Naunyn-Schmiedeberg's

Archivesof Pharmacology

Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 6 5 - 7 3 (1977)

9 by Springer-Verlag 1977

Alterations of Rat Serum Lipoproteins and Lecithin-Cholesterol-Acyltransferase Activity in Praseodymium-Induced Liver Damage O. GRAJEWSKI, B. VON LEHMANN, H.-R. ARNTZ, P. ARVELA*, and E. OBERDISSE Pharmakologisches Institut der Freien Universitfit Berlin, Thielallee 69/73, D-1000 Berlin 33

Summary. During liver damage induced by i.v. injection of 10 mg/kg praseodymium nitrate (Pr) marked alterations of the serum lipids are observed in female Wistar rats. The triglycerides (TG) decrease to about 50 ~ of the control values after 2 days. Total phospholipids (TPL) and cholesterol (C) show a parallel time course: a 50 ~o decrease 2 and 4 days after Pr is followed by a 50 ~ increase at 4 days. The changes in the TPL are mainly due to alterations of phosphatidylcholine (PC) concentration, while the decrease in the concentration of C during the first 2 days is caused by a decrease of the esterified cholesterol (EC), whereas unesterified cholesterol (UC) is responsible for the subsequent increase of C. The fractionation of the serum lipoproteins (Lp) by sequential ultracentrifugation demonstrates a different response to Pr in each Lp class. At 2 days the very low density lipoprotein (VLDL) concentration decreases by 50 ~. Experiments with Triton WR 1339 show an inhibition of hepatic TG secretion at this time. On the other hand, the LDL increases to more than four times the controls, 4 days after Pr. The accumulating LDL are abnormally rich in UC and PC. Up to 2 days after Pr the high density lipoprotein (HDL) content declines to about 20 ~ of the concentration in control animals. During the subsequent recovery the lipid pattern alters, as UC and TPL are increased; the HDL2 are particularly involved. Concomitant with the changes in the lipid patterns of LDL and H D L the activity of serum lecithin-cholesterol-acyltransferase (LCAT) decreases to about 15 ~, 3 days after Pr. From our results we conclude: (1) After Pr the VLDL, H D L and LCAT secretion of the liver is inhibited. Send offprint requests to E. Oberdisse at the above address

*

Fellow of the Alexander von Humboldt-Stiftung

(2) In the LDL, HDL, and less in the VLDL, UC and PC-rich Lp occur. This results in an alteration of the lipid pattern of the Lp. (3) The alteration of the lipid pattern is mainly due to the diminution of LCAT activity.

Key words." Liver damage - Praseodymium - Serum lipoproteins - Hepatic lipoprotein secretion - Lecithin-cholesterol-acyltransferase.

INTRODUCTION The liver plays a major role in the synthesis and catabolism of the serum lipoproteins (serum-Lp) which are carriers of triglycerides (TG), phospholipids (TPL) and esterified and unesterified cholesterol (EC and UC) in the blood (Margolis and Capuzzi, 1972; Stein et al., 1972; Hamilton and Kayden, 1974; Eisenberg and Levy, 1975; Jackson et al., 1975). The fact that changes in the serum-Lp pattern can be observed regularly during human liver diseases indicates the functional importance of this organ in lipid metabolism (Smith et al., 1967; Papadopoulos and Charles, 1970; Barclay, 1972; Baumgarten et al., 1974; Picard et al., 1974). Though many experiments have been carried out on this subject it is still not known which biochemical disturbances lead to the changes in concentration and structure of the different lipoprotein fractions. Moreover, it is obvious that dissolved and structurallybound Lp exchange with each other, not only in the liver but elsewhere so that changes in one of these compartments cannot be without consequences for the other (Barclay and Skipski, 1975; Eisenberg and Levy, 1975; Jackson et al., 1975). Alterations in the serum-Lp after experimental lesion of the liver have so far received little attention

66

(Lombardi and Ugazio, 1965; Sabesin et al., 1975) and this may be a reason for the lack of understanding of these processes. In order to extend our knowledge of the pathophysiology of the serum lipoprotein metabolism during experimental liver damage, we began to study the changes of the serum-Lp in the course of the liver lesion induced by praseodymium. This substance belongs to the group of rare earth metals, the salts of which after i.v. injection of small doses cause reproducible liver damage, manifested by liver cell necroses, leucocyte and histiocyte infiltration and a pronounced fatty degeneration of the liver (Fischler and Roeckl, 1938; Bruce et al., 1963; Magnusson, 1963; Gross et al., 1974). The primary site of action of the rare earths is through inhibition of RNA-synthesizing mechanisms at the nuclear level (Oberdisse et al., 1974; Sarkander and Brade, 1976; v. Lehmann et al., 1976).

MATERIALS AND METHODS Animals. Female Wistar rats, weighing from 160--180 g, received standard diet (Atromin ~) and tap water ad libitum. Reagents. Praseodymium nitrate pentahydrate (Pr(NO3)3 x 5 H20) was obtained from Schuchardt (MiJnchen). Phospholipids were purchased from Koch-Light Laboratories Ltd. (Colnbrook, Bucks.). They all derived from natural sources and were substantially pure as checked by thin layer chromatography. Triton WR 1339, 2mercaptoethanol, and 5,5'-dithio-2-nitrobenzoic acid (Ellman's reagent) were bought from Serva Feinbiochemica GmbH & Co. KG (Heidelberg), bovine serum albumin from Behringwerke (Frankfurt a. M). All other reagents were of analytical quality and supplied by Merck (Darmstadt). Pretreatment. Praseodymium nitrate pentahydrate was dissolved in 0.9 ~ sodium chloride solution and injected i.v. in a dose of 10 rag/ kg body weight (injection volume: 0.5 ml/100 g body weight). This dose corresponds to 3 mg/cation/kg. Preparation of Serum-Lipoproteins. Serum-Lp were obtained by sequential preparative ultracentrifugation carried out at 4~ in a Spinco ultracentrifuge model Lso (Beckman GmbH, Mtinchen) using a rotor 50 Ti (Havel et al., 1955; Hatch and Lees, 1968). For the isolation of very low density lipoproteins (VLDL, d < 1.006 g/ ml) including chylomicrons, four volumes of the pooled sera of five animals were overlaid with 4.5 volumes of a 0.15 M NaC1 solution containing 0.25 mM EDTA at pH 7.4 (d = 1.006 g/ml) and centrifuged for 1.5x 108 gmin (105000 g for 20 h). The top Lp layer was removed by suction, the tube refilled and the centrifugation repeated twice. The second and third top layers were combined with the first; the bottom fraction was used for the subsequent isolation of the Lp classes of higher densities. The solution densities of aliquots of the remaining bottom portions were increased successively to d = 1.063 and d = 1.21 g/ml by addition of appropriate volumes of NaC1/NaBr solutions (Hatch and Lees, 1968). Each fraction obtained was washed twice in salt solutions of the same density as for the isolation process, the densities being controlled by pycnometry. The average centrifugal force for preparation of low density lipoproteins (LDL, 1.006 < d < 1.063) was 2 x 10s groin and for preparation of high density lipoproteins (HDL, 1.063

Naunyn-Schmiedeberg's Arch. Pharmacol. 30I (t977) < d < 1.21) 2.5 x 10s gmin. In one experiment the HDL were subfractionated into HDL2 (1.063 1.21) remained in the HDL infranatant and was extracted for lipids without further separation from the serum proteins.

Lipid Extraction and Chemical Determinations. Serum lipids and Lp were extracted with chloroform/methanol 2:1 (v:v) and purified according to Folch et al. (1957). Aliquots of the purified extracts were evaporated for the determination of TG according to Eggstein and Kreutz (1966), TPL according to Raheja et al. (1973), and C as well as EC and UC according to Zak (1965). The quantitative separation of the individual pbospholipids (PL), lysophosphatidylcholine (LPC), sphingomyeline (S), phosphatidylethanolamine (PE), phosphatidylcholine (PC) phosphatidyl-serine (PS) and phosphatidylinositol (PI) was performed as described by Peter and Wolf (1973) by means of one-dimensional thin layer chromatography and fluorimetrical scanning of the phospholipid spots. Determination of Protein. The protein content of the Lp was determined according to Lowry et al. (1951) with bovine serum albumin as a standard. Statistical Calculations. Mean values and standard errors of the mean (S.E.M.) were calculated in each group. Using Student's t test, differences were considered to be significant if P was less than 0.01.

RESULTS Figure I shows the concentrations and respective time courses of TG, TPL, and C in the serum after i.v. injection of praseodymium. The difference between the course of the TG-concentration and the course of the TPL- and C-concentrations is conspicuous. While the TG-content of the serum drops to a minimum after 2 d and returns to the initial value 1 d later, the concentrations of TPL and C show a clearly biphasic course. A decrease of about 50 ~o is seen after 1 d and is followed by an increase of about 50 ~ after 4 d. With the TPL, this is mainly due to a change in the concentration of PC, while the drop of C is caused by a decrease in EC, the subsequent rise on the other hand is produced mainly by an increase in UC. As the time course of the serum lipids only demonstrates the sum of all changes in the different serum-Lp fractions, VLDL, LDL, HDL and VHDL were subsequently tested individually. Their main constituents are shown as parameters for the changes in these fractions. The diminution of the concentration of the TG-rich VLDL at first corresponds approximately to that of the serum-TG, but after a transient increase on the 4th day after praseodymium it shows a new decrease which can still be observed after a further 3 days (Fig. 2), although at this time only slight lesions of the liver parenchyma are found histologically and biochemically (Magnusson, 1963; v. Lehmann et al., 1976). As inhibition of the TG-release from the liver has been described for several experimental lesions of the liver (Barclay, 1972; Margolis and

O. Grajewski et al. : Lipoproteins and LCAT during Experimental Liver Disease

67

g/I

g/,

gl

117

/",,, L

TG

1,0-

1j5-

q7-

0,9-

Fig. 1 Time course (days) of the concentrations of serum triglycerides (TG), total phospholipids (TPL), phosphatidylcholine (PC), total cholesterol (C), esterified and unesterifled cholesterol (EC and UC) after i.v. injection of praseodymium nitrate. Each value represents the mean of eight individual determinations of unpooled sera, Co = untreated animals

111

qg.

as0,4-

0~7i

9

i

%"",,

'\

/

" '-t C EC

,

,

,

CO

1

2

;

, 4

OTI" "'r

lUC

LPC

; 5

I 0,5-

0~

0,3-

0~"

~...

o,1.

0,6-

TG

0,2-

,

o~.

/

:

-..

o#.

aaa2ol-

VLDL

~

L

o,7-

o,6-

0,7-

0,1- ~

?

o,8-

g 0,8-

TPL

1/3-

~ TPL ............ ~ C

Fig. 2. Time course (days) of the triglyceride (TG), total cholesterol (C), and total phospholipid (TPL) content of serum very low density lipoproteins (VLDL) after i.v. injection of praseodymium nitrate. Each value is the mean of 5 individual determinations. Co = untreated animals

Capuzzi, 1972; Stein et al., 1972), we examined the TGsecretion of the liver by means of experiments with the detergent Triton WR 1339. After i.v. administration this substance prevents the catabolism of the serum-TG and thereby leads to a hypertriglyceridemia (Friedman and Byers, 1957; Otway and Robinson, 1967 a). The increase in TG-concentration in the serum therefore is a measure for the TG-secretion of the liver

d

(Otway and Robinson, 1967b; Recknagel, 1967). In our experiments the TG-concentration in the control animals rose from 0.85 • 0.05 g/1 (Fig. l) to 10.7 • 0.5g/1 (Fig. 3). Lombardi and Recknagel (1962) have already demonstrated the validity of Triton experiments in experimental liver injury due to ethionine, cerium, phosphorus and choline deficient diet. The inhibition of the TG-release, measured as the decrease of TG-concentration in serum, is most pronounced 2 d after administration of Pr and in contrast to the concentration of the serum-TG and the VLDL-TG, it returns to the initial value 4 d after the injection of Pr (Fig. 3). In the course of the liver damage due to Pr the LDL react quite differently from the VLDL (Fig. 4). A short, but significant decrease I d after administration of the rare earth is followed by a steep increase to threefold the control level with C and to fourfold with TPL, after 4 d. These changes are mainly the expression of increases in UC and PC, both of which increase to about eightfold (Tables 1, 2). At this time the LDL lipids are particularly rich in PC and UC, the proportion of UC increasing from 7 to 15 ~, and that of PC increasing from 15 to 28 ~. Subsequent to the large increase of both TPL and C of the LDL, an increase in LDL-TG is observed in the late phase of liver damage so that at this stage the TG form the greater part of the LDL-lipids. The protein content is not shown but changes only relatively slightly. The serum-HDL concentration is clearly decreased during the 2 d following the injection of Pr, all components of the H D L being equally affected (Fig. 5). The subsequent increase is characterized by the fact that the composition of the fraction changes (Fig. 5, Tables i and 2). The TPL, and in particular the PC, increase much faster than do C and protein. Thus, the TPL become the relatively greatest fraction of the H D L

68

Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977)

gTG/L 12-1

Cl

o/

LDL

q~80/4-

q3-

q2-

J

\

7

TPL--

011-

I

Co

I

1

I

I

2

3

I

4

I

5

I

6d

Fig. 3. Influence of pretreatment with praseodymium nitrate on serum triglyceride (TG) concentration after i.v. injection of Triton W R 1339 (500 mg/kg dissolved in 0.9 % NaC1). The time of praseodymium nitrate injection before the rats were killed is indicated on the abscissa. Triton W R 1339 was always given 90 rain before the animals were killed. Each value represents the mean of eight determinations. Co = Triton-treated control animals. The fall in TG concentration compared with Co indicates a diminished TG secretion

Fig.4. Time course (days) of the triglyceride (TG), total cholesterol (C), and total phospholipid (TPL) content in the serum low density lipoproteins (LDL) after i.v. injection of praseodymium nitrate. Each value is the mean of 5 independent determinations. Co = untreated animals

Table 1. Alteration of serum-Lp phospholipid content and pattern 4 days after injection of praseodymium. The phospholipid concentration is indicated in rag/i, the phospholipid pattern in ~o of total phospholipids. Each value is the mean -- S.E.M. of 4 determinations obtained from the pooled sera of 5 animals LPC

S

PC

PI

PE

VLDL Co mg/l %

3.5 • 0.2 4.2

4.5 4- 0.9 5.4

67.5 i-_ 1.9 80.3

5.0 +_ 1.4 5.9

2.4 4-_ 0.1 2.9

Pr mg/1

3.9 4- 0,4 1.6

5.4 -- 0.3 3.5

134.5 4- 5.1 88.3

2.9 • 0.2 2.0

5.5 4- 0,3 3.6

Comg/1

3.9 4- 0.3 4.4

13.8 -- 1.0 15.5

2.1

5.1 • 0.5 5.7

3.8 4- 0.2 4.3

Pr mg/1 ~o

9.2 -- 1,3 2.2

36.0 +_ 0.9 8.6

341.5 • 81.5

3.8

ll.3 ,, 0.4 2.7

20.9 ,, 4.6 5.0

Co mg/1

21.2 -- 1.2 4.3 7.3 4- 0.7 1.2

39.4 ,, 0.9 8.0 54.1 ,, 2.6 8.7

387.0 ,, 4.5 78.5 490.3 __ 11.5 79.3

32.5 ,, 1.1 6.6 45.5 _+ 5.3 7.4

15.3 _+ 0.5 3.1 21.1 ,, 2.0 3.4

LDL

HDL

Pr mg/1 %

62.8 -70.5

Abbreviations: LPC = lysophosphatidylchotine; S = sphingomyeIin; PC = phosphatidylcholine; PI = phosphatidylinositol; PE = phosphatidylethanolamine; VLDL = very low density lipoprotein; LDL = low density lipoprotein; H D L = high density lipoprotein

O. Grajewski et al. : Lipoproteins and LCAT during Experimental Liver Disease

69

Table 2. Changes of serum-Lp composition 4 days after i.v. injection of praseodymium. The lipid and protein concentrations are indicated in rag/1. The indicated percentages represent the contribution to the total fraction. The other experimental conditions were the same as described for Table 1 TG VLDLCo

LDL

EC

UC

TPL

Prot

750 • 51 71

45 • 4.2

5

21 • 2.0

2

95 • 13 8.9

153 • 11 14

Pr

480• 58

63*

36• 4.3

4*

40_+ 4.8

4

98 • 10 12

176 • 10 21

Co

91 • 24

9

98 • 26

5

28 • 7

3

89_+ 23

Pr ~

229 • 11" 18

121 • 19 10

186 • 31" 15 22 • 2.4

3

3

79 • 21

2

419 • 69* 34

288 • 23

9*

305 • 10 32.9

322 • 11 34.7

539 • 61" 50

264 • 20 25

HDL2 Co

40 • 4.3

2

238 • 14 25.7

Pr ~/o

37 • 3.4

3

102 • 10" 9.5

HDL3 Co

43 • 4.5

3

188 • 20

8

17 • 1.8

2

231 _+ 9 24

472 • 12 50

Pr

34 • 8

4

44• 12

6*

I8 • 5

3

124 • 15" 33

158 • 42

131 • 13" 12

8*

Abbreviations: TG = triglycerides; EC = ester• cholesterol; UC : unesterified cholesterol; TPL = total phospholipids; Prot = protein; VLDL = very low density lipoprotein, LDL = low density lipoprotein, HDL2/HDL3 = high density lipoprotein, HDL2 = lower density than HDL3 * P<0.001 vs. controlanimals

0,l8

j

HDL

j ?

PROT

. . . , . . . . , - " ' "

i

q6 o,5

"

i

., ,:

TPL C

o~ q3o,2 o,1-

c'o

4

5

~

,i

g

g

§ d

Fig. 5. Time course (days) of the total cholesterol (C), total phospholipid (TPL), and protein (prot) content in the high density lipoproteins (HDL) after i.v. injection of praseodymium nitrate. Each value represents the mean of 5 independent determinations. Co = untreated animals

for some time. If the UC is determined, a reaction similar to that of the TPL or of the PC is found. The highest concentrations of PC and UC in the H D L are attained 4 d after administration of Pr. At this time the UC amounts to 5 0 - 6 0 ~ of the HDL-C, while it is normally only 10 ~. If the H D L are subdivided into HDL2 and HDLa (Table 2), it becomes evident that these processes occur mainly in the HDL2, i.e. in the HDL-fraction that has the lower density of the two. In the HDL3 only a relative increase in UC and TPL can be found, but not an absolute increase. Changes in the LDL- and HDL-lipids which are comparable to the findings observed here, occur with cholestase and also when there is an inborn disturbance of the lipid metabolism, in which case there is a Iack of an enzyme formed by the liver and normally occurring in the serum, lecithin-cholesterin-acyltransferase (Seidel et al., 1969; Glomset et al., 1970; Norum et al., 1971 ; Seidel et al., 1972). This enzyme transfers a fatty acid from position 2 of the PC to the hydroxyl group of the C. For this reason we determined the activity of this enzyme both by the method of Glomset (1969) and by that of Stokke and Norum (1971). The main difference between the two methods is that the first uses homologous cofactor containing substrate, while the second uses autologous substrate, the cofactor content of which may be variable. However,

Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977)

70

g/i o~ VHDL TPL

q2-

o,I l

c'o

4

i

;

;

;

;

d

Fig. 6. Time course (days) of the total phospholipid (TPL) content of the very high density lipoproteins (VHDL) after i.v. injection of praseodymium nitrate. Each value is the mean of 5 independent determinations. Co = untreated animals

there are usually no significant differences between the results obtained with the two methods; in this case the enzyme activity calculated according to Stokke and Norum (1971) was 40.8 + 1.4 gmole/1/h as compared with 3 3 . 9 _ 1.2 i,tmole/1/h obtained by the method of Glomset (1969). Our assays of LCAT-activity according to Stokke and Norum (1971) showed an 80-90~o decrease during the period of 2 - 4 d after Pr. These results are published in an earlier paper (see Lehmann et al., 1977). Using the Glomset method we obtained a similar time course for LCAT-activity, except that the decrease was less pronounced I d and 5 d after the application of Pr. In no assay was substrate availability a limiting factor, as in the serum of Pr-treated animals only about 1 - 2 ~o of the substrate becomes esterified. The product of the LCAT-reaction, lysophosphatidylcholine (LPC), which occurs almost exclusively in the VHDL-fraction where it is bound to albumin and represents about 80 ~o of TPL, shows a corresponding time course (Fig. 1 and 6).

DISCUSSION The changes in the serum-Lp after i.v. administration of Pr resemble in many respects the findings obtained

in human parenchymatous lesions of the liver (Smith et al., 1967; Papadopoulos and Charles, 1970; Barclay, 1972; Seidel et al., 1972; Baumgarten et al., 1974; Hartmann et al., 1974). As was the case after Pr, a decrease in VLDL and H D L as well as an increase in the LDL in the acute phase of the liver damage were reported. The results obtained in animal experiments after carbontetrachloride and galactosamine poisoning point in a similar direction (Lombardi and Ugazio, 1965; Sabesin et al., 1975). Our study has the advantage of showing the complete time course of the serumLp alterations from the start of the hepatic degeneration almost to the end of the regeneration processes (v. Lehmann et al., 1976). It clearly demonstrates that different stages of hepatic failure correspond to different serum-Lp patterns and helps in understanding the controversial results of earlier investigations. Moreover, our results show that in the course of liver disease, investigations of serum lipids without fractionation of serum-Lp supply very limited information, as each Lp-fraction behaves differently. The cause of the changes in the concentrations of VLDL and H D L after administration of Pr is probably a decreased hepatic secretion of these lipoproteins. The assays with Triton WR 1339 clearly showed an inhibition of the TG-release from the liver during the 3 days following the administration of Pr. These results complement those of similar experiments by Lombardi and Recknagel (1962) and by Snyder and Kyker (1964) and explain one cause of the massive accumulation of TG in the liver after Pr. However, a distinct inhibition of secretion must also be supposed for the HDL, as the formation of this lipoprotein fraction also expresses a Special functional performance of the liver (Margolis and Capuzzi, 1972; Stein et al., 1972; Eisenberg and Levy, 1975). Changes that are demonstrable both biochemically and with the electronmicroscope occur at the rough and smooth membranes of the endoplasmic reticulum soon after the administration of the rare earth metal (Magnusson, 1963; v. Lehmann et al., 1975); thus cell structures that are decisive for the synthesis and the intracellular Lp-transport are altered (Hamilton and Kayden, 1974; Eisenberg and Levy, 1975; Glaumann et al., 1975). Analogous pathobiochemical mechanisms are certainly responsible for the decreased activity of the LCAT in the serum, as this enzyme is exclusively formed by the liver and its activity is reduced during liver diseases (Gjone and Norum, 1970; Osuga and Portman, 1971; Simon and Boyer, 1970; Simon and Scheig, 1970; Kattermann and Wolfrum, 1970; Calandra et al., 1971). It seems inconceivable that the decrease of the LCAT-activity can be the result of a primary deficiency of H D L which contain the physio-

O. Grajewski et al. : Lipoproteins and LCAT during Experimental Liver Disease

logical activators of this enzyme (Soutar et al., 1975), as there is a distinct difference in the time courses of LCAT-activity and HDL-concentration. Moreover, the decreased activity can also be observed in the cofactor containing Glomset-assay. Therefore, the diminution of the LCAT-activity is much more likely to be due to the inhibition of hepatic LCAT-secretion. The relatively longlasting fall in LCAT-activity in the serum leads to a change in the composition of the HDL in the late phase of liver damage, i.e. 3 - 5 days after administration of Pr, as this Lp-fraction in particular is the preferred substrate of this enzyme (Marcel and Vezina, 1974). During the first 2 days after Pr, the activities of HDL and LCAT diminish almost equally so that the lack of enzyme activity does not produce clear effect. An increase in TPL and C which is especially caused by PC and UC, is typical in the inborn deficiency in this enzyme (Glomset et al., 1970; Norum et al., 1971), as well as the occurrence of a Lp rich in PC and UC which is associated with the HDL2 (Norum et al., 1971). Similar alterations of the HDL and the LCATactivity were recently reported after galactosamine poisoning of female rats (Sabesin et al., 1975). Among all lipoproteins we examined, the LDL are the fraction showing the most conspicuous changes, the causes of which can, however, only be explained in part. The shifts in the lipid pattern of this fraction, which is mainly characterized by an increase in PC and UC, lead to the conclusion that in the course of Prinduced liver damage, a lipoprotein occurs which is equivalent to the Lp X appearing in man during intraand extrahepatic cholestasis (Seidel et al., 1969). The results presented in this paper show such a clear time relation between the TPL and the C in the LDL and the decrease of the LCAT-activity that a causal relation between these parameters is indicated. Though the contrasting behaviour between the LDL and the LCAT-activity was also evident in several other investigations, apart from the inherited deficiency of this enzyme, no underlying biochemical mechanism was discovered (Glomset and Norum, 1973 ; McIntyre et al., 1974; Ritland and Gjone, 1975). A simple enzyme-substrate relation between LDL and LCAT is improbable for several reasons (Glomset and Norum, 1973), while on the other hand shifts of lipid and protein content as well as of the physical properties of the different Lp can occur as a consequence of the LCAT-catalysed reaction (Norum et al., 1975). It is evident that Lp secreted by the liver are different from those normally found in the plasma and that the secreted forms are rapidly metabolized and transformed in the blood (Marsh, 1974; Noel and Rubinstein, 1974). Possibly the Lp-changes after Pr are the result of a mechanism suggested by Hamilton and

71

Kayden (1974), who proposed an alteration in the transformation of the nascent Lp secreted by the liver to the physiological serum-Lp during liver failure. This is confirmed by the fact that LCAT takes part in this transformation. Apart from the increase of the TPL and C in the LDL fraction, an increase of the TG in the LDL occurs at the time when the decrease of the VLDL-TG becomes conspicuous in the late stage of the liver damage due to Pr. Apparently a disturbance of the VLDL-metabolism occurs when the TG-secretion of the liver returns to normal. This disturbance leads to the occurrence of TG-rich particles of intermediary density which is supposedly caused by a deficiency in hepatic lipoprotein lipase (Mfiller et al., 1974; Bolzano et al., 1975). Preliminary assays in which an oral TG-load was given, showed that the hypertriglyceridemia occurring post-resorptively persists much longer in animals treated with Pr than it does in untreated controls. Our studies on the disturbances of the Lp metabolism in the course of Pr-induced liver damage reveal that several factors are of importance for the development of these changes. These are inhibition of hepatic VLDL, HDL, and LCAT secretion as well as a disturbance of TG-utilisation. However these alterations do not occur simultaneously in the course of the liver damage. The available results do not allow a decision, as to whether it is possible to assess the severity and the stage of a liver damage from a study of these parameters. It seems that at least the HDL are of some value in this respect (Blomhoff et al., 1974; Baumgarten et al., 1974; Skrede et al., 1975; Solberg et al., 1975; Thalassinos et al., 1975; v. Lehmann et al., 1976). The observed changes, however, reveal that the model of Pr-induced liver injury is well suited for studying causes and consequences of the disturbed Lp metabolism during experimental liver damage.

REFERENCES Barclay, M. : Lipoprotein class distribution in normal and diseased states. In: Blood lipids and lipoproteins : Quantitation, composition, and metabolism (G. J. Nelson, ed.), pp. 585-704. New York-Toronto : Wiley Interscience 1972 Barclay, M., Skipski, V. P.: Lipoproteins in relation to cancer. Progr. Biochem. Pharmacol. 10, 76-111 (1975) Baumgarten, M., Wienbeck, M., Engelhardt, A., Martini, G. A.: Serumlipoproteine bei akuter Virushepatitis. Klin. Wschr. 52, 617-623 (1974) Blomhoff, J. P., Skrede, S., Ritland, S.: Lecithin:cholesterol acyltransferase and plasma proteins in liver disease. Clin. Chim. Acta 53, 197--207 (1974) Bolzano, K., Krempler, F., Sandhofer, F. : "Hepatic '~ and "extrahepatic" triglyceride lipase activity in post-heparin plasma of normals and patients with cirrhosis of the liver. Horm. Metab. Res. 7, 238-24I (1975)

72 Bruce, D. W., Hietbrink, B. E., Du Bois, K. P.: The acute mammalian toxicity of rare-earth nitrates and oxides. Toxieok appl. Pharmacol. 5, 750- 759 (1963) Calandra, S., Martin, M. J., McIntyre, N. : Plasma lecithin: cholesterol acyltransferase activity in liver disease. Europ. J. clin. Invest. 1, 352- 360 (1971) Eggstein, M., Kreutz, F. H. : Eine neue Bestimmung der Neutralfette im Blutserum nnd Gewebe. 1. Mitteilung: Prinzip, Durchffihrung und Besprechung der Methode. Klin. Wschr. 44, 262267 (1966) Eisenberg, S., Levy, R. I. : Lipoproteins and lipoprotein metabolism. In: Handbook of experimental pharmacology, New series, Vol. 41 (D. Kritchevsky, ed.), pp. 191-213. Berlin-HeidelbergNew York, Springer 1975 Fischler, F., Roeckl, K. W. : Ober experimentelle Beeinflussung der Leberfunktion und der anatomischen Leberstruktur durch Einwirkung seltener Erden. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 189, 4 - 2 1 (1938) Folch, J., Lees, M., Stanley, G. H. S. : A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509 (1957) Friedman, M., Byers, S. O. : Mechanism underlying hypercholesterinemia induced by Triton WR 1339. Amer. J. Physiol. 190, 439 - 445 (1957) Gjone, E., Norum, K. : Plasma lecithin:cholesterol acyltransferase and erythrocyte lipids in liver disease. Acta Med. Scand. 187, 153 - 161 (1970) Glaumann, H., Bergstrand, A., Ericsson, J. L. E.: Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver. J. Cell Biol. 64, 356-377 (1975) Glomset, J. A.: Plasma cholesterol esterifying enzyme. Meth. Enzymol. 15, 543 - 548 (1969) Glomset, J. A., Norum, K. R. : The metabolic role of lecithin: cholesterol aeyltransferase. Perspectives from pathology. Adv. Lipid Res. 11, 1 - 6 5 (1973) Glomset, J. A., Norum, K. R., King, W.: Plasma lipoproteins in familial lecithin:cholesterol acyltransferase deficiency: Lipid composition and reactivity in vitro. J. clin. Invest. 49, 18271837 (1970) Gross, U., Werneke, M., Beckenbach, H., Oberdisse, E.: Pathomorphologie der pharmakologisch beeinflul3ten Lanthanid-induzierten Leberdystrophie. Verb. dtsch. Ges. Path. 58, 521 (1974) Hamilton, R. L., Kayden, H. J.: The liver and the formation of normal and abnormal plasma lipoproteins. In: The liver, normal and abnormal functions (F. F. Becket, ed.), pp. 531572. New York: Marcel Dekker Inc. 1974 Hartmann, L., Ollier, M. P., Fiessinger, J.N., Nebut, M.: Abnormality of the structure and composition of plasma lipoproreins (HDL) during different types of hepatitis. Biomedicine 21, 480 - 485 (1974) Hatch, F. T., Lees, R. S. : Practical methods for plasma lipoprotein analysis. Adv. Lipid Res. 6, 2 - 6 8 (1968) Havel, J. R , Eder, H. A., Bragdon, J. H.: The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. clin. Invest. 34, 1345-1353 (1955) Jackson, R. L., Morrisett, J. D., Gotto, A. M., Jr.: Lipoprotein structure and metabolism. Physiol. Rev. 55, 250-316 (1975) Kattermann, R., Wolfrum, D. I. : Cholesterinstoffwechsel und Lecithin-Cholesterin-Acyltransferase im Plasma bei experimenteller Hepatitis und Cholestase an der Ratte. Z. klin. Chem. Biochem. 8, 413-419 (1970) v. Lehmann, B., Oberdisse, E., Grajewski, O., Arntz, H.-R. : Subcellular distribution of phospholipids during liver damage induced by rare earths. Arch. Toxicol. 34, 89-101 (1975) v. Lehmann, B., Grajewski, O., Arntz, H.-R., Oberdisse, E. : Correlation between serum high density lipoprotein content and liver

Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977) function during experimental hepatic degeneration and regeneration. Acta Hepato-Gastroenterol. 24, 328-333 (1976) v. Lehmann, B., Grajewski, O,, Arntz, H.-R., Oberdisse, E. : Correlation between serum lecithin-cholesterol-acyltransferase activity and erythrocyte lipid content during experimental liver damage. Naunyn-Schmiedeberg's Arch. Pharmacol. 298, 211216 (1977) Lombardi, B., Recknagel, R. O. : Interference with secretion of triglycerides by the liver as a common factor in toxic liver injury. Amer. J. Path. 40, 571- 586 (1962) Lombardi, B., Ugazio, B. : Serum lipoproteins in rats with carbon tetrachloride-induced fatty liver. J. Lipid Res. 6, 498-505 (1965) Lowry, O. H., Rosebrough, H. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Magnusson, G. : The behaviour of certain lanthanons in rats. Acta Pharmacol. Toxicol, (Kbh) 20, Suppl. 3, 1 - 9 5 (1963) Marcel, Y. L., Vezina, C.: Lecithin:cholesterol acyltransferase of human plasma. Role of chylomicrons, very low, and high density lipoproteins in the reaction. Scan& J. clin. Lab. Invest. 33, Suppl. 137, 4 5 - 4 8 (1974) Margolis, S., Capuzzi, D. : Serum-lipoprotein synthesis and metabolism. In: Blood lipid and lipoproteins: Quantitation, composition, and metabolism (G. J. Nelson, ed.), pp. 825-880. New York-Toronto:Wiley Interscience 1972 Marsh, J. R. : Lipoproteins in a non recirculating perfusate of rat liver. J. Lipid Res. 15, 544-550 (1974) McIntyre, N., Calandra, S., Pearson, A. J. : Lipid and lipoprotein abnormalities in liver disease. The possible role of lecithin:cholesterol acyltransferase deficiency. Scand. J. clin. Lab. Invest. 33, Suppl. 137, 115-120 (1974) Mtiller, P., Fellin, R., Lambrecht, J., Agostini, B., Wieland, H., Rost, W., Seidel, D. : Hypertriglyceridaemia secondary to liver disease. Europ. J. clin. Invest. 4, 419-428 (1974) Noel, S. P., Rubinstein, D. : Secretion of apolipoproteins in very low density and high density lipoproteins by perfnsed rat liver. J. Lipid Res. 15, 301--308 (1974) Norum, K. R., Glomset, J. A., Nichols, A. V., Forte, T.: Plasma lipoproteins in familial lecithin:cholesterol acyltransferase deficiency: Physical and ckemical studies of low and high density lipoproteins. J. clin. Invest. 50, 1131-1140 (1971) Norum, K. R., Glomset, J. A., Nichols, A. V., Forte, T., Albers, J.J., King, W. C., Mitchell, C. D., Applegate, K. R., Gong, E. L., Cabana, V., Gjone, E. : Plasma lipoproteins in familial lecithin:cholesterol acyltransferase deficiency: Effects of incubation with lecithin:cholesterol acyltransferase in vitro. Scan& J. Lab. Invest. 35, Suppl. 142, 31-55 (1975) Oberdisse, E., Winkler, R., Grajewski, O., v. Lehmann, B., Arntz, H.-R. : Pharmakologische Untersuchungen zum Mechanismus der Praseodym-ausgel6sten LeberscMdigung. Verh. dt. Ges. inn. Med. 80, 1556-1558 (1974) Osuga, T., Portman, O. W.: Origin and disappearance of plasma lecithin:cholesterol acyltransferase. Amer. J. Physiol. 220, 735-741 (1971) Otway, S., Robinson, D. S. : The effect of a non-ionic detergent (Triton WR 1339) on the removal of triglyceride fatty acids from the blood of the rat. J. Physiol. 190, 309-319 (1967a) Otway, S., Robinson, D. S.: The use of a non-ionic detergent (Triton WR 1339) to determine rates of triglyceride entry into the circulation of the rat under different physiological conditions. J. Physiol. (Lond.) 190, 321- 332 (1967b) Papadopoulos, N. M., Charles, M. A.: Serum lipoprotein patterns in liver disease. Proc. Soc. exp. biol. Med. 134, 797-799 (1970) Peter, H. W., Wolf, U. : A new method for the in situ determination of phospholipids after thin-layer separation. The phospholipid content of Ca-ATPase and Na, K-ATPase from human erythro-

O. Grajewski et al. : Lipoproteins and LCAT during Experimental Liver Disease cyte in comparison with phospholipid content of human erythrocytes. J. Chromatography 82, 1 5 - 30 (1973) Picard, J., Veissi6re, D., Bertrand, F. : Lipoprot6ines s6riques anormales dans l'h6patite virale cholostatique. Clin. Chim. Acta 51, 5 - 1 2 (1974) Raheja, R. K., Kaur, C., Singh, A., Bhatia, I. S. : New colorimetric method for the quantitative estimation of phospholipids without acid digestion. J. Lipid Res. 14, 695-697 (1973) RecknageI, R. O. : Carbon tetrachloride hepatotoxicity. Pharmacol. Rev. 19, 145-208 (1967) Ritland, S., Gjone, E.: Quantitative studies of lipoprotein-X in familial lecithin:cholesterol acyltransferase deficiency and during cholesterol esterification. Clin. Chim. Acta 59, 109-119 (1975) Sabesin, S. M., Kuiken, L. B., Ragland, J.B.: Lipoprotein and lecithin:cholesterol acyltransferase changes in galactosamineinduced rat liver injury. Science 190, 1302-1304 (1975) Sarkander, H.-I., Brade, W. P. : On the mechanism of lanthanideinduced liver toxicity. Arch. Toxicol. 36, 1 - 1 7 (1976) Seidel, D., Alaupovic, P., Furman, R. H. : A lipoprotein characterizing obstructive jaundice. I. Method for quantitative separation and identification of lipoprotein in jaundiced subjects. J. clin. Invest. 48, 1211-1223 (1969) Seidel, G., Greten, H., Geisen, H. P., Wengeler, H., Wieland, H. : Further aspects on the characterization of high and very low density lipoproteins in patients with liver disease. Europ. J. clin. Invest. 2, 359-364 (1972) Simon, J. B., Boyer, J. C. : Production of lecithin: cholesterol acyltransferase by the isolated perfused rat liver. Biochim. Biophys. Acta (Amst.)218, 549-551 (1970) Simon, J. B., Scheig, R.: Serum cholesterol esterification in liver diseases. Importance of lecithin: cholesterol acyltransferasel New Engl. J. Med. 283, 841-846 (1970)

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Skrede, S., Blomhoff, J. P., Elgj6, K., Gjone, E. : Serum proteins in diseases of the liver. Scand. J. clin. Lab. Invest. 35, 399-406 (1975) Smith, S. C., Scheig, R., Klatskin, G., Levy, R. I. : Lipoprotein abnormalities in liver disease. Clin. Res. 15, 330-336 (1967) Snyder, F., Kyker, G. C. : Triglyceride accumulation and release in the rare-earth fatty liver. Proc. Soc. exp. biol. Med. 116, 890893 (1964) Solberg, H.E., Skrede, S., Blomhoff, J.P.: Diagnosis of liver diseases by laboratory results and discriminant analysis. Identification of best combinations of laboratory tests. Scand. J. clin. Lab. Invest. 35, 713-721 (1975) Soutar, A. K., Garner, G. W., Baker, H. N., Parrow, J. T., Jackson, R. L., Gotto, A. M., Smith, L. C. : Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin : cholesterol acyltransferase. Biochemistry 14, 3057- 3064 (1975) Stein, O., Bar-On, H., Stein, Y.: Lipoproteins and the liver. In: Progr. Liver Disease, Vol. 4 (H. Popper, F. Schaffner, eds.), pp. 45-62. New York-London: Grune and Stratton 1972 Stokke, K. T., Norum, K. R. : Determination of lecithin : cholesterol acyltransferase in human blood plasma. Scand. J. clin. Lab. Invest. 27, 21-27 (1971) Thalassinos, N., Hatzionnou, J., Scliros, Ph., Kanaghinis, T., Anastasiou, C., Crocos, P., Thomopoulos, D., Gardikas, C. : Plasma atpha-lipoprotein pattern in acute viral hepatitis. Amer. J. dig. Dis. 20, 149-155 (1975) Zak, B. : Total and free cholesterol. In: Standard methods of clinical chemistry, Vol. 5 (S. Meites, ed.), pp. 79-89. New YorkLondon: Academic Press 1965

Received March 28/Accepted August 22, 1977