Biochimica et Itiophysh'a Acla. 11186( 1991 ) 225-2211 ~'~ ItJgl Elsevier Science Publishers B.V, All rights reserved 11111|5-27h0/91/$11351~
225
IIBALIP 53772
Plasma lecithin-cholesterol acyltransferase activity and cholesterol and phospholipid levels in premature newborn infants Sushil K. Jain a n d J a v i e r J. Diaz Dt,partmetzl of Pediatrics, Lotlisianu Slate Unicer'ity St'htnd of Mt,dicine. Shrereport. L,4 (U.S.A.) (Received 211 March 1991)
Key words; Lecithin-cholesterol acyllransferase: I lypcrchtdesh:rolemia; Ilyperpht~spholipidemia; Premalurity: (Newborn inlant)
lecithin-cholesterol acyltransferase (LCAT) activity has been suggested to play an important role in the regulation of lipid metabolism. The present study was undertaken to examine any relationship between LCAT activity and altered cholesterol levels in plasma of full-term and preterm newborn infants. Plasma total, free and esterifled cholesterol, total phospholipid and LCAT activity (cholesterol esterifled, n m o l / m l per h) were determined in placental cord blood. There was a significant negative relationship between total cholesterol levels and gestational age. The increased cholesterol with prematurity was due to both free and esterified cholesterol. There was aLo a significant negative relationship between LCAT activity and free cholesterol levels but not between LCAT activity and total cholesterol and esterified cholesterol levels. There was no relationship between esterifled-to-free cholesterol ratio and LCAT activity. Tota; phospholipid was not significantly related to either gestational age or LCAT activity. This study suggests that reduced LCAT activity may be one of the factors that result in the accumulation of cholesterol in premature infants.
Introduction The role of LCAT in the regulation of lipid metabolism has been extensively studied [I-5]. LCAT esterifies free cholesterol to cholesterol ester, utilizing beta fatty acid of the phosphatidylcholine molecule. Deficiency of LCAT in both humans and experimental animals has been shown to bc associated with hypereholesterolemia and hyperphospholipidemia and fat accumulation in the tissues [6-12}. Other studies have reported the occmrence of hypcrlipidemia and fat accumulation in certain tissues of premature newborn infants [ 13,14]. Previous studies have shown that plasma cholesterol levc!s are lower in newborns than in adults [15-21] and. farther, that cholesterol levels arc higher [18,20-22] and similar [19,23] in the cord plasma of premature infaat:, than in the cord plasma of full-term newborns. On the other hand, other investigators have reported lower plasma LCAT activity in newborns than in adults [24-27], and much lower levels in premature
Ck~rrespondence: S.K. Jain, Department of Pediatrics. I,SLI Medical ('enter. 151H Kings Highway, Shreveport. LA 71130, U.S.A.
infants than in full-term newborns [27-29]. The present study was undertaken to examine any relationship between cholesterol and phospholipid levels and LCAT activity, and ~,~t,vvi~n:~l~,- in c,~r,! pl?,sma of full-term a~:d premature newborn infimts. Materials and Methods Approval by the Mcdical Center Review Committee on Human Expclimentation was obtained for this study. Blood samples were collected at random. Placental cord blood samples collected into EDTA tubes, immediately after the deliver),, were utilized fi~r the entire study. Blood was cen~.rifugcd at l()00 x g at 4 ° C for l0 rain. Plasma was aspiratcd with a Pasteur pipette and frozen at below --2t)°C. All anabscs wcrc performed on frozen plasma samples. Gestational ages of newborn infimts were obtained from the log book of the delivery room after completion of all determinations. The cord blood samples wcrc of I I white (5 males. 6 females) and 21~bhtek ( 13 males, 16 females) newborns. Plasma LCAT ass,,,y was started in the middle of the study, therefore data with LCAT assay arc from only 2t) (8 white, 5 males and 3 female.~: 21 blacks. 10 males and l I females) cord blood samples.
226 Plasma I+CAT ttlt'asurel:wnl,~ LCAT assaywas performed as described by GIomset [I]. Substratu for LCAT was prepared by incubating 10 #Ci of 4-[m~C]cholcsterol (spee. act. 59.4 mCi/mmol: New England Nuclear, Boston, MA) with 10 ml of heat-inactivated normal human plasma (55"(" for 30 rain) in a shaking water bath for 3 h. Heat-inactivated plasma containing [l~Cleholcsterol was then centrifuged for l0 min at 150(X)×g in a refrigerated centrifuge, mid the clear supernatant was used an substrate for enzyme assay. To 10-ml capacity glass tubes, 201)/,tl of substrate and 50/,tl of test plasma were each added. Control tubes contained only substrate. Tubes wcre il c , b a t e d in a water bath at 3 7 ° C for 2 h. A time cour,;e of 2 h was chosen because preliminary experiments showed that cholesterol esterification was linear for 2 h in thi'~ assay. At the end of incubation, the reaction was stopped by adding 2 ml of methanol and mixing. Tubes were allowed to stand at loom temperalure for 15 rain during which they were mixed at leant three times. Then 2 ml of chloroform were added, and tubes were allowed to ,~-md at room temperature for another 15 min with intermittent mixing. The tubes were then centrifuged at It)00 x g for l0 min. Because the amount of plasma :sample used wus .,anall, there was only one solvent layer (supcrnatant) and protein residue at bottom of the tube. The supernatant was then decanted into another 10-ml glass tube and dried with a slrcam of nitrogen while keeping the tubes in z, water bath at about 40 ° C. The dried lipid extract was dissolved in 101) # l of chloroform. The free and esterifled cholesten)l in the lipid extract were separated by "ILC on silica gel G plates (Brinkman Instruments, Wcstbury, NY) with a solvent system consisting of petroleum e t h e r / c h e r / a c e t i c acid (90: l : l, v/v). On some plates, standard cholesterol and eholesteral ester were also spotted as a marker. Alter developing the "I LC p h t c , ;t war, dried and then exposed to iodine vapors to visualize cholesterol and chole~:terol enter spots, l.ipid spots were encircled with :l needle or lead pencil. The plate was left at room temperature for aboul a h or preferably overnight to get rid of iouinc, which may thcr,~isc cause quenching of radioactive counts. The free cholesterol and cholesterol ester spots were scraped into cnunting vials and 5 ml of counting fluid was added (Ncw England Nuclear). 14C radioactivity was determined with a Beckman LS-250 liquid scintillation system. LCAT way expressed as nmol of eslerificd cholesterol / m l per h. Plasma lipids were extracted by the method of Folch ct al. [30], using the solvent system c h l o r o f o r m / methanol (2 : I, v/v). Cholesterol and cholesterol esters were separated as described above. Cholesterol way determined by the method of Zlatkis c t a l . [31]. Phospholipid-phosphorus way quamitatcd after its hydrolysis with H2SO 4 by the method of Fiske and Subbarow
[32]. Statistica: analv~cs were carried ovt using EPIS. TAT statistical soltwarc with tin IBM PC XT computer. A P value of less than (I.I)5 was considered statistically significant. Results
Fig. I illustrates the effect of gestatkmal age on the total, free, attd esterified cholesterol in plasma of newborn infants Levels of plasma cholesterol increased with a decrease in gestational age. This increase was due to an increase in both free and esterified cholesterol. The correlation coefficients ( r ) for gestational age and total ( r = - 0 . 3 8 , P < 0 . 0 2 ) , free ( r = - 0 . 4 2 ,
"~ E
120 100 r
-0.36(p,
0.03)
80)
60 40
"~
20
I¢I
0
J
i
i
"
!
24
!,o 50
E
•--~
•
40
r
-0.42(p.
0.01)
20
~
10
LL
0 24
26
20
30
32
34
36
38
40
42
140 120-
t 3
lOO.
'~
8o
•u¢
6o
~0
4e
o 0
I'.-
•
~. :~.p.~ :..
r
-038(p
0,02)
" : "~--.__~
| ',,,
20 0 24
i
i
i
i
i
i
i
i
]
26
28
30
32
34
36
30
40
42
G e s t a t i o n a l A qe (weeks) [:ig I I;fh:ct ot guMalh)nal age on the total, l rc¢ and cstcri[h:d cholc'~tcrol in cord pla~,ma o1' 40 newborn mlanls. Note statistically *,ignit,~:~,,nt relationship bctwuen increased plasma cholesterol luvels and prematurily.
227 P < 0 . 0 1 ) , and esterificd cholesterol ( r = - 0 . 3 6 , P < 0.03) were all statistically significant. As stated in Materials and Methods, the LCAT assay was started in the middle of this study, so we have LCAT data on only 29 samples. When these 29 samples were used, r of gestational age with total cholesterol was - 0 . 3 0 ( P < 0. I 1 ), free cholesterol was - 0.40 ( P < 0.03), and with esterified cholesterol - 0,27 ( P < 0.15). Thus, correlation between gestational age and total cholesterol was significant only with larger sample populations and with free cholesterol in both sample populations.
3.$ r0.31(#'
'1' 3.0 O -
0.11
2.5
%
2.0 ~J
I.S
O
U. •;
uJ
1,0 o.s
0.0 0
2
4
6
8
10
12
14
16
LCAT (truiNg choleslefo(tllll~l|l~ mlJhr) 90-
3.5
80
o
? ~,
r
7O
-0.17(p,
0.4)
0 (J
4O
= tm
3O
-L~
20
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o
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~
:LO.
~
1.5
26
24
2
4
6
8
10
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14
16
g
1,4 • :
16
28 30 32
077 (p ,
34
36 3:8 40 42
.1/
/
0.00001)
E
5O
/,
/ ;
/
4o r ~ -0.38 (p
0.04)
•
•°
6
~
v
/ I
0
24
o
4
6
8
10
i:
4 2
U ..,I
10
2
•
/
8
20
12
14
16
18
26
28 30 32
34
36 38
40
42
G e s t a t i o ~ l Age (weexl)
120
F i g 3 Relationship of gcslalioaal age with IX'AT activity (bottom
?
panel) and esteril'led-lo-frcc ctLolc~,tt'r,.~,l latio (nliddlc panel), arld belwe,,.'n [.(YVI" aclivil.~ and eslcrilied-lo-lrce eh~,}lg~,,Icrol talit) (lop), in c,~}rd pl;iMrla ill ~,i lli'Wbtlrn il~,al'llS, NOB.' ~,ignilicant rel.'llion,,hip bel'*cen t.('AT dehcicncy and prcmalurily and lack ol any relalion,,hip hclwccn csler-lo-l'rce chol,..'slerol ratio am.l L ( ' A T aetivily and geMal;orlal ilgC,.
100 r
-0.24 ( p
0..
80
so (j
I
o.5
:.
E
u.
.:.
0
~o
30
o~
/ .::/
,7. ~.o
•
10
?
o
~ .1
0
r - O. IS (D " 0.5/
3.o,
4o
m m
o I,-.
20 ,,, ,,
0 0
2
4
6
8
10
12
14
15
L C A T ( n m o l l l c l l o l e l l l O r o l oster0hed~ ml/hr} Fig. 2. Relationship between I X ' A T acti;'ily and B~lal. free, and ester cholesterol levels in cord plasma of 29 newborn infilnl~,. Nol¢ statistically signifkant relation between free cholesterol levels and LCAT activity.
Fig. 2 shows In- relationship between cord plasma LCAT activity and total, fi'ee and esterified cholcsterol levels. There was a significant relationship between LCAT and free choh',~tcrol ( r = - 0 . t 8 , P < l)04); however, the rclatiof,:'hi),, between LCAT and total cholcstcrol {r = -0.24, P < 0.2) and LCAT and cholesterol cster ( r = -{).17, P
225
IIBALIP 53772
Plasma lecithin-cholesterol acyltransferase activity and cholesterol and phospholipid levels in premature newborn infants Sushil K. Jain a n d J a v i e r J. Diaz Dt,partmetzl of Pediatrics, Lotlisianu Slate Unicer'ity St'htnd of Mt,dicine. Shrereport. L,4 (U.S.A.) (Received 211 March 1991)
Key words; Lecithin-cholesterol acyllransferase: I lypcrchtdesh:rolemia; Ilyperpht~spholipidemia; Premalurity: (Newborn inlant)
lecithin-cholesterol acyltransferase (LCAT) activity has been suggested to play an important role in the regulation of lipid metabolism. The present study was undertaken to examine any relationship between LCAT activity and altered cholesterol levels in plasma of full-term and preterm newborn infants. Plasma total, free and esterifled cholesterol, total phospholipid and LCAT activity (cholesterol esterifled, n m o l / m l per h) were determined in placental cord blood. There was a significant negative relationship between total cholesterol levels and gestational age. The increased cholesterol with prematurity was due to both free and esterified cholesterol. There was aLo a significant negative relationship between LCAT activity and free cholesterol levels but not between LCAT activity and total cholesterol and esterified cholesterol levels. There was no relationship between esterifled-to-free cholesterol ratio and LCAT activity. Tota; phospholipid was not significantly related to either gestational age or LCAT activity. This study suggests that reduced LCAT activity may be one of the factors that result in the accumulation of cholesterol in premature infants.
Introduction The role of LCAT in the regulation of lipid metabolism has been extensively studied [I-5]. LCAT esterifies free cholesterol to cholesterol ester, utilizing beta fatty acid of the phosphatidylcholine molecule. Deficiency of LCAT in both humans and experimental animals has been shown to bc associated with hypereholesterolemia and hyperphospholipidemia and fat accumulation in the tissues [6-12}. Other studies have reported the occmrence of hypcrlipidemia and fat accumulation in certain tissues of premature newborn infants [ 13,14]. Previous studies have shown that plasma cholesterol levc!s are lower in newborns than in adults [15-21] and. farther, that cholesterol levels arc higher [18,20-22] and similar [19,23] in the cord plasma of premature infaat:, than in the cord plasma of full-term newborns. On the other hand, other investigators have reported lower plasma LCAT activity in newborns than in adults [24-27], and much lower levels in premature
Ck~rrespondence: S.K. Jain, Department of Pediatrics. I,SLI Medical ('enter. 151H Kings Highway, Shreveport. LA 71130, U.S.A.
infants than in full-term newborns [27-29]. The present study was undertaken to examine any relationship between cholesterol and phospholipid levels and LCAT activity, and ~,~t,vvi~n:~l~,- in c,~r,! pl?,sma of full-term a~:d premature newborn infimts. Materials and Methods Approval by the Mcdical Center Review Committee on Human Expclimentation was obtained for this study. Blood samples were collected at random. Placental cord blood samples collected into EDTA tubes, immediately after the deliver),, were utilized fi~r the entire study. Blood was cen~.rifugcd at l()00 x g at 4 ° C for l0 rain. Plasma was aspiratcd with a Pasteur pipette and frozen at below --2t)°C. All anabscs wcrc performed on frozen plasma samples. Gestational ages of newborn infimts were obtained from the log book of the delivery room after completion of all determinations. The cord blood samples wcrc of I I white (5 males. 6 females) and 21~bhtek ( 13 males, 16 females) newborns. Plasma LCAT ass,,,y was started in the middle of the study, therefore data with LCAT assay arc from only 2t) (8 white, 5 males and 3 female.~: 21 blacks. 10 males and l I females) cord blood samples.
226 Plasma I+CAT ttlt'asurel:wnl,~ LCAT assaywas performed as described by GIomset [I]. Substratu for LCAT was prepared by incubating 10 #Ci of 4-[m~C]cholcsterol (spee. act. 59.4 mCi/mmol: New England Nuclear, Boston, MA) with 10 ml of heat-inactivated normal human plasma (55"(" for 30 rain) in a shaking water bath for 3 h. Heat-inactivated plasma containing [l~Cleholcsterol was then centrifuged for l0 min at 150(X)×g in a refrigerated centrifuge, mid the clear supernatant was used an substrate for enzyme assay. To 10-ml capacity glass tubes, 201)/,tl of substrate and 50/,tl of test plasma were each added. Control tubes contained only substrate. Tubes wcre il c , b a t e d in a water bath at 3 7 ° C for 2 h. A time cour,;e of 2 h was chosen because preliminary experiments showed that cholesterol esterification was linear for 2 h in thi'~ assay. At the end of incubation, the reaction was stopped by adding 2 ml of methanol and mixing. Tubes were allowed to stand at loom temperalure for 15 rain during which they were mixed at leant three times. Then 2 ml of chloroform were added, and tubes were allowed to ,~-md at room temperature for another 15 min with intermittent mixing. The tubes were then centrifuged at It)00 x g for l0 min. Because the amount of plasma :sample used wus .,anall, there was only one solvent layer (supcrnatant) and protein residue at bottom of the tube. The supernatant was then decanted into another 10-ml glass tube and dried with a slrcam of nitrogen while keeping the tubes in z, water bath at about 40 ° C. The dried lipid extract was dissolved in 101) # l of chloroform. The free and esterifled cholesten)l in the lipid extract were separated by "ILC on silica gel G plates (Brinkman Instruments, Wcstbury, NY) with a solvent system consisting of petroleum e t h e r / c h e r / a c e t i c acid (90: l : l, v/v). On some plates, standard cholesterol and eholesteral ester were also spotted as a marker. Alter developing the "I LC p h t c , ;t war, dried and then exposed to iodine vapors to visualize cholesterol and chole~:terol enter spots, l.ipid spots were encircled with :l needle or lead pencil. The plate was left at room temperature for aboul a h or preferably overnight to get rid of iouinc, which may thcr,~isc cause quenching of radioactive counts. The free cholesterol and cholesterol ester spots were scraped into cnunting vials and 5 ml of counting fluid was added (Ncw England Nuclear). 14C radioactivity was determined with a Beckman LS-250 liquid scintillation system. LCAT way expressed as nmol of eslerificd cholesterol / m l per h. Plasma lipids were extracted by the method of Folch ct al. [30], using the solvent system c h l o r o f o r m / methanol (2 : I, v/v). Cholesterol and cholesterol esters were separated as described above. Cholesterol way determined by the method of Zlatkis c t a l . [31]. Phospholipid-phosphorus way quamitatcd after its hydrolysis with H2SO 4 by the method of Fiske and Subbarow
[32]. Statistica: analv~cs were carried ovt using EPIS. TAT statistical soltwarc with tin IBM PC XT computer. A P value of less than (I.I)5 was considered statistically significant. Results
Fig. I illustrates the effect of gestatkmal age on the total, free, attd esterified cholesterol in plasma of newborn infants Levels of plasma cholesterol increased with a decrease in gestational age. This increase was due to an increase in both free and esterified cholesterol. The correlation coefficients ( r ) for gestational age and total ( r = - 0 . 3 8 , P < 0 . 0 2 ) , free ( r = - 0 . 4 2 ,
"~ E
120 100 r
-0.36(p,
0.03)
80)
60 40
"~
20
I¢I
0
J
i
i
"
!
24
!,o 50
E
•--~
•
40
r
-0.42(p.
0.01)
20
~
10
LL
0 24
26
20
30
32
34
36
38
40
42
140 120-
t 3
lOO.
'~
8o
•u¢
6o
~0
4e
o 0
I'.-
•
~. :~.p.~ :..
r
-038(p
0,02)
" : "~--.__~
| ',,,
20 0 24
i
i
i
i
i
i
i
i
]
26
28
30
32
34
36
30
40
42
G e s t a t i o n a l A qe (weeks) [:ig I I;fh:ct ot guMalh)nal age on the total, l rc¢ and cstcri[h:d cholc'~tcrol in cord pla~,ma o1' 40 newborn mlanls. Note statistically *,ignit,~:~,,nt relationship bctwuen increased plasma cholesterol luvels and prematurily.
227 P < 0 . 0 1 ) , and esterificd cholesterol ( r = - 0 . 3 6 , P < 0.03) were all statistically significant. As stated in Materials and Methods, the LCAT assay was started in the middle of this study, so we have LCAT data on only 29 samples. When these 29 samples were used, r of gestational age with total cholesterol was - 0 . 3 0 ( P < 0. I 1 ), free cholesterol was - 0.40 ( P < 0.03), and with esterified cholesterol - 0,27 ( P < 0.15). Thus, correlation between gestational age and total cholesterol was significant only with larger sample populations and with free cholesterol in both sample populations.
3.$ r0.31(#'
'1' 3.0 O -
0.11
2.5
%
2.0 ~J
I.S
O
U. •;
uJ
1,0 o.s
0.0 0
2
4
6
8
10
12
14
16
LCAT (truiNg choleslefo(tllll~l|l~ mlJhr) 90-
3.5
80
o
? ~,
r
7O
-0.17(p,
0.4)
0 (J
4O
= tm
3O
-L~
20
"o
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• coo
o
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~
:LO.
~
1.5
26
24
2
4
6
8
10
12
14
16
g
1,4 • :
16
28 30 32
077 (p ,
34
36 3:8 40 42
.1/
/
0.00001)
E
5O
/,
/ ;
/
4o r ~ -0.38 (p
0.04)
•
•°
6
~
v
/ I
0
24
o
4
6
8
10
i:
4 2
U ..,I
10
2
•
/
8
20
12
14
16
18
26
28 30 32
34
36 38
40
42
G e s t a t i o ~ l Age (weexl)
120
F i g 3 Relationship of gcslalioaal age with IX'AT activity (bottom
?
panel) and esteril'led-lo-frcc ctLolc~,tt'r,.~,l latio (nliddlc panel), arld belwe,,.'n [.(YVI" aclivil.~ and eslcrilied-lo-lrce eh~,}lg~,,Icrol talit) (lop), in c,~}rd pl;iMrla ill ~,i lli'Wbtlrn il~,al'llS, NOB.' ~,ignilicant rel.'llion,,hip bel'*cen t.('AT dehcicncy and prcmalurily and lack ol any relalion,,hip hclwccn csler-lo-l'rce chol,..'slerol ratio am.l L ( ' A T aetivily and geMal;orlal ilgC,.
100 r
-0.24 ( p
0..
80
so (j
I
o.5
:.
E
u.
.:.
0
~o
30
o~
/ .::/
,7. ~.o
•
10
?
o
~ .1
0
r - O. IS (D " 0.5/
3.o,
4o
m m
o I,-.
20 ,,, ,,
0 0
2
4
6
8
10
12
14
15
L C A T ( n m o l l l c l l o l e l l l O r o l oster0hed~ ml/hr} Fig. 2. Relationship between I X ' A T acti;'ily and B~lal. free, and ester cholesterol levels in cord plasma of 29 newborn infilnl~,. Nol¢ statistically signifkant relation between free cholesterol levels and LCAT activity.
Fig. 2 shows In- relationship between cord plasma LCAT activity and total, fi'ee and esterified cholcsterol levels. There was a significant relationship between LCAT and free choh',~tcrol ( r = - 0 . t 8 , P < l)04); however, the rclatiof,:'hi),, between LCAT and total cholcstcrol {r = -0.24, P < 0.2) and LCAT and cholesterol cster ( r = -{).17, P
