/l, glycerol tri-[ l-14Cloleate (The Radiochemical Centre, Amersham, Bucks., U.K.), 0.04 pCi/pmol, and soya-bean lecithin (Sigma), 1 g/l (final concentration), was prepared by using the microtip attachment of a Branson Sonifier Cell Disruptor 200 (Branson Sonic Power Co., Danburry, Conn., U.S.A.) for 4 min at setting 3, whilst packed in ice. A stock solution of defatted bovine serum albumin (Sigma), 160 g/l in Tris-chloride (0.8 mol/l) buffer, pH 8.6, was made up each month. The incubation mixture contained 0-1 ml of substrate emulsion, 0-05 ml of stock albumin solution, 0.02 ml of the column fraction being assayed and water to a total volume of 0.2 ml. Thus the final concentration of triolein in the mixture was 11.3 mmol/l. A portion (0.02 ml) of pooled plasma drawn from healthy fasting subjects without prior injection of heparin, and inactivated by heating at 56OC for 1 h, was included, at the expense of water, for the assay of lipoprotein lipase.
I 0
,
1
10
20
30
40
50
60
Time (min)
FIG.1. Mean activity of plasma lipases at different time intervals after administration of heparin (50 i.u./kg body weight): e--., hepatic lipase from healthy subjects; e - - 0 , hepatic lipase from uraemic patients; A-A, lipoprotein lipase from healthy subjects; A---A, lipoprotein iipase from uraemic patients. Enzyme fractions were incubated with 11-3 mmol of triolein at pH 8.6, with 20 pl of added plasma for lipoprotein lipase assay. NEFA, Non-esterifled fatty acids.
G . A. Crawford, E . Savdie and J. H . Stewart
158
7
8
9
10
PH FIG.2. Mean activity of plasma lipases at different p H values: M, hepatic lipase from healthy subjects; 0---., hepatic lipase from uraemic patients; A-4 lipoprotein lipase from healthy subjects; A---A, lipoprotein lipase from uraemic patients. The activity was measured I5 min after intravenous administration of heparin (50 i.u./kg body weight). Enzyme fractions were incubated with 11.3 mmol of trioleidl with 20 pI of added plasma for lipoprotein lipase assay. A substrate blank was subtracted for each p H value.
Poobd, heat-inactivated pre-heparin plasma, from fasting normal subjects, added to the incubation medium was slightly inhibitory for hepatic lipase and generally stimulating for lipoprotein lipase (Fig. 3). However, this stimulation was significantly (P < 0-05)greater for normal lipoprotein lipase than for the enzyme from uraemic
patients, when 20 pl or more of pooled plasma was added. Both hepatic and lipoprotein lipase appeared to conform to Michaelis-Menten enzyme-substrate kinetics, and the substrate concentration used in the definitive assays was shown not to be rate-limiting for either enzyme (Fig. 4). Mean apparent Michaelis constants calculated from Lineweaver-Burk plots were 0.8 ( r = 0.98) and 3-0( r = 0-98) mmol of triolein/l for normal subjects, and 0.3 ( r = 0.77) and 5.0 (r = 0.96) mmol/l in uraemic subjects, for hepatic lipase and lipoprotein lipase respectively. With optimum assay conditions the within-run coefficient of variation was 12% for hepatic lipase and 7% for lipoprotein lipase (four portions assayed in duplicate with different columns). When post-heparin plasma was divided into eight portions, stored frozen and assayed on separate days spread over a 3 month period, activity did not decline significantly despite a between-run coefficient of variation for hepatic lipase of 22% and for lipoprotein lipase of 31%. This rather large variation was attributable to day-to-day differences in the substrate emulsion, which we were unable to eliminate despite rigidly standardized conditions of preparation. The variation was monitored by including a standard plasma source of enzyme activity with each assay, and rejecting results in which the standard’s activity was more than 10% above or below the expected value.
0
-
.-> m y
g
i
.-I
0
,
, 10
,
, 20
,
, 30
Plasma (/.11/0,2rnl of assay medium)
FIG.3. Mean activity of plasma lipases (as a percentage of that activity with no added plasma) with increasing amounts of pooled plasma added to the incubation medium: + 0 . hepatic lipase from healthy subjects; O---O, hepatic lipase from uraemic patients; A-A, lipoprotein lipase from healthy subjects; A---A, lipoprotein lipase from uraemic patients. The activity was measured 15 min aRer intravenous administration of heparin (50 i.u./kg body weight). Enzyme fractions were incubated with 11.3 mmol of triolein/l at pH 8.6.
Plasma lipases in chronic renal failure
159
concentration) heated pooled plasma. Fatty acids released were extracted by the method of Dole (1956) and measured by the spectrophotometric method of Duncombe (1963).
5
-
v
Statistical calculations Results were analysed by conventional parametric statistical methods including linear regression analysis and Student’s t-test (Snedecor & Cochran, 1967).
Triolein (pnol)
FIG.4. Mean activity of plasma lipases with increasing hepatic lipase from substrate concentration: W, hepatic -.lipase , from uraemic healthy subjects; +patients; A-4 lipoprotein lipase from healthy subjects; A--- A, lipoprotein lipase from uraemic patients. Activity was measured 15 min after intravenous administration of heparin (50 i.u./kg body weight). Enzyme fractions were incubated at pH 8.6 with addition of 20 pl of pooled plasma for lipoprotein lipase measurement. K,, Apparent Michaelis constant.
Enzyme assay with Intralipid Fractions from the heparin-Sepharose columns containing enzyme activity were incubated with an unlabelled Intralipid (Vitrum, Stockholm, Sweden) substrate mixture by the method of Boberg & Carlson (1964). The incubation conditions were modiied from those of Boberg & Carlson as follows: (i) incubation at 28OC, (ii) samples taken at 20 and 65 min for fatty acid estimation, (iu) assay of hepatic lipase at pH 9.3, (iv) assay of lipoprotein lipase at pH 8-6 with addition of 10% (final
Results
Results with “Cltriolein substrate Twelve male and 18 female patients with undialysed chronic renal failure, eight male and seven female renal transplant recipients and 17 male and 15 female healthy control subjects were so studied (Table 1). Male and female patients with chronic renal failure had elevated fasting serum triacylglycerol concentrations and expanded plasma volumes, but body weights and serum cholesterol concentrations which were not significantly different from those of control subjects. In addition to slightly elevated serum creatinine concentrations, the male and female transplant recipients had significantly raised serum triacylglycerols, but normal serum cholesterol values, body weights and plasma volumes. Hepatic lipase (Table 3a and Fig. 5). Hepatic lipase activity tended to be higher in males than in females, but significantly so only in controls (P
