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Physical-chemical changes in serum lipoproteins during incubation of human serum
87
BIOCHIMICA ET BIOPHYSICA ACTA BBA 35263
P H Y S I C A L - C H E M I C A L CHANGES IN SERUM L I P O P R O T E I N S D U R I N G INCUBATION OF HUMAN SERUM
A. V. N I C H O L S , E. L. C O G G I O L A , L. C. J E N S E N AND E. H. Y O K O Y A M A
Donner Laboratory, Lawrence Radiation Laboratory, University of California, Berheley, Calif. 94720
(U.S.A.) (Received M a y ist, 1968)
SUMMARY
Incubation of human serum for 24 h at 37 ° resulted in a marked shift in the ultracentrifugal distribution of the high-density lipoprotein (HDL) fraction. The serum concentrations of the F°1.20 0-3.5 (HDL3) decreased while those of the F°1.20 3.5-9.0 (HDLz) increased. Inhibition of serum fatty acid transferase activity by p-hydroxymercuribenzoate did not inhibit the incubation-induced change in H D L distribution. A significant reduction in the protein content of the HDL, isolated from incubated serum, was detected. These data suggest that the shift in HDL distribution may result from an initial dissociation of protein from H D L a followed by an association of relatively lipid-rich lipoprotein residues to give HDLe-type lipoproteins. Small order increases in S°f rates of the major peak of the S°), 0-20 fraction were also observed, during incubation of serum, in the presence as well as in the absence of transferase activity. The magnitude of the increase in sf5 rate was found to be directly related to the level of S°), 20-400 lipoproteins in the serum and to be greater in the presence of transferase activity.
INTRODUCTION
Incubation of human serum or plasma for 24 h at 37 ° results in changes in both the chemical composition of serum lipids as well as in changes in distribution of serum lipids among the maj or classes of serum lipoproteins. The change in serum lipid composition results from an enzymatic transesterification of unesterified cholesterol by fatty acids from lecithin and leads to a net increase in serum esterified cholesterol and A b b r e v i a t i o n s : H D L , h i g h - d e n s i t y lipoprotein; V L D L , v e r y l o w - d e n s i t y lipoprotein; L D L , l o w - d e n s i t y lipoprotein; V H D L , v e r y h i g h - d e n s i t y lipoprotein. * F l o t a t i o n r a t e s described b y F°m.~0 v a l u e s are r a t e s in S v e d b e r g u n i t s for lipoproteins in a m e d i u m of d 1.2o g / m l (NaC1-NaBr, 26 °, 52 64o rev./min) a n d are corrected for c o n c e n t r a t i o n a n d J o h n s t o n - O g s t o n effects. F l o t a t i o n r a t e i n t e r v a l s d e s i g n a t e d b y F°l.20 o 3.5 a n d F°l.20 3.5-9.o c o r r e s p o n d a p p r o x i m a t e l y to H D L s u b c l a s s e s H D L 3 a n d HDL2, respectively, p r e v i o u s l y described b y DE LALLA AND GOFMAN TM.
Biochim. Biophys. Acta, 168 (1968) 87-94
88
A.V. NICHOLS et al.
lysolecithin 1. The change in lipid distribution among lipoproteins results from a net transfer of some triglycerides from the very low-density lipoproteins (VLDL, d < I.OO6 g/ml) to the low-density lipoproteins (LDL, d I .oo6-1.o63 g/ml) and high-density lipoproteins (HDL, d 1.o63-1.2o g/ml) coupled with a reciprocal transfer of some cholesterol esters from the LDL and H D L to the VLDL during incubation 2. The lysolecithin resulting from the transesterification reaction has also been observed to accumulate in the serum protein fraction (d > 1.21 g/ml) 3. Although detailed studies have been performed on various aspects of the chemical changes in serum lipoproteins during incubation of serum, little information is available on the effects of incubation on the ultracentrifugal properties of the major classes of serum lipoproteins. The present study was undertaken to describe the changes in ultracentrifugal patterns following incubation and to relate them to available chemical information.
METHODS AND MATERIALS
Sampling Blood was drawn into sterile glass containers from non-fasting healthy males, ages 26-47. The serum was removed and after addition of penicillin and streptomycin (o.12 and 0.38 mg/ml, respectively) was stored under N 2 at 4 ° prior to use.
Incubation Aliquots of serum were incubated in sealed 6-ml cellulose nitrate tubes ordinarily used for preparative ultracentrifugation. Prior to use tubes were exposed to ultraviolet light and the assembled stainless steel caps were autoclaved. The incubations were performed in a constant-temperature water bath for 24 h at 37 °. Control samples were kept in identical containers at 4 ° for 24 h. In some incubation experiments, the activity of serum fatty acid transferase was inhibited b y the addition (0.338 mg/ml) of p-hydroxymercuribenzoatO (Sigma Chemical Co., St. Louis, Mo.).
Ultracentrifugal analyses Immediately following the incubation period, aliquots of incubated and nonincubated serum were subjected to preparative and analytic ultracentrifugation procedures for determination of serum concentrations of low- and high-density lipoproteins 4. In some instances, specific lipoprotein fractions were isolated by standard preparative ultracentrifugation techniques 5. All salt solutions used in the ultracentrifugal procedures were prepared with double-distilled water and contained o.Io g/1 of EDTA. Computer-derived profiles of lipoprotein distributions were obtained according to programs and procedures developed by JENSEN, RICH AND LINDGREN6.
Lipid and protein analyses Serum and lipoprotein lipids were extracted, chromatographed on silicic acid, and quantified b y infrared spectroscopy according to methods described earlier ~. The weight percentage of protein of H D L was calculated from N, C and H values determined by elemental NCH analysis 8. The contribution of phospholipid N to the above values was estimated from phospholipid phosphorus values determined by the method of BARTLETT 9.
Biochim. Biophys. Acta, 168 (1968) 87-94
89
CHANGES IN SERUM LIPOPROTEINS TABLE I EFFECT OF 2 4 h INCUBATION OF SERUM ON CONCENTRATIONSOF FRACTIONS F°l.g0 o-3. 5 AND F°l.*0 3.5-9.0 ( m g / i o o ml).
Subject No. I 2 3 4 5 6
F°1.2o0-3.5
HIGH-DENSITYLIPOPROTEIN
F°t.2o 3.5-9 .0
Non-incubated
Incubated
Non-incubated
Incubated
243 4205 -43064225 ± 226 238
159 4115 419°± 145 4144 156
lO2 21 81 42 86 31
192 98 181 94
21" 26 12 4
8 4 7 8
4444-
4 7 2 2
444±
4 7 3 4
177 132
" W h e r e analyses were performed in duplicate, s t a n d a r d deviations are given.
RESULTS AND DISCUSSION
The effect of incubation of serum on the concentrations of H D L lipoproteins is shown in Table I. The concentrations of the F°t.20 o-3.5 decreased markedly while those of the F°1.20 3.5-9.o increased. However, there was no significant change in the total H D L concentration following incubation of serum.
20
F1,20 ° rate (Svedberg units) 9
FI.~2orate (Svedberg units) 0
9
20
I
0 ...... t-.J
]
I
L
I
i
:
i ~'"r'-~j...I li l I L
control\ /
,Lilt ii'i'/1, I l l l l l l l l
50mg]lO0
:
cantrol~'.,./ I I I I L
"/ I
I
I
J
[
I
L
i I
I
t
I
I
L I
lOOmg/lOOImll I
I
[ I
]
5 0 m g ] l O 0 rn
I l l [ i ] ] l l
I
Fig. I. C o m p a r i s o n of the average ultracentrifugal profiles of H D L f r o m n o n - i n c u b a t e d and inc u b a t e d sera (upper). E a c h of the lipoprotein profiles s h o w n is an average profile d r a w n by comp u t e r f r o m the individual profiles of 6 h u m a n subjects. D i a g r a m below is a c o m p u t e r - d e r i v e d differential plot obtained b y s u b t r a c t i n g the average lipoprotein profile of H D L , f r o m non-inc u b a t e d sera, f r o m the average lipoprotein profile of H D L , f r o m incubated sera. Fig. 2. Comparison of the average ultracentrifugM profiles of H D L f r o m n o n - i n c u b a t e d and inc u b a t e d sera, containing p - h y d r o x y m e r e u r i b e n z o a t e (upper). Sera were f r o m the same 6 subjects studied in Fig. I. D i a g r a m below is a c o m p u t e r - d e r i v e d differential plot obtained b y s u b t r a c t i n g the average lipoprotein profile of H D L , f r o m n o n - i n c u b a t e d sera, f r o m the average lipoprotein profile of H D L , f r o m incubated sera.
Biochim. Biophys. $cta, 168 (I968) 87~) 4
90
A . V . NICHOLS
et al.
The effect of incubation on the average ultracentrifugal lipoprotein profile of the H D L of 6 subjects is shown in Fig. I. The ultracentrifugal profile of HDL from incubated serum has a faster flotation rate for the major peak than the profile of HDL from non-incubated serum. To check if the HDL shift might possibly be due to an increase in the density of serum, occurring during incubation, we measured the background salt concentrations following preparative ultracentrifugation of the serum by refractometry. No increase in background salt concentrations was observed. Fig. I also shows a computer-derived plot of the difference between the average HDL profiles for the 6 sera before and after incubation. Decreases in the average HDL profile occur in the range of F°a.~0 o-3.o and the increases occur in the F°1.20 3.0-9.0. Since the flotation ranges _F°1.200-3. 5 and F°1.20 3.5 9 .0 have been used previously to designate the established subclasses HDL 3 and HDL2, respectively, we have used these ranges to report the concentration changes in HDL rather than the F°1.20 0-3.0 and F°1.20 3.0-9.0 ranges. Following incubation, all sera showed a substantial transesterification of unesterified cholesterol. The average increase in serum cholesterol esters was 4 6 4- 12 nag per IOO ml. GLOMSET$ has suggested that a major source of the fatty acid for the transesterification is the lecithin of the HDL. Statistical evaluation of our data for the 6 subjects also suggests a correlation (r = 0. 9 at a significance level of 0.05 )between the increase in cholesterol ester concentration in incubated serum and the concentration of the F°1.2o 0-3.5 (HDL3) in non-incubated serum. Our statistical correlation is based on a very small sample and certainly the series should be expanded for a definitive determination of correlation. s T rote (Svedberg units)
400
100
20
0
2.0, Without p ~ h y d r o x y m e r c u r i b e n z o o te
1.6 /
No p-hydroxymercuribenzoote
/"
1.2
I
i
/
i
sI
....... Control --Incuboted
'
f
/
J
•
X'~ ~ ~
>~
0.4
c
o~ o-0.4
o-Hydroxymer
i~i~!!!I~(ii~!i!il!i~i!i!i!i 100 ~g / 100 ml
o f /
.;c o =~o~, -o.e 7
-
i
Withp-hydroxymercuribenzoate
5()
100
150
200
250
2oncn. of s~ 20-400 class (rng 100 rnl)
Fig. 3. C o m p a r i s o n of t h e a v e r a g e u l t r a c e n t r i f u g M profiles of S°f o 400 l i p o p r o t e i n s f r o m noni n c u b a t e d a n d i n c u b a t e d sera, w i t h o u t (upper) a n d w i t h (lower) p - h y d r o x y m e r c u r i b e n z o a t e . Sera were f r o m t h e s a m e 6 s u b j e c t s in Fig. i. FFig. 4- R e g r e s s i o n lines s h o w i n g t h e regression of the c h a n g e in S°f r a t e of t h e m a j o r p e a k of t h e S°)* 0 - 2 0 class (following i n c u b a t i o n of serum, w i t h a n d w i t h o u t p - h y d r o x y m e r c u r i b e n z o a t e ) on t h e c o n c e n t r a t i o n of t h e S°y 20 4o0 class in t h e n o n - i n c u b a t e d serum. W h e r e d u p l i c a t e m e a s u r e m e n t s were p e r f o r m e d t h e m e a n v a l u e s are p l o t t e d w i t h lines s h o w i n g i one s t a n d a r d d e v i a t i o n .
Biochim. Biophys. Acta, 168 (1968) 87-94
9I
CHANGES IN S E R U M L I P O P R O T E I N S
To evaluate the possible influence of transferase activity on the observed shift in H D L ultracentfifugal profiles, sera were incubated wherein p-hydroxymercuribenzoate was added to inhibit transferase activity. Fig. 2 shows the average ultracentrifugal profiles and the differential plot for the H D L distributions from the 6 incubated sera now containing p-hydroxymercuribenzoate. A comparable shift in H D L distribution was observed for the inhibited sera as for the inhibitor-free sera. With p-hydroxymercuribenzoate present, a net decrease in total H D L concentration following incubation was detected. No significant esterification of cholesterol was found in these sera during incubation (average increase in cholesterol esters was 3 /c 6 mg per IOO ml). The effect of incubation of serum on the ultracentrifugal profiles of the S°I o- 400 lipoproteins was also investigated. Fig. 3 shows the average computer-derived plots for the S°), 0-400* lipoproteins of the six subjects following incubation of their sera with and without p-hydroxymercuribenzoate. The increases in mean concentration of the S°), o-2o lipoproteins following incubation of both series could not be proved TABLE
II
EFFECT OF 24 h INCUBATION ON SERU~ CONCENTRATIONS AND COMPOSITION OF LIPIDS OF O--20 LIPOPROTEINS
THE
Sj
6 - m l a l i q u o t s o f s e r u m f r o m a n o n - f a s t i n g m a l e s u b j e c t w e r e i n c u b a t e d for 24 h. C o n t r o l s w e r e k e p t a t 4 ° for s a m e p e r i o d . S f 0 - 2 0 l i p o p r o t e i n s w e r e i s o l a t e d b y p r e p a r a t i v e u l t r a c e n t r i f u g a t i o n . A n a l y s e s w e r e p e r f o r m e d i n d u p l i c a t e a n d s t a n d a r d d e v i a t i o n s a r e r e p o r t e d . C o n c e n t r a t i o n of p - h y d r o x y m e r c u r i b e n z o a t e w a s 0.338 m g / m l . L i p i d c o n c e n t r a t i o n s a r e r e p o r t e d as m g p e r i o o m l ill s e r u m a n d v a l u e s i n p a r e n t h e s e s a r e p e r c e n t a g e s of t o t a l l i p i d .
Sample conditions
Cholesterol esters
Phospho- Triglylipicls cerides
Unester- Unesterified ified cholesfatty terol acids
Non-incubated
I17 i 2 (44) 136 ± o (45)
80 ~- 2 (3 ° ) 79 ± i (26)
4° ~ i (15) 68 Jc o (22)
30 3_ I (II) 2o J- i (7)
0. 4 ~ o . o l
267 :~ 2
o.6 ± o.o2
3o4 zE i
4o~ o (I5) 45 ~z i (17)
33 ± i (I2) 29 -~ i (II)
0. 4=~ o
265i
0.5 i
271 ~- 6
incubated
Total lipid
p- Hydroxymercuribenzoate added Non-incubated Incubated
112 ~ 7 (4 2 ) 116 i i (43)
8o± 2 (3 o ) 80 ± 3 (29)
0.02
9
significant from the available data. The apparent increase in mean flotation rate of the major peak in the S°), o-2o range, after incubation of inhibitor-fr ee serum, also was not statistically significant. Although the changes in flotation rates were not significant, a significant relationship was observed between the changes in flotation rate and serum concentrations of S°f 2o-4oo lipoproteins. Fig. 4 shows the regression lines for the regression of the incubation-induced change in S°y rate of the S°y o-2o lipoproteins, from inhibited and non-inhibited sera, on the concentration of S°y 2o-4oo in the sera * F l o t a t i o n r a t e s d e s c r i b e d b y S°f v a l u e s a r e r a t e s i n S v e d b e r g u n i t s for l i p o p r o t e i n s i n a m e d i u m o f d I.O63 g / m l (NaC1, 26 °, 52 64o r e v . / m i n ) a n d a r e r a t e s c o r r e c t e d for c o n c e n t r a t i o n a n d J o h n s t o n O g s t o n effects.
Biochim. Biophys. Acta, 168 (1968) 8 7 - 9 4
92
A.v. NICHOLS et al.
determined prior to incubation. For sera containing elevated concentrations of S°j 20-400 lipoproteins, the shift in S°/rate of the S°~0-20 major peak is greater in noninhibited than in inhibited sera. From the above data, the changes in S°/0-20 flotation rates appear to have some relationship to the presence of transferase activity whereas the marked shift in H D L distribution does not. Table I I shows the changes in lipid concentration and composition of S¢ 0-20 lipoproteins isolated from a VLDL-rich serum which was incubated with and without p-hydroxymercuribenzoate. The triglyceride concentration in this serum was 214 mg/Ioo ml. The very large increase in triglyceride content of the S 1 0-20 in the presence of transferase activity suggests that transesterification is an important factor in the transfer of VLDL-triglyceride in vitro. In the absence of transferase activity, triglyceride transfer is also observed but to a much lower extent than in the non-inhibited serum. The apparent lack of change in the phospholipid content of the S/0-20 following incubation of sera containing elevated concentrations of triglyceride has been frequently noted and will be described in a subsequent report. No consistent changes were observed in the S°l 20-40o distributions of the sera studied in this investigation. Since the serum S°: 20-400 concentrations of several of our subjects were relatively low, further studies are needed to evaluate the effect of serum incubation on S°/20-400 lipoprotein profiles. The apparent shift of the H D L profile to higher flotation rates is of interest in light of reports of changes in ultracentrifugal recovery and immunochemical properties of H D L following exposure of H D L or serum to various physical and chemical conditions. SCANU AND GRANDA11 subjected H D L 2 and H D L a to successive preparative recentrifugations and observed significant reductions in protein recovery for these fractions. They ascribed the losses to a degradation of the H D L 2 and H D L a with the formation of more dense lipoprotein species which appeared either in the H D L 3 or the d > 1.20 g/ml ultracentrifugal fractions. The species appearing in the d > 1.20 g/ml fraction were very low in lipid content and were designated by them as very highdensity lipoproteins (VHDL). Immunochemical studies by SCAI~U AND GRANDA showed that the VHDL, derived from H D L 2 or HDLa, and the protein moiety obtained from delipidation of HDL2 or H D L a had identical immunochemical properties. They proposed the hypothesis that native H D L consists of a relatively lipid-rich lipoprotein moiety in labile association with a lipid-poor V H D L moiety. I-IAYASHI, LINDGREN AND NICHOLSTM were able to dissociate a lipid-poor protein from H D L b y mild ether treatment; and LEVY AND FREDRICKSONla obtained immunochemical evidence for the release of a lipid-poor protein moiety from H D L in serum by freeze-thawing, storage or addition of urea. Ether treatment, in addition to producing a lipid-poor protein also shifts the distribution of the H D L in the direction of higher flotation rates. Fig. 5 shows this effect of ether on the H D L distribution. This shift is highly similar to that observed following incubation of serum, and the mechanisms responsible m a y be comparable. Incubation of serum thus m a y lead to the dissociation of H D L into V H D L and relatively lipid-rich lipoprotein moieties. The latter moieties m a y subsequently associate to yield species with faster flotation rates. Our data would suggest that during incubation the dissociating species are primarily H D L a. HAYASHI, LINDGREN AND NICHOLS also showed the formation of lipoprotein species with flotation rates in the S°: 0-20 range after ether treatment of HDL. This observation suggests even further dissociation of protein from the H D L and subsequent association of the more lipid-rich lipoprotein residues. In our own incubation studies an increase in the Biochim. Biophys. Acta, 168 (1968) 87-94
93
CHANGES IN SERUM L I P O P R O T E I N S
concentration of the S°S o-2o was indicated but was not found significant. This trend towards increased values is suggestive and m a y indicate the presence of all of the possible processes of dissociation and association observed during exposure of H D L to ether. To test whether protein dissociation from H D L during incubation of serum can be detected, protein analyses were performed on H D L ultracentrifugally isolated from an incubated serum sample and its control. Prior to protein analysis, the H D L fractions were subjected to dialysis against 0.202 M NaC1 to remove any low molecular weight nitrogenous contaminants. Triplicate analyses showed a mean percentage protein content of 52.1% (based on individual values of 52.0, 52.2 and 52.0%) and
F~,20 rote(Svedberg units)
20
--~jl'i-,.,..2• 10 " I I\ I 1 1 I !:' Ofl
Ether-treated NDL
-
/
Control F4DLX\ ./ I k I L I L I L I I Fig. 5. L i p o p r o t e i n p r o f i l e s o f a t o t a l I t D L f r a c t i o n b e f o r e a n d a f t e r e t h e r t r e a t m e n t 1=. T h e s e profiles do not represent the actual concentrations of HDL encountered in this experiment but are presented to show the characteristics of the change in HDL distribution following ether exposure.
49.1% (based on individual values of 49.2, 48.8 and 49.3%) for the H D L fractions separated from control and incubated serum aliquots, respectively. This significant reduction in protein content is in reasonable agreement with estimated reductions in protein content calculated from our ultracentrifugal analyses of the shift in H D L distribution and available information on the chemical compositions of HDL~ and HDL~. These data support the hypothesis that the shift in H D L distribution during incubation of serum results from an initial dissociation of lipid-poor protein from the H D L followed b y an association of the relatively lipid-rich lipoprotein residues. The latter products comprise the faster floating species in the analytic ultracentrifuge. Factors influencing the above dissociations and associations during incubation of serum are under investigation. Furthermore, although comparable protein dissociation would appear to occur from the H D L in sera with and without transferase inhibitor, this does not rule out some possible relationship between such H D L protein and transferase activity in serum. In light of earlier reports~, 14 suggesting the detection of transferase activity in ultracentrifugally isolated HDL, we are currently investigating the possible relationship oftransferase activity to proteins which are in labile association with HDL. Biochirn. Biophys. Acta, i 6 8 (1968) 8 7 - 9 4
94
A . V . NICHOLS et al,
ACKNOWLEDGEMENTS T h e a u t h o r s w i s h t o t h a n k Dr. F. T. LINDGREN for p r o v i d i n g t h e p r o t e i n a n a l y s e s a n d t o a c k n o w l e d g e t h e v a l u a b l e t e c h n i c a l a s s i s t a n c e o f P. SHAFER AND W . H o . T h i s w o r k w a s s u p p o r t e d in p a r t b y P u b l i c H e a l t h S e r v i c e R e s e a r c h G r a n t H E 1 0 8 7 8 - 0 2 f r o m t h e N a t i o n a l H e a r t I n s t i t u t e , U.S. P u b l i c H e a l t h S e r v i c e , a n d b y t h e A t o m i c Energy Commission, REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14
J. A. C*LOMSET,Biochim. Biophys. Acta, 65 (1962) 128. A. V. NICHOLS AND L. SMITH, J. Lipid Res., 6 (1965) 206. J- A. GLOMSET, Biochim. Biophys. Aeta, 7° (1963) 389. A. M. EWlN~, N. K. FREEMAN AND F. T. LINDGREN, Advan. LipidRes., 3 (1965) 25. F. T. LINDGREN, A. V. NICHOLS AND R. D. WILLS, Am. J. Clin. Nutr., 9 (1961) 13. L. C. JENSEN, T. H. RICH AND F. T. LINDGREN, Donner Laboratory Semiannual R e p o r t Biology and Medicine, Lawrence Radiation Laboratory, UCRL 18066, Fall, 1967. N. K. FREEMAN, F. T. LINDGREN AND A. V. NICHOLS, in R. T. HOLMAN, W. O. LUNDBERG and T. WALKIN, Progress in the Chemistry of Fats and other Lipids, Vol. 6, MacMillan (Pergamon), New York, 1963, p. 215. F. T. HATCH, N. K. FREEMAN, L. C. JENSEN, G. R. STEVENS AND F. T. LINDGREN, Lipids, 2 (1967) 183. G. R. BARTLETT, J. Biol. Chem., 234 (1959) 466. O. F. DE LALLA AND J. W. GOFMAN, in D. GLICK, Methods in Biochemical Analysis, ¥ol. I Wiley (Interscience), New York, 1954, p. 459. A. SCANU AND J. L. GRANDA, Biochemistry, 5 (1966) 446. S. HAYASHI, F. LINDGREN AND A. NICHOLS, J, Am. Chem. Soc., 81 (1959) 3793. R. I. LEvy AND D. S. FREDRICKSON, J. Clin. Invest., 44 (1965) 426. ~¥. J. Lossow, S. N. SHAH AND I, L. CHAIKOFF, Biochim. Biophys. Acta, 116 (1966) 172.
Biochim. Biophys..4cta, 168 (1968) 87-94
BIOCHIMICA ET BIOPHYSICA ACTA BBA 35263
P H Y S I C A L - C H E M I C A L CHANGES IN SERUM L I P O P R O T E I N S D U R I N G INCUBATION OF HUMAN SERUM
A. V. N I C H O L S , E. L. C O G G I O L A , L. C. J E N S E N AND E. H. Y O K O Y A M A
Donner Laboratory, Lawrence Radiation Laboratory, University of California, Berheley, Calif. 94720
(U.S.A.) (Received M a y ist, 1968)
SUMMARY
Incubation of human serum for 24 h at 37 ° resulted in a marked shift in the ultracentrifugal distribution of the high-density lipoprotein (HDL) fraction. The serum concentrations of the F°1.20 0-3.5 (HDL3) decreased while those of the F°1.20 3.5-9.0 (HDLz) increased. Inhibition of serum fatty acid transferase activity by p-hydroxymercuribenzoate did not inhibit the incubation-induced change in H D L distribution. A significant reduction in the protein content of the HDL, isolated from incubated serum, was detected. These data suggest that the shift in HDL distribution may result from an initial dissociation of protein from H D L a followed by an association of relatively lipid-rich lipoprotein residues to give HDLe-type lipoproteins. Small order increases in S°f rates of the major peak of the S°), 0-20 fraction were also observed, during incubation of serum, in the presence as well as in the absence of transferase activity. The magnitude of the increase in sf5 rate was found to be directly related to the level of S°), 20-400 lipoproteins in the serum and to be greater in the presence of transferase activity.
INTRODUCTION
Incubation of human serum or plasma for 24 h at 37 ° results in changes in both the chemical composition of serum lipids as well as in changes in distribution of serum lipids among the maj or classes of serum lipoproteins. The change in serum lipid composition results from an enzymatic transesterification of unesterified cholesterol by fatty acids from lecithin and leads to a net increase in serum esterified cholesterol and A b b r e v i a t i o n s : H D L , h i g h - d e n s i t y lipoprotein; V L D L , v e r y l o w - d e n s i t y lipoprotein; L D L , l o w - d e n s i t y lipoprotein; V H D L , v e r y h i g h - d e n s i t y lipoprotein. * F l o t a t i o n r a t e s described b y F°m.~0 v a l u e s are r a t e s in S v e d b e r g u n i t s for lipoproteins in a m e d i u m of d 1.2o g / m l (NaC1-NaBr, 26 °, 52 64o rev./min) a n d are corrected for c o n c e n t r a t i o n a n d J o h n s t o n - O g s t o n effects. F l o t a t i o n r a t e i n t e r v a l s d e s i g n a t e d b y F°l.20 o 3.5 a n d F°l.20 3.5-9.o c o r r e s p o n d a p p r o x i m a t e l y to H D L s u b c l a s s e s H D L 3 a n d HDL2, respectively, p r e v i o u s l y described b y DE LALLA AND GOFMAN TM.
Biochim. Biophys. Acta, 168 (1968) 87-94
88
A.V. NICHOLS et al.
lysolecithin 1. The change in lipid distribution among lipoproteins results from a net transfer of some triglycerides from the very low-density lipoproteins (VLDL, d < I.OO6 g/ml) to the low-density lipoproteins (LDL, d I .oo6-1.o63 g/ml) and high-density lipoproteins (HDL, d 1.o63-1.2o g/ml) coupled with a reciprocal transfer of some cholesterol esters from the LDL and H D L to the VLDL during incubation 2. The lysolecithin resulting from the transesterification reaction has also been observed to accumulate in the serum protein fraction (d > 1.21 g/ml) 3. Although detailed studies have been performed on various aspects of the chemical changes in serum lipoproteins during incubation of serum, little information is available on the effects of incubation on the ultracentrifugal properties of the major classes of serum lipoproteins. The present study was undertaken to describe the changes in ultracentrifugal patterns following incubation and to relate them to available chemical information.
METHODS AND MATERIALS
Sampling Blood was drawn into sterile glass containers from non-fasting healthy males, ages 26-47. The serum was removed and after addition of penicillin and streptomycin (o.12 and 0.38 mg/ml, respectively) was stored under N 2 at 4 ° prior to use.
Incubation Aliquots of serum were incubated in sealed 6-ml cellulose nitrate tubes ordinarily used for preparative ultracentrifugation. Prior to use tubes were exposed to ultraviolet light and the assembled stainless steel caps were autoclaved. The incubations were performed in a constant-temperature water bath for 24 h at 37 °. Control samples were kept in identical containers at 4 ° for 24 h. In some incubation experiments, the activity of serum fatty acid transferase was inhibited b y the addition (0.338 mg/ml) of p-hydroxymercuribenzoatO (Sigma Chemical Co., St. Louis, Mo.).
Ultracentrifugal analyses Immediately following the incubation period, aliquots of incubated and nonincubated serum were subjected to preparative and analytic ultracentrifugation procedures for determination of serum concentrations of low- and high-density lipoproteins 4. In some instances, specific lipoprotein fractions were isolated by standard preparative ultracentrifugation techniques 5. All salt solutions used in the ultracentrifugal procedures were prepared with double-distilled water and contained o.Io g/1 of EDTA. Computer-derived profiles of lipoprotein distributions were obtained according to programs and procedures developed by JENSEN, RICH AND LINDGREN6.
Lipid and protein analyses Serum and lipoprotein lipids were extracted, chromatographed on silicic acid, and quantified b y infrared spectroscopy according to methods described earlier ~. The weight percentage of protein of H D L was calculated from N, C and H values determined by elemental NCH analysis 8. The contribution of phospholipid N to the above values was estimated from phospholipid phosphorus values determined by the method of BARTLETT 9.
Biochim. Biophys. Acta, 168 (1968) 87-94
89
CHANGES IN SERUM LIPOPROTEINS TABLE I EFFECT OF 2 4 h INCUBATION OF SERUM ON CONCENTRATIONSOF FRACTIONS F°l.g0 o-3. 5 AND F°l.*0 3.5-9.0 ( m g / i o o ml).
Subject No. I 2 3 4 5 6
F°1.2o0-3.5
HIGH-DENSITYLIPOPROTEIN
F°t.2o 3.5-9 .0
Non-incubated
Incubated
Non-incubated
Incubated
243 4205 -43064225 ± 226 238
159 4115 419°± 145 4144 156
lO2 21 81 42 86 31
192 98 181 94
21" 26 12 4
8 4 7 8
4444-
4 7 2 2
444±
4 7 3 4
177 132
" W h e r e analyses were performed in duplicate, s t a n d a r d deviations are given.
RESULTS AND DISCUSSION
The effect of incubation of serum on the concentrations of H D L lipoproteins is shown in Table I. The concentrations of the F°t.20 o-3.5 decreased markedly while those of the F°1.20 3.5-9.o increased. However, there was no significant change in the total H D L concentration following incubation of serum.
20
F1,20 ° rate (Svedberg units) 9
FI.~2orate (Svedberg units) 0
9
20
I
0 ...... t-.J
]
I
L
I
i
:
i ~'"r'-~j...I li l I L
control\ /
,Lilt ii'i'/1, I l l l l l l l l
50mg]lO0
:
cantrol~'.,./ I I I I L
"/ I
I
I
J
[
I
L
i I
I
t
I
I
L I
lOOmg/lOOImll I
I
[ I
]
5 0 m g ] l O 0 rn
I l l [ i ] ] l l
I
Fig. I. C o m p a r i s o n of the average ultracentrifugal profiles of H D L f r o m n o n - i n c u b a t e d and inc u b a t e d sera (upper). E a c h of the lipoprotein profiles s h o w n is an average profile d r a w n by comp u t e r f r o m the individual profiles of 6 h u m a n subjects. D i a g r a m below is a c o m p u t e r - d e r i v e d differential plot obtained b y s u b t r a c t i n g the average lipoprotein profile of H D L , f r o m non-inc u b a t e d sera, f r o m the average lipoprotein profile of H D L , f r o m incubated sera. Fig. 2. Comparison of the average ultracentrifugM profiles of H D L f r o m n o n - i n c u b a t e d and inc u b a t e d sera, containing p - h y d r o x y m e r e u r i b e n z o a t e (upper). Sera were f r o m the same 6 subjects studied in Fig. I. D i a g r a m below is a c o m p u t e r - d e r i v e d differential plot obtained b y s u b t r a c t i n g the average lipoprotein profile of H D L , f r o m n o n - i n c u b a t e d sera, f r o m the average lipoprotein profile of H D L , f r o m incubated sera.
Biochim. Biophys. $cta, 168 (I968) 87~) 4
90
A . V . NICHOLS
et al.
The effect of incubation on the average ultracentrifugal lipoprotein profile of the H D L of 6 subjects is shown in Fig. I. The ultracentrifugal profile of HDL from incubated serum has a faster flotation rate for the major peak than the profile of HDL from non-incubated serum. To check if the HDL shift might possibly be due to an increase in the density of serum, occurring during incubation, we measured the background salt concentrations following preparative ultracentrifugation of the serum by refractometry. No increase in background salt concentrations was observed. Fig. I also shows a computer-derived plot of the difference between the average HDL profiles for the 6 sera before and after incubation. Decreases in the average HDL profile occur in the range of F°a.~0 o-3.o and the increases occur in the F°1.20 3.0-9.0. Since the flotation ranges _F°1.200-3. 5 and F°1.20 3.5 9 .0 have been used previously to designate the established subclasses HDL 3 and HDL2, respectively, we have used these ranges to report the concentration changes in HDL rather than the F°1.20 0-3.0 and F°1.20 3.0-9.0 ranges. Following incubation, all sera showed a substantial transesterification of unesterified cholesterol. The average increase in serum cholesterol esters was 4 6 4- 12 nag per IOO ml. GLOMSET$ has suggested that a major source of the fatty acid for the transesterification is the lecithin of the HDL. Statistical evaluation of our data for the 6 subjects also suggests a correlation (r = 0. 9 at a significance level of 0.05 )between the increase in cholesterol ester concentration in incubated serum and the concentration of the F°1.2o 0-3.5 (HDL3) in non-incubated serum. Our statistical correlation is based on a very small sample and certainly the series should be expanded for a definitive determination of correlation. s T rote (Svedberg units)
400
100
20
0
2.0, Without p ~ h y d r o x y m e r c u r i b e n z o o te
1.6 /
No p-hydroxymercuribenzoote
/"
1.2
I
i
/
i
sI
....... Control --Incuboted
'
f
/
J
•
X'~ ~ ~
>~
0.4
c
o~ o-0.4
o-Hydroxymer
i~i~!!!I~(ii~!i!il!i~i!i!i!i 100 ~g / 100 ml
o f /
.;c o =~o~, -o.e 7
-
i
Withp-hydroxymercuribenzoate
5()
100
150
200
250
2oncn. of s~ 20-400 class (rng 100 rnl)
Fig. 3. C o m p a r i s o n of t h e a v e r a g e u l t r a c e n t r i f u g M profiles of S°f o 400 l i p o p r o t e i n s f r o m noni n c u b a t e d a n d i n c u b a t e d sera, w i t h o u t (upper) a n d w i t h (lower) p - h y d r o x y m e r c u r i b e n z o a t e . Sera were f r o m t h e s a m e 6 s u b j e c t s in Fig. i. FFig. 4- R e g r e s s i o n lines s h o w i n g t h e regression of the c h a n g e in S°f r a t e of t h e m a j o r p e a k of t h e S°)* 0 - 2 0 class (following i n c u b a t i o n of serum, w i t h a n d w i t h o u t p - h y d r o x y m e r c u r i b e n z o a t e ) on t h e c o n c e n t r a t i o n of t h e S°y 20 4o0 class in t h e n o n - i n c u b a t e d serum. W h e r e d u p l i c a t e m e a s u r e m e n t s were p e r f o r m e d t h e m e a n v a l u e s are p l o t t e d w i t h lines s h o w i n g i one s t a n d a r d d e v i a t i o n .
Biochim. Biophys. Acta, 168 (1968) 87-94
9I
CHANGES IN S E R U M L I P O P R O T E I N S
To evaluate the possible influence of transferase activity on the observed shift in H D L ultracentfifugal profiles, sera were incubated wherein p-hydroxymercuribenzoate was added to inhibit transferase activity. Fig. 2 shows the average ultracentrifugal profiles and the differential plot for the H D L distributions from the 6 incubated sera now containing p-hydroxymercuribenzoate. A comparable shift in H D L distribution was observed for the inhibited sera as for the inhibitor-free sera. With p-hydroxymercuribenzoate present, a net decrease in total H D L concentration following incubation was detected. No significant esterification of cholesterol was found in these sera during incubation (average increase in cholesterol esters was 3 /c 6 mg per IOO ml). The effect of incubation of serum on the ultracentrifugal profiles of the S°I o- 400 lipoproteins was also investigated. Fig. 3 shows the average computer-derived plots for the S°), 0-400* lipoproteins of the six subjects following incubation of their sera with and without p-hydroxymercuribenzoate. The increases in mean concentration of the S°), o-2o lipoproteins following incubation of both series could not be proved TABLE
II
EFFECT OF 24 h INCUBATION ON SERU~ CONCENTRATIONS AND COMPOSITION OF LIPIDS OF O--20 LIPOPROTEINS
THE
Sj
6 - m l a l i q u o t s o f s e r u m f r o m a n o n - f a s t i n g m a l e s u b j e c t w e r e i n c u b a t e d for 24 h. C o n t r o l s w e r e k e p t a t 4 ° for s a m e p e r i o d . S f 0 - 2 0 l i p o p r o t e i n s w e r e i s o l a t e d b y p r e p a r a t i v e u l t r a c e n t r i f u g a t i o n . A n a l y s e s w e r e p e r f o r m e d i n d u p l i c a t e a n d s t a n d a r d d e v i a t i o n s a r e r e p o r t e d . C o n c e n t r a t i o n of p - h y d r o x y m e r c u r i b e n z o a t e w a s 0.338 m g / m l . L i p i d c o n c e n t r a t i o n s a r e r e p o r t e d as m g p e r i o o m l ill s e r u m a n d v a l u e s i n p a r e n t h e s e s a r e p e r c e n t a g e s of t o t a l l i p i d .
Sample conditions
Cholesterol esters
Phospho- Triglylipicls cerides
Unester- Unesterified ified cholesfatty terol acids
Non-incubated
I17 i 2 (44) 136 ± o (45)
80 ~- 2 (3 ° ) 79 ± i (26)
4° ~ i (15) 68 Jc o (22)
30 3_ I (II) 2o J- i (7)
0. 4 ~ o . o l
267 :~ 2
o.6 ± o.o2
3o4 zE i
4o~ o (I5) 45 ~z i (17)
33 ± i (I2) 29 -~ i (II)
0. 4=~ o
265i
0.5 i
271 ~- 6
incubated
Total lipid
p- Hydroxymercuribenzoate added Non-incubated Incubated
112 ~ 7 (4 2 ) 116 i i (43)
8o± 2 (3 o ) 80 ± 3 (29)
0.02
9
significant from the available data. The apparent increase in mean flotation rate of the major peak in the S°), o-2o range, after incubation of inhibitor-fr ee serum, also was not statistically significant. Although the changes in flotation rates were not significant, a significant relationship was observed between the changes in flotation rate and serum concentrations of S°f 2o-4oo lipoproteins. Fig. 4 shows the regression lines for the regression of the incubation-induced change in S°y rate of the S°y o-2o lipoproteins, from inhibited and non-inhibited sera, on the concentration of S°y 2o-4oo in the sera * F l o t a t i o n r a t e s d e s c r i b e d b y S°f v a l u e s a r e r a t e s i n S v e d b e r g u n i t s for l i p o p r o t e i n s i n a m e d i u m o f d I.O63 g / m l (NaC1, 26 °, 52 64o r e v . / m i n ) a n d a r e r a t e s c o r r e c t e d for c o n c e n t r a t i o n a n d J o h n s t o n O g s t o n effects.
Biochim. Biophys. Acta, 168 (1968) 8 7 - 9 4
92
A.v. NICHOLS et al.
determined prior to incubation. For sera containing elevated concentrations of S°j 20-400 lipoproteins, the shift in S°/rate of the S°~0-20 major peak is greater in noninhibited than in inhibited sera. From the above data, the changes in S°/0-20 flotation rates appear to have some relationship to the presence of transferase activity whereas the marked shift in H D L distribution does not. Table I I shows the changes in lipid concentration and composition of S¢ 0-20 lipoproteins isolated from a VLDL-rich serum which was incubated with and without p-hydroxymercuribenzoate. The triglyceride concentration in this serum was 214 mg/Ioo ml. The very large increase in triglyceride content of the S 1 0-20 in the presence of transferase activity suggests that transesterification is an important factor in the transfer of VLDL-triglyceride in vitro. In the absence of transferase activity, triglyceride transfer is also observed but to a much lower extent than in the non-inhibited serum. The apparent lack of change in the phospholipid content of the S/0-20 following incubation of sera containing elevated concentrations of triglyceride has been frequently noted and will be described in a subsequent report. No consistent changes were observed in the S°l 20-40o distributions of the sera studied in this investigation. Since the serum S°: 20-400 concentrations of several of our subjects were relatively low, further studies are needed to evaluate the effect of serum incubation on S°/20-400 lipoprotein profiles. The apparent shift of the H D L profile to higher flotation rates is of interest in light of reports of changes in ultracentrifugal recovery and immunochemical properties of H D L following exposure of H D L or serum to various physical and chemical conditions. SCANU AND GRANDA11 subjected H D L 2 and H D L a to successive preparative recentrifugations and observed significant reductions in protein recovery for these fractions. They ascribed the losses to a degradation of the H D L 2 and H D L a with the formation of more dense lipoprotein species which appeared either in the H D L 3 or the d > 1.20 g/ml ultracentrifugal fractions. The species appearing in the d > 1.20 g/ml fraction were very low in lipid content and were designated by them as very highdensity lipoproteins (VHDL). Immunochemical studies by SCAI~U AND GRANDA showed that the VHDL, derived from H D L 2 or HDLa, and the protein moiety obtained from delipidation of HDL2 or H D L a had identical immunochemical properties. They proposed the hypothesis that native H D L consists of a relatively lipid-rich lipoprotein moiety in labile association with a lipid-poor V H D L moiety. I-IAYASHI, LINDGREN AND NICHOLSTM were able to dissociate a lipid-poor protein from H D L b y mild ether treatment; and LEVY AND FREDRICKSONla obtained immunochemical evidence for the release of a lipid-poor protein moiety from H D L in serum by freeze-thawing, storage or addition of urea. Ether treatment, in addition to producing a lipid-poor protein also shifts the distribution of the H D L in the direction of higher flotation rates. Fig. 5 shows this effect of ether on the H D L distribution. This shift is highly similar to that observed following incubation of serum, and the mechanisms responsible m a y be comparable. Incubation of serum thus m a y lead to the dissociation of H D L into V H D L and relatively lipid-rich lipoprotein moieties. The latter moieties m a y subsequently associate to yield species with faster flotation rates. Our data would suggest that during incubation the dissociating species are primarily H D L a. HAYASHI, LINDGREN AND NICHOLS also showed the formation of lipoprotein species with flotation rates in the S°: 0-20 range after ether treatment of HDL. This observation suggests even further dissociation of protein from the H D L and subsequent association of the more lipid-rich lipoprotein residues. In our own incubation studies an increase in the Biochim. Biophys. Acta, 168 (1968) 87-94
93
CHANGES IN SERUM L I P O P R O T E I N S
concentration of the S°S o-2o was indicated but was not found significant. This trend towards increased values is suggestive and m a y indicate the presence of all of the possible processes of dissociation and association observed during exposure of H D L to ether. To test whether protein dissociation from H D L during incubation of serum can be detected, protein analyses were performed on H D L ultracentrifugally isolated from an incubated serum sample and its control. Prior to protein analysis, the H D L fractions were subjected to dialysis against 0.202 M NaC1 to remove any low molecular weight nitrogenous contaminants. Triplicate analyses showed a mean percentage protein content of 52.1% (based on individual values of 52.0, 52.2 and 52.0%) and
F~,20 rote(Svedberg units)
20
--~jl'i-,.,..2• 10 " I I\ I 1 1 I !:' Ofl
Ether-treated NDL
-
/
Control F4DLX\ ./ I k I L I L I L I I Fig. 5. L i p o p r o t e i n p r o f i l e s o f a t o t a l I t D L f r a c t i o n b e f o r e a n d a f t e r e t h e r t r e a t m e n t 1=. T h e s e profiles do not represent the actual concentrations of HDL encountered in this experiment but are presented to show the characteristics of the change in HDL distribution following ether exposure.
49.1% (based on individual values of 49.2, 48.8 and 49.3%) for the H D L fractions separated from control and incubated serum aliquots, respectively. This significant reduction in protein content is in reasonable agreement with estimated reductions in protein content calculated from our ultracentrifugal analyses of the shift in H D L distribution and available information on the chemical compositions of HDL~ and HDL~. These data support the hypothesis that the shift in H D L distribution during incubation of serum results from an initial dissociation of lipid-poor protein from the H D L followed b y an association of the relatively lipid-rich lipoprotein residues. The latter products comprise the faster floating species in the analytic ultracentrifuge. Factors influencing the above dissociations and associations during incubation of serum are under investigation. Furthermore, although comparable protein dissociation would appear to occur from the H D L in sera with and without transferase inhibitor, this does not rule out some possible relationship between such H D L protein and transferase activity in serum. In light of earlier reports~, 14 suggesting the detection of transferase activity in ultracentrifugally isolated HDL, we are currently investigating the possible relationship oftransferase activity to proteins which are in labile association with HDL. Biochirn. Biophys. Acta, i 6 8 (1968) 8 7 - 9 4
94
A . V . NICHOLS et al,
ACKNOWLEDGEMENTS T h e a u t h o r s w i s h t o t h a n k Dr. F. T. LINDGREN for p r o v i d i n g t h e p r o t e i n a n a l y s e s a n d t o a c k n o w l e d g e t h e v a l u a b l e t e c h n i c a l a s s i s t a n c e o f P. SHAFER AND W . H o . T h i s w o r k w a s s u p p o r t e d in p a r t b y P u b l i c H e a l t h S e r v i c e R e s e a r c h G r a n t H E 1 0 8 7 8 - 0 2 f r o m t h e N a t i o n a l H e a r t I n s t i t u t e , U.S. P u b l i c H e a l t h S e r v i c e , a n d b y t h e A t o m i c Energy Commission, REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14
J. A. C*LOMSET,Biochim. Biophys. Acta, 65 (1962) 128. A. V. NICHOLS AND L. SMITH, J. Lipid Res., 6 (1965) 206. J- A. GLOMSET, Biochim. Biophys. Aeta, 7° (1963) 389. A. M. EWlN~, N. K. FREEMAN AND F. T. LINDGREN, Advan. LipidRes., 3 (1965) 25. F. T. LINDGREN, A. V. NICHOLS AND R. D. WILLS, Am. J. Clin. Nutr., 9 (1961) 13. L. C. JENSEN, T. H. RICH AND F. T. LINDGREN, Donner Laboratory Semiannual R e p o r t Biology and Medicine, Lawrence Radiation Laboratory, UCRL 18066, Fall, 1967. N. K. FREEMAN, F. T. LINDGREN AND A. V. NICHOLS, in R. T. HOLMAN, W. O. LUNDBERG and T. WALKIN, Progress in the Chemistry of Fats and other Lipids, Vol. 6, MacMillan (Pergamon), New York, 1963, p. 215. F. T. HATCH, N. K. FREEMAN, L. C. JENSEN, G. R. STEVENS AND F. T. LINDGREN, Lipids, 2 (1967) 183. G. R. BARTLETT, J. Biol. Chem., 234 (1959) 466. O. F. DE LALLA AND J. W. GOFMAN, in D. GLICK, Methods in Biochemical Analysis, ¥ol. I Wiley (Interscience), New York, 1954, p. 459. A. SCANU AND J. L. GRANDA, Biochemistry, 5 (1966) 446. S. HAYASHI, F. LINDGREN AND A. NICHOLS, J, Am. Chem. Soc., 81 (1959) 3793. R. I. LEvy AND D. S. FREDRICKSON, J. Clin. Invest., 44 (1965) 426. ~¥. J. Lossow, S. N. SHAH AND I, L. CHAIKOFF, Biochim. Biophys. Acta, 116 (1966) 172.
Biochim. Biophys..4cta, 168 (1968) 87-94