Atherosclerosis,
22 (1975) 425430
425
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A STUDY PROTEIN
B.
PERSSON,
OF DIFFERENT LIPASE ACTIVITY
U. SMITH
First and Second (Sweden)
Medical
AND
METHODS FOR THE IN HUMAN ADIPOSE
ASSAY TISSUE
OF
LIPO-
B. LARSSON
Departments,
Sahlgrenska
Sjukhuset,
University
of Gteborg,
Giiteborg
(Received December 23rd, 1974) (Revised received March 13th, 1975) (Accepted March 13th, 1975)
SUMMARY
A method
for the assay of lipoprotein
lipase activity
(LPLA)
in heparin
eluates
of needle biopsies of adipose tissue is presented. A serum activated phosphatidestabilized emulsion of labelled triolein has been used as substrate. This method and a previously described method using heparin eluates as enzyme source and a commercial triglyceride emulsion, Ediol, as substrate, showed a high degree of correlation (correlation coefficient = 0.94) when parallel determinations were performed on biopsies from 16 subjects. Further, the Ediol method similarly correlated well with a method for LPLA assay, previously described by Nilsson-Ehle and Belfrage, on acetone-ether extracts of adipose tissue (correlation coefficient = 0.88; 19 subjects).
Key words : Adipose tissue - Enzyme analysis - Lipoprotein lipase
INTRODUCTION
A method for the determination of lipoprotein lipase activity (LPLA) in heparin eluates of human subcutaneous adipose tissue, using a triglyceride emulsion (Ediol) as substrate, was reported in 19661. This technique has been used in several studies of normal individuals and patients with hyperlipidemia2-4. Major findings of these studies have been that the LPLA, expressed per unit wet weight, is higher in women than in men3, that it decreases with increasing obesity2.3 and that it is low in isolated hypertriglyceridemia, Type IV according to the WHO-classification3. However, questions have been raised as to the physiological significance of studies with heparin eluates as well as to the specificity of the above-mentioned method for the enzyme
426
B. PERSSON,
lipoprotein
lipase1*2+9.
Recently,
methods
have
been
U. SMITH,
described
B. LARSSON
claiming
higher
enzyme specificityS*7. Therefore, in order to evaluate the different techniques, a comparison has been made between our previously described assay system1 and two new methods, present
one described
by Nilsson-Ehle
and Belfrages
and one described
in the
communication.
MATERIAL
AND METHODS
Needle biopsy specimens of subcutaneous adipose tissue from the gluteal region were obtained from healthy subjects and hyperlipidemic patients after an overnight fast as previously describedlpg. The biopsies were collected in Ringer’s solution and remained there at room temperature for a period not exceeding 20 min. One part of the specimens was taken for an elution procedure with Ringer’s solution containing heparin, insulin and glucose as previously describedlss. About 100 mg adipose tissue per ml eluting solution was used. Another part was homogenized and extracted with acetone-ether and further analyzed Nilsson-Ehle and Belfrages. After incubation and removal
for LPLA according of the biopsies
to the method
the heparin
described
by
eluates were analyzed
for LPLA with either or both of the two methods; with Ediol as substrate as previously used139 or with triolein labelled in the fatty acid moiety as described below. Eluate-triolein method Tri[aH]oleylglycerol was obtained from the Radiochemical Centre, Amersham, England and, if necessary, purified according to Jensen et al.1° to a radiopurity of 99.5 % as determined
by thin-layer
chromatography.
The labelled product
was diluted
with unlabelled triolein to a specific activity of about 4 mCi/mmole. An emulsion of about 65 mA4 triolein was prepared with 1.2% phosphatide solution* by repeated short sonications at 4°C with a Branson sonifier for a total period of 1 min. This emulsion is stable, but a short sonication was repeated each day. A new emulsion was prepared each week. For the assay of LPLA the following components were first mixed: 5 vol. Tris-buffer, 0.3 A4 with pH 8.6; 2 vol. 20% bovine serum albumin (Armour, Eastbourne, England) in ammonium sulphate, 0.25 M, titrated to pH 8.6 with ammonium hydroxide; 4 vol. pooled serum; 1 vol. triolein substrate prepared as described above. After IO-30 min 10 vol. of the heparin eluatet of the adipose tissue was added and the mixture was incubated for 45 min at 37°C in a metabolic shaker. In most cases the final assay volume was 1.1 ml. Controls, including eluate from a medium incubated in the absence of adipose tissue, followed the same procedure. Released fatty acids were extracted and radioactivity determined according to Belfrage and Vaughan11 as modified by Nilsson-Ehle and Belfrage5. Enzymatic activity
* Generously supplied by AB Vitrum, Stockholm, same as in “Intralipid” but without glycerol.
Sweden. The phosphatide
emulsion is the
ASSAY OF LIPOPROTEIN
was expressed
LIPASE ACTIVITY
427
IN ADIPOSE TISSUE
as mU per g adipose tissue (wet weight). One mU represents
the release
of one nmole fatty acid per min. Additional
enzyme
source
In preliminary experiments a crude lipoprotein lipase preparation from raw cow milk prepared according to Kornla was used. An immunological cross-reaction between the bovine milk and human lipoprotein lipases has recently been shownis, indicating that the bovine enzyme may be used as a good model for these enzymes in human tissues. experiments). RESULTS
This is also in agreement
with our own experience
(unpublished
AND DISCUSSION
Optimal conditions for the assay of LPLA with the labelled triolein emulsion were tested with the milk enzyme. Fig. 1A shows the effect of increasing the substrate concentration at two different serum concentrations. At the lower serum concentration, the decrease in FFA release with increasing substrate concentration, was seen in repeated experiments. In the assay, the higher serum concentration (18 %) and a triolein concentration of about 3 mM have been used. Fig. 1B shows the requirements of serum and albumin for optimal assay conditions. Figure 1C shows the linear FFA release with time at two substrate concentrations and Fig. 1D the linear relation between amount of enzyme and fatty acid release. The eluate method
for LPLA with Ediol as substratei
has been compared
with
the eluate method with labelled triolein as substrate in 16 subjects and the former method with the method of Nilsson-Ehle and Belfrage5 in 19 subjects. It is evident from Fig. 2 that in both instances there is a close correlation between the determined activities in spite of several differences in substrate character and adipose tissue handling. Thus, at least in the fasting state, the eluted LPLA seems to be well representative of the total tissue activity of the enzyme at the time of sampling, as also previously reported’j. The result of the comparisons between the Ediol method and the methods using highly purified triolein substrates does not indicate the presence of any quantitatively significant amount of a specific monoglyceridase in the adipose eluates acting on the monostearate emulsifier component of Ediol. This is in accordance with previous findingsss, while Elkeles7 recently reported the hydrolysis of mono-palmitin, in a gum-arabic stabilized dispersion, by heparin as well as nonheparin eluates of adipose tissue. In the latter study the activity was not affected by the absence of serum or presence of 0.5 M sodium chloride, in contrast to previous findings in heparin eluates with emulsified mono-olein as substrate (Refs. 8, 14 and Persson, B., unpublished observations). The importance of the physical state of the lipid substrate has repeatedly been stressed in discussions of lipase specificities15-i7 and differences therein may be one explanation of the apparent differences between the findings of Elkeles7 and others s*s,i*. Slight differences in the elution procedures may also be of importancei.
428
B.
PERSSON, U. SMITH, B. LARSSON
r a6
B
A 075
15
0.45
6
3
t8
36
PER CENT ALBUMIN
mM TRGLYCERIDES mU 25. . g 20. 15
MINUTES
PERCENT ENZYME
Fig. 1. Assay of LPLA with a labelled triolein emulsion as substrate. Basal conditions for the assay as described under MATERIAL AND METHODS with milk lipoprotein lipase as enzyme source. Enzymatic activity has been expressed as mU or fatty acid release per 0.2 ml of the assay medium. A: Increasing substrate concentration at different serum concentrations, ?? ??18% and 0 - - - 0 9 %. The former serum concentration and an about 3 mM triglyceride concentration has been chosen in the assay system. B: Serum and albumin requirement for the assay. At the zero point neither serum nor albumin was added, while at the following two points 1 % and 9 % of serum was added, respectively. At the last two points 9% of serum as well as bovine albumin were added. Indicated albumin concentrations include albumin in serum. An about 2.5 ‘A albumin concentration has been chosen in the assay system. C: Release of labelled oleic acid with time at two different substrate concentrations: ?? 03 6mM.0 -0 indicates spontaneous fatty acid release at a 6 mM substrate mMand * --+ concentration, without any added enzyme. D: Release of labelled oleic acid with different amounts of a lipoprotein lipase preparation added to the assay system. “Per cent enzyme” = per cent of most active enzyme preparation used in the experiment. There was an obvious difference in activity levels between the eluate-Ediol method and the two methods using [sH]triolein as substrate (Fig. 2). The first method yielded 5 to 7 times higher activity than the two other methods. The activity with Ediol as substrate has previously been shown to be slightly lower than that obtained with a phosphatide-stabilized emulsion of purified coconut oil triglycerides but about
ASSAY OF LIPOPROTEIN LIPASE ACTIVITY IN ADIPOSE TISSUE
429
.
Fig. 2. Relations between the eluate-Ediol method for LPLA assay and two methods using labelled triolein as substrate. Filled circles correspond to parallel determinations with the eluate-Ediol method and the method described by Nilsson-Ehle and Belfrages on adipose tissue biopsies from 19 subjects. The regression line (- - -) is based on the following equation: y = 0.234 x - 5.58; correlation coefficient = 0.88. Open circles correspond to a similar comparison between the eluate-Ediol method and the eluate-triolein method on biopsies from 16 subjects. Regression line (--): equation y = 0.17x - 3.54; correlation coefficient = 0.94. The point of intersection between the x and y axes is located within the 5% confidence limits of y for both regression lines. There is no statistically significant difference between the slopes of the two lines.
twice the activity obtained with Intralipid (= phosphatide-stabilized soya bean oil emulsion)s. The composition of the triglyceride substrate therefore seems to be of importance for the hydrolyzing capacity of the adipose tissue lipoprotein lipase. One conclusion that can be drawn from the present investigation is that the recently described methods for LPLA assay determine the same type of enzyme activity as the eluate - Ediol method. Thus, claims of higher enzyme specificity of these methods cannot be validated. They are useful, however, due to the small amount of adipose tissue required and also because of simplified methodology. Thus, methods are available for larger scale use in clinical laboratories allowing for instance genetic studies of hyperlipidemic states, which might well prove more valuable in this respect than classification according to lipid electrophoresis.
REFERENCES 1 PERSSON, B., BI~RNTORP, P. AND Hook, B., Lipoprotein lipase activity in human adipose tissue, Part 1 (Conditions for release and relationship to triglycerides in serum), Metabolism, 15 (1966) 730. 2 PERSON, B., SCHRBDER, G. AND HOOD, B., Lipoprotein lipase activity in human adipose tissue: Assay methods - Relations to the serum triglyceride level in a normolipidemic population. The effect of ethyl chlorophenoxy-isobutyrate, Atherosclerosis, 16 (1972) 37.
430
B. PERSSON,
U. SMITH, B. LARSSON
3 PERSSON,B., Lipoprotein lipase activity of human adipose tissue in different types of hyperlipidemia, Actu Med. Stand., 193 (1973) 447. 4 PERSSON,B., Lipoprotein lipase activity of human adipose tissue in health and in some diseases with hyperlipidemia as a common feature, Acta Med. Scund., 194 (1973) 457. 5 NILSSON-EHLE,P. AND BELFRAGE,P., Rapid determination of lipoprotein lipase activity in human adipose tissue, Clin. Chim. Acta, 42 (1972) 383. 6 NILSSON-EHLE,P., Human lipoprotein lipase activity - Comparison of assay methods, Clin. Chim. Acta, 54 (1974) 283. 7 ELKELES,R. S., Lipoprotein lipase in human adipose tissue, Clin. Sci. Molec. Med., 46 (1974) 753. 8 PERSSON,B. AND Hook, B., Characterization of lipoprotein lipase activity eluted from human adipose tissue, Atherosclerosis, 12 (1970) 241. 9 PERSSON,B., Lipoprotein Lipase Activity in Human Adipose Tissue - With Special Reference to the Relation between the Enzyme Activity and the Serum Triglyceride Level, Thesis, Goteborg, 1972. 10 JENSEN,R. G., MARKS, T. A., SAMPUGNA,J., QUINN, J. G. AND CARPENTER,P. L., Purification of triglycerides with an alumina column, Lipids, 1 (1955) 451. 11 BELFRAGE,P. AND VAUGHAN, M., Simple liquid-liquid partition system for isolation of labelled oleic acid from mixtures with glycerides, .I. Lipid Res., 10 (1969) 341. 12 KORN, E. D., The lipoprotein lipase of cow’s milk, J. Lipid Res., 3 (1962) 246. 13 HERNELL,O., EGELRUD,T. AND OLIVECRONA,T., Serum-stimulated lipases (lipoprotein lipases) Immunological crossreaction between the bovine and the human enzymes, Biochim. Biophys. Acta, 381 (1975) 233. 14 SHAFRIR, E. AND BIALE, Y., Effect of experimental hypertriglyceridaemia on tissue and serum lipoprotein lipase activity, Eur. J. Clin. Invest., 1 (1970) 19. 15 MORGAN, R. G. H., BARROWMAN,J., FILIPEK-WENDER,H. AND BORGSTROM,B., The lipolytic enzymes of rat pancreatic juice, Biochim. Biophys. Acta, 146 (1967) 314. 16 DATTA, D. V. AND WIGGINS, H. S., New effects of sodium chloride and protamine on human postheparin lipoprotein lipase activity, Proc. Sot. Exp. Biol. Med., 115 (1964) 788. 17 HAVEL, R. J., FIELDING,C. J., OLIVECRONA,T., SHORE,V. G., FIELDING,PH. E. AND EGELRUD,T., Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoprotein lipase from different sources, B&ochemistry, 12 (1973) 1828.
22 (1975) 425430
425
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A STUDY PROTEIN
B.
PERSSON,
OF DIFFERENT LIPASE ACTIVITY
U. SMITH
First and Second (Sweden)
Medical
AND
METHODS FOR THE IN HUMAN ADIPOSE
ASSAY TISSUE
OF
LIPO-
B. LARSSON
Departments,
Sahlgrenska
Sjukhuset,
University
of Gteborg,
Giiteborg
(Received December 23rd, 1974) (Revised received March 13th, 1975) (Accepted March 13th, 1975)
SUMMARY
A method
for the assay of lipoprotein
lipase activity
(LPLA)
in heparin
eluates
of needle biopsies of adipose tissue is presented. A serum activated phosphatidestabilized emulsion of labelled triolein has been used as substrate. This method and a previously described method using heparin eluates as enzyme source and a commercial triglyceride emulsion, Ediol, as substrate, showed a high degree of correlation (correlation coefficient = 0.94) when parallel determinations were performed on biopsies from 16 subjects. Further, the Ediol method similarly correlated well with a method for LPLA assay, previously described by Nilsson-Ehle and Belfrage, on acetone-ether extracts of adipose tissue (correlation coefficient = 0.88; 19 subjects).
Key words : Adipose tissue - Enzyme analysis - Lipoprotein lipase
INTRODUCTION
A method for the determination of lipoprotein lipase activity (LPLA) in heparin eluates of human subcutaneous adipose tissue, using a triglyceride emulsion (Ediol) as substrate, was reported in 19661. This technique has been used in several studies of normal individuals and patients with hyperlipidemia2-4. Major findings of these studies have been that the LPLA, expressed per unit wet weight, is higher in women than in men3, that it decreases with increasing obesity2.3 and that it is low in isolated hypertriglyceridemia, Type IV according to the WHO-classification3. However, questions have been raised as to the physiological significance of studies with heparin eluates as well as to the specificity of the above-mentioned method for the enzyme
426
B. PERSSON,
lipoprotein
lipase1*2+9.
Recently,
methods
have
been
U. SMITH,
described
B. LARSSON
claiming
higher
enzyme specificityS*7. Therefore, in order to evaluate the different techniques, a comparison has been made between our previously described assay system1 and two new methods, present
one described
by Nilsson-Ehle
and Belfrages
and one described
in the
communication.
MATERIAL
AND METHODS
Needle biopsy specimens of subcutaneous adipose tissue from the gluteal region were obtained from healthy subjects and hyperlipidemic patients after an overnight fast as previously describedlpg. The biopsies were collected in Ringer’s solution and remained there at room temperature for a period not exceeding 20 min. One part of the specimens was taken for an elution procedure with Ringer’s solution containing heparin, insulin and glucose as previously describedlss. About 100 mg adipose tissue per ml eluting solution was used. Another part was homogenized and extracted with acetone-ether and further analyzed Nilsson-Ehle and Belfrages. After incubation and removal
for LPLA according of the biopsies
to the method
the heparin
described
by
eluates were analyzed
for LPLA with either or both of the two methods; with Ediol as substrate as previously used139 or with triolein labelled in the fatty acid moiety as described below. Eluate-triolein method Tri[aH]oleylglycerol was obtained from the Radiochemical Centre, Amersham, England and, if necessary, purified according to Jensen et al.1° to a radiopurity of 99.5 % as determined
by thin-layer
chromatography.
The labelled product
was diluted
with unlabelled triolein to a specific activity of about 4 mCi/mmole. An emulsion of about 65 mA4 triolein was prepared with 1.2% phosphatide solution* by repeated short sonications at 4°C with a Branson sonifier for a total period of 1 min. This emulsion is stable, but a short sonication was repeated each day. A new emulsion was prepared each week. For the assay of LPLA the following components were first mixed: 5 vol. Tris-buffer, 0.3 A4 with pH 8.6; 2 vol. 20% bovine serum albumin (Armour, Eastbourne, England) in ammonium sulphate, 0.25 M, titrated to pH 8.6 with ammonium hydroxide; 4 vol. pooled serum; 1 vol. triolein substrate prepared as described above. After IO-30 min 10 vol. of the heparin eluatet of the adipose tissue was added and the mixture was incubated for 45 min at 37°C in a metabolic shaker. In most cases the final assay volume was 1.1 ml. Controls, including eluate from a medium incubated in the absence of adipose tissue, followed the same procedure. Released fatty acids were extracted and radioactivity determined according to Belfrage and Vaughan11 as modified by Nilsson-Ehle and Belfrage5. Enzymatic activity
* Generously supplied by AB Vitrum, Stockholm, same as in “Intralipid” but without glycerol.
Sweden. The phosphatide
emulsion is the
ASSAY OF LIPOPROTEIN
was expressed
LIPASE ACTIVITY
427
IN ADIPOSE TISSUE
as mU per g adipose tissue (wet weight). One mU represents
the release
of one nmole fatty acid per min. Additional
enzyme
source
In preliminary experiments a crude lipoprotein lipase preparation from raw cow milk prepared according to Kornla was used. An immunological cross-reaction between the bovine milk and human lipoprotein lipases has recently been shownis, indicating that the bovine enzyme may be used as a good model for these enzymes in human tissues. experiments). RESULTS
This is also in agreement
with our own experience
(unpublished
AND DISCUSSION
Optimal conditions for the assay of LPLA with the labelled triolein emulsion were tested with the milk enzyme. Fig. 1A shows the effect of increasing the substrate concentration at two different serum concentrations. At the lower serum concentration, the decrease in FFA release with increasing substrate concentration, was seen in repeated experiments. In the assay, the higher serum concentration (18 %) and a triolein concentration of about 3 mM have been used. Fig. 1B shows the requirements of serum and albumin for optimal assay conditions. Figure 1C shows the linear FFA release with time at two substrate concentrations and Fig. 1D the linear relation between amount of enzyme and fatty acid release. The eluate method
for LPLA with Ediol as substratei
has been compared
with
the eluate method with labelled triolein as substrate in 16 subjects and the former method with the method of Nilsson-Ehle and Belfrage5 in 19 subjects. It is evident from Fig. 2 that in both instances there is a close correlation between the determined activities in spite of several differences in substrate character and adipose tissue handling. Thus, at least in the fasting state, the eluted LPLA seems to be well representative of the total tissue activity of the enzyme at the time of sampling, as also previously reported’j. The result of the comparisons between the Ediol method and the methods using highly purified triolein substrates does not indicate the presence of any quantitatively significant amount of a specific monoglyceridase in the adipose eluates acting on the monostearate emulsifier component of Ediol. This is in accordance with previous findingsss, while Elkeles7 recently reported the hydrolysis of mono-palmitin, in a gum-arabic stabilized dispersion, by heparin as well as nonheparin eluates of adipose tissue. In the latter study the activity was not affected by the absence of serum or presence of 0.5 M sodium chloride, in contrast to previous findings in heparin eluates with emulsified mono-olein as substrate (Refs. 8, 14 and Persson, B., unpublished observations). The importance of the physical state of the lipid substrate has repeatedly been stressed in discussions of lipase specificities15-i7 and differences therein may be one explanation of the apparent differences between the findings of Elkeles7 and others s*s,i*. Slight differences in the elution procedures may also be of importancei.
428
B.
PERSSON, U. SMITH, B. LARSSON
r a6
B
A 075
15
0.45
6
3
t8
36
PER CENT ALBUMIN
mM TRGLYCERIDES mU 25. . g 20. 15
MINUTES
PERCENT ENZYME
Fig. 1. Assay of LPLA with a labelled triolein emulsion as substrate. Basal conditions for the assay as described under MATERIAL AND METHODS with milk lipoprotein lipase as enzyme source. Enzymatic activity has been expressed as mU or fatty acid release per 0.2 ml of the assay medium. A: Increasing substrate concentration at different serum concentrations, ?? ??18% and 0 - - - 0 9 %. The former serum concentration and an about 3 mM triglyceride concentration has been chosen in the assay system. B: Serum and albumin requirement for the assay. At the zero point neither serum nor albumin was added, while at the following two points 1 % and 9 % of serum was added, respectively. At the last two points 9% of serum as well as bovine albumin were added. Indicated albumin concentrations include albumin in serum. An about 2.5 ‘A albumin concentration has been chosen in the assay system. C: Release of labelled oleic acid with time at two different substrate concentrations: ?? 03 6mM.0 -0 indicates spontaneous fatty acid release at a 6 mM substrate mMand * --+ concentration, without any added enzyme. D: Release of labelled oleic acid with different amounts of a lipoprotein lipase preparation added to the assay system. “Per cent enzyme” = per cent of most active enzyme preparation used in the experiment. There was an obvious difference in activity levels between the eluate-Ediol method and the two methods using [sH]triolein as substrate (Fig. 2). The first method yielded 5 to 7 times higher activity than the two other methods. The activity with Ediol as substrate has previously been shown to be slightly lower than that obtained with a phosphatide-stabilized emulsion of purified coconut oil triglycerides but about
ASSAY OF LIPOPROTEIN LIPASE ACTIVITY IN ADIPOSE TISSUE
429
.
Fig. 2. Relations between the eluate-Ediol method for LPLA assay and two methods using labelled triolein as substrate. Filled circles correspond to parallel determinations with the eluate-Ediol method and the method described by Nilsson-Ehle and Belfrages on adipose tissue biopsies from 19 subjects. The regression line (- - -) is based on the following equation: y = 0.234 x - 5.58; correlation coefficient = 0.88. Open circles correspond to a similar comparison between the eluate-Ediol method and the eluate-triolein method on biopsies from 16 subjects. Regression line (--): equation y = 0.17x - 3.54; correlation coefficient = 0.94. The point of intersection between the x and y axes is located within the 5% confidence limits of y for both regression lines. There is no statistically significant difference between the slopes of the two lines.
twice the activity obtained with Intralipid (= phosphatide-stabilized soya bean oil emulsion)s. The composition of the triglyceride substrate therefore seems to be of importance for the hydrolyzing capacity of the adipose tissue lipoprotein lipase. One conclusion that can be drawn from the present investigation is that the recently described methods for LPLA assay determine the same type of enzyme activity as the eluate - Ediol method. Thus, claims of higher enzyme specificity of these methods cannot be validated. They are useful, however, due to the small amount of adipose tissue required and also because of simplified methodology. Thus, methods are available for larger scale use in clinical laboratories allowing for instance genetic studies of hyperlipidemic states, which might well prove more valuable in this respect than classification according to lipid electrophoresis.
REFERENCES 1 PERSSON, B., BI~RNTORP, P. AND Hook, B., Lipoprotein lipase activity in human adipose tissue, Part 1 (Conditions for release and relationship to triglycerides in serum), Metabolism, 15 (1966) 730. 2 PERSON, B., SCHRBDER, G. AND HOOD, B., Lipoprotein lipase activity in human adipose tissue: Assay methods - Relations to the serum triglyceride level in a normolipidemic population. The effect of ethyl chlorophenoxy-isobutyrate, Atherosclerosis, 16 (1972) 37.
430
B. PERSSON,
U. SMITH, B. LARSSON
3 PERSSON,B., Lipoprotein lipase activity of human adipose tissue in different types of hyperlipidemia, Actu Med. Stand., 193 (1973) 447. 4 PERSSON,B., Lipoprotein lipase activity of human adipose tissue in health and in some diseases with hyperlipidemia as a common feature, Acta Med. Scund., 194 (1973) 457. 5 NILSSON-EHLE,P. AND BELFRAGE,P., Rapid determination of lipoprotein lipase activity in human adipose tissue, Clin. Chim. Acta, 42 (1972) 383. 6 NILSSON-EHLE,P., Human lipoprotein lipase activity - Comparison of assay methods, Clin. Chim. Acta, 54 (1974) 283. 7 ELKELES,R. S., Lipoprotein lipase in human adipose tissue, Clin. Sci. Molec. Med., 46 (1974) 753. 8 PERSSON,B. AND Hook, B., Characterization of lipoprotein lipase activity eluted from human adipose tissue, Atherosclerosis, 12 (1970) 241. 9 PERSSON,B., Lipoprotein Lipase Activity in Human Adipose Tissue - With Special Reference to the Relation between the Enzyme Activity and the Serum Triglyceride Level, Thesis, Goteborg, 1972. 10 JENSEN,R. G., MARKS, T. A., SAMPUGNA,J., QUINN, J. G. AND CARPENTER,P. L., Purification of triglycerides with an alumina column, Lipids, 1 (1955) 451. 11 BELFRAGE,P. AND VAUGHAN, M., Simple liquid-liquid partition system for isolation of labelled oleic acid from mixtures with glycerides, .I. Lipid Res., 10 (1969) 341. 12 KORN, E. D., The lipoprotein lipase of cow’s milk, J. Lipid Res., 3 (1962) 246. 13 HERNELL,O., EGELRUD,T. AND OLIVECRONA,T., Serum-stimulated lipases (lipoprotein lipases) Immunological crossreaction between the bovine and the human enzymes, Biochim. Biophys. Acta, 381 (1975) 233. 14 SHAFRIR, E. AND BIALE, Y., Effect of experimental hypertriglyceridaemia on tissue and serum lipoprotein lipase activity, Eur. J. Clin. Invest., 1 (1970) 19. 15 MORGAN, R. G. H., BARROWMAN,J., FILIPEK-WENDER,H. AND BORGSTROM,B., The lipolytic enzymes of rat pancreatic juice, Biochim. Biophys. Acta, 146 (1967) 314. 16 DATTA, D. V. AND WIGGINS, H. S., New effects of sodium chloride and protamine on human postheparin lipoprotein lipase activity, Proc. Sot. Exp. Biol. Med., 115 (1964) 788. 17 HAVEL, R. J., FIELDING,C. J., OLIVECRONA,T., SHORE,V. G., FIELDING,PH. E. AND EGELRUD,T., Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoprotein lipase from different sources, B&ochemistry, 12 (1973) 1828.
