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Lipoproteins in neonates
April 1974 The Journal o f P E D I A T R I C S 5 8 5
Lipoproteins in neonates Hertha R. Cress, Ph.D., Reda M. Shaher, M.D., Ph.D., Martin H. Greenberg, M.D., and Robert Laffin, Ph.D., Albany, N. Y.
D E SP t T E T H E F ACT that hyperlipoproteinemia has received great clinical attention, there is a scarcity of information on the normal distribution of lipids and lipoproteins or screening tests for reliable detection of hyperlipoproteinemia in children. 1,2 Kwiterovich and associates 3 and Glueck and associates 4 have suggested that hyperlipoproteinemia can be diagnosed at birth by elevated levels of umbilical cord cholesterol or low-density lipoprotein cholesterol. However, Darmady and associates 5 stated that serum cholesterol estimations in cord blood cannot be used as a screening test for the diagnosis of familial hypercholesterolemia because babies with elevated cholesterol had values distributed throughout the normal range when re-examined at one year of age. The purpose of the present study was to measure cord blood lipoproteins of healthy infants, and also to investigate if lipoprotein patterns might be useful in detecting hyperlipoproteinemia at birth. MATERIALS
AND METHODS
Lipoprotein agarose gel eleetrophoresis. The lipoprotein electrophoresis method of Noble 6 was carried out. The agarose gel strips were fixed, dried, and stained with Sudan Black B. The optical density of the dye solution was read in a spectrophotometer, and if the optical density react at 590 fell below 13, the solution was discarded. The Spinco Model RB analytrol provided densitometric scanning of stained electrophoretograms with automatic integration of the peaks of optical density. Variations in staining and technique were standardized against control strips containing known concentrations of human From the Division of Pediatric Cardiology and the Division o f Newborn Services, Department o f Pediatrics, Albany Medical Center Hospital and Albany Medical College. This investigation was supported by the Heart Association of Eastern New York.
serum, cholesterol, and triglyceride. Reproducibility of the method was determined by replicate analyses of individual serum samples. Linearity between lipoprotein amount and dye uptake was demonstrated for lipoprotein fractions by determining values for sera of known concentration and dilutions of sera. IgA determinations. A test to check for maternal contribution to cord blood samples was performed by utilizing the t e c h n i q u e of d o u b l e d i f f u s i o n in gel. Monospecific anti-human IgA (Meloy Laboratories) was used to form a precipitate with known human IgA. When a cord blood sample containing IgA was placed in a well adjacent to the known sample, a reaction of complete identity was observed. The system was sufficiently sensitive to detect IgA levels greater than 5 mg. per cent. Neonates. Blood samples were obtained from 138 male and 132 female unselected normal healthy infants. Umbilical cord blood was obtained promptly at the time of delivery after the cord was divided between clamps. Approximately 10 ml. of blood was collected from the placental end of the cord, transferred to a sterile tube, and refrigerated at 4 ~ C. Data were stored on an on-line IBM computer 360 model 50. RESULTS Distinct bands for beta, pre-beta, alpha, and pre-alpha l i p o p r o t e i n were s h a r p l y v i s i b l e on the electrophoretograms. The two fastest-moving fractions, alpha and pre-alpha lipoprotein, were grouped together and designated alpha lipoprotein complex. After this, a pre-beta lipoprotein fraction was usually seen, well separated from the beta-lipoprotein band. Chylomicrons, found in sera of normal nonfasting individuals and some hyperlipoproteinemia patients, 2 were never observed in the neonates. Values for individual lipoproteins were obtained from densitometer scans of electrophoresis strips,
VoL 84, No. 4, pp. 585-587
586
Cress et al.
The Journal of Pediatrics April 1974
Table I. Lipoproteins in umbilical cord sera
138 male neonates
Range I dye I
I I
I I
•
132female neonates
Per cent total dye
Lipoprotein 10-70 5-55 5-95
6.71 3.23 11.93
3.85 3.33 6.71
33.02 13.23 53.22
j t
PREBETA LP
ALPHA LP
LP
I
3
i4
6
INCREASED BETA LIPOPROTEIN
BETA
PRE-
ALPHA
LP
BETA LP
LP
6
3
8
9
PREBETALp
ALPHA LP
I
i
17
•
]
•
I
Per cent total dye
S.D.
Beta Pre-beta Alpha
BETA LP
I Range dye I
10
i~
INCREASED PRE-BETA LIPOPROTEIN
!
NORMAL LIPOPROTEIN
BETA LP
PREBETA LP
ALPHA LP
13
14
t5
INCREASED BETA AND PRE-BETA LIPOPROTEIh
Fig. 1. Densitometric scans and integration tracings of electrophoretograms from a normal neonate and neonates with increased lipoproteins. The vertical lines delineate the lipoprotein zones. The number of integration units within each zone is expressed in dye units.
13.72 9.07 16.27
15-85 5-60 5-85
6.90 3.09 11.78
4.10 2.63 6.54
33.04 13.63 53.35
• S.D. 14.1 9.2 15,4
lipoprotein made up 53 per cent of the total lipoproteins. Although alpha lipoprotein was usually found separated into alpha and pre-alpha lipoprotein, 21 boys and 24 girls did not have detectable amounts of pre-alpha lipoprorein. Beta lipoprotein represented 33 per cent of total lipoproteins, and a pre-beta lipoprotein content of 13 per cent of total lipoprotein was found in both male and female infants. However, no detectable pre-beta lipoprotein was seen in 12 boys and 8 girls. Two of the male neonates demonstrated two pre-beta lipoprotein bands. Cord blood lipoprotein levels greater than cut-off limits (derived experimentally but selected arbitrarily as m e a n cord blood lipoprotein + 2 S.D.) were identified as elevated. Seven male neonates and four female neonates exceeded the cut-off limits for beta lipoprotein, and five boys and six girls exceeded the cut-olTlimits for pre-beta lipoprotein. Two boys and two girls exceeded the cut-off limits for both beta and pre-beta lipoprotein. A normal pattern of low amounts of lipoproteins, compared with densitometer scans showing increased beta lipoprotein, or increased pre-beta lipoprotein, or a pattern in which both beta and pre-beta lipoprotein were increased, is shown in Fig. 1. A correlation was not observed between values for lipoproteins and birth weight. Five of 110 neonatal sera had detectable amounts o f lgA antibody, and one of these had an increased amount of betalipoprotein. DISCUSSION
and dye uptake of individual fractions was expressed as per cent of total dye uptake of each serum sample. The results of lipoprotein determinations are given in Table I, A statistically significant difference between boys and girls in mean concentrations of each lipoprotein was not found. Amounts of lipoproteins were low and in somewhat different distribution than found in adults. Alpha
The preliminary results indicate that, although cord blood lipoproteins were low in the majority of neonates, some had elevated values that could be detected by agarose gel electrophoresis. The finding of a lipoprotein pattern of (1) increased beta-lipoprotein, (2) increased pre-beta lipoprotein, or (3) both beta and pre-betalipoprotein increased in umbilical cord serum raises the question as to whether or not this is an early expression
Volume 84 Number 4
o f type II or IV hyperlipoproteinemia. H o w e v e r , it remains to be established if the elevated lipoproteins r e p r e s e n t a transient or genetic h y p e r l i p o p r o t e i n e m i a u n t i l f o l l o w - u p s t u d i e s i n c l u d i n g c h o l e s t e r o l and triglyceride determinations of these neonates and their relatives h a v e been performed. The authors thank Mr. John Ryan for assistance with the statistical computations. REFERENCES 1. Levy, R. I., and Rifkind, B. M.: Diagnosis and management of hyperlipoproteinemia in infants and children, Am. J. Cardiol. 31: 547, 1973.
Lipoproteins in neonates
587
2. Beaumont, J. L., Carlson, L. A., Cooper, G. R., et al.: Classification of hyperlipidaemias, Bull. W. H. O. 43: 891, 1970. 3. Kwiterovich, P. O., Levy, R. I., and Fredrickson, D. S.: Neonatal diagnosis of familial type II hyperlipoproteinemia, Lancet 1: 118, 1973. 4. Glueck, C. J., Heckman, F., Schoenfeld, M., Steiner, P., and Pearce, W.: Neonatal familial type I1 hyperlipoproteinemia: Cord blood cholesterol in 1800 births, Metabolism 20: 597, 1971. 5. Darmady, J. M., Fosbrooke, A. S., and Lloyd, J. K.: Prospective study of serum cholesterol levels during first year of life, Br. Med. J. 2: 685, 1972. 6. Noble, R. P.: Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res. 9: 693, 1968.
Lipoproteins in neonates Hertha R. Cress, Ph.D., Reda M. Shaher, M.D., Ph.D., Martin H. Greenberg, M.D., and Robert Laffin, Ph.D., Albany, N. Y.
D E SP t T E T H E F ACT that hyperlipoproteinemia has received great clinical attention, there is a scarcity of information on the normal distribution of lipids and lipoproteins or screening tests for reliable detection of hyperlipoproteinemia in children. 1,2 Kwiterovich and associates 3 and Glueck and associates 4 have suggested that hyperlipoproteinemia can be diagnosed at birth by elevated levels of umbilical cord cholesterol or low-density lipoprotein cholesterol. However, Darmady and associates 5 stated that serum cholesterol estimations in cord blood cannot be used as a screening test for the diagnosis of familial hypercholesterolemia because babies with elevated cholesterol had values distributed throughout the normal range when re-examined at one year of age. The purpose of the present study was to measure cord blood lipoproteins of healthy infants, and also to investigate if lipoprotein patterns might be useful in detecting hyperlipoproteinemia at birth. MATERIALS
AND METHODS
Lipoprotein agarose gel eleetrophoresis. The lipoprotein electrophoresis method of Noble 6 was carried out. The agarose gel strips were fixed, dried, and stained with Sudan Black B. The optical density of the dye solution was read in a spectrophotometer, and if the optical density react at 590 fell below 13, the solution was discarded. The Spinco Model RB analytrol provided densitometric scanning of stained electrophoretograms with automatic integration of the peaks of optical density. Variations in staining and technique were standardized against control strips containing known concentrations of human From the Division of Pediatric Cardiology and the Division o f Newborn Services, Department o f Pediatrics, Albany Medical Center Hospital and Albany Medical College. This investigation was supported by the Heart Association of Eastern New York.
serum, cholesterol, and triglyceride. Reproducibility of the method was determined by replicate analyses of individual serum samples. Linearity between lipoprotein amount and dye uptake was demonstrated for lipoprotein fractions by determining values for sera of known concentration and dilutions of sera. IgA determinations. A test to check for maternal contribution to cord blood samples was performed by utilizing the t e c h n i q u e of d o u b l e d i f f u s i o n in gel. Monospecific anti-human IgA (Meloy Laboratories) was used to form a precipitate with known human IgA. When a cord blood sample containing IgA was placed in a well adjacent to the known sample, a reaction of complete identity was observed. The system was sufficiently sensitive to detect IgA levels greater than 5 mg. per cent. Neonates. Blood samples were obtained from 138 male and 132 female unselected normal healthy infants. Umbilical cord blood was obtained promptly at the time of delivery after the cord was divided between clamps. Approximately 10 ml. of blood was collected from the placental end of the cord, transferred to a sterile tube, and refrigerated at 4 ~ C. Data were stored on an on-line IBM computer 360 model 50. RESULTS Distinct bands for beta, pre-beta, alpha, and pre-alpha l i p o p r o t e i n were s h a r p l y v i s i b l e on the electrophoretograms. The two fastest-moving fractions, alpha and pre-alpha lipoprotein, were grouped together and designated alpha lipoprotein complex. After this, a pre-beta lipoprotein fraction was usually seen, well separated from the beta-lipoprotein band. Chylomicrons, found in sera of normal nonfasting individuals and some hyperlipoproteinemia patients, 2 were never observed in the neonates. Values for individual lipoproteins were obtained from densitometer scans of electrophoresis strips,
VoL 84, No. 4, pp. 585-587
586
Cress et al.
The Journal of Pediatrics April 1974
Table I. Lipoproteins in umbilical cord sera
138 male neonates
Range I dye I
I I
I I
•
132female neonates
Per cent total dye
Lipoprotein 10-70 5-55 5-95
6.71 3.23 11.93
3.85 3.33 6.71
33.02 13.23 53.22
j t
PREBETA LP
ALPHA LP
LP
I
3
i4
6
INCREASED BETA LIPOPROTEIN
BETA
PRE-
ALPHA
LP
BETA LP
LP
6
3
8
9
PREBETALp
ALPHA LP
I
i
17
•
]
•
I
Per cent total dye
S.D.
Beta Pre-beta Alpha
BETA LP
I Range dye I
10
i~
INCREASED PRE-BETA LIPOPROTEIN
!
NORMAL LIPOPROTEIN
BETA LP
PREBETA LP
ALPHA LP
13
14
t5
INCREASED BETA AND PRE-BETA LIPOPROTEIh
Fig. 1. Densitometric scans and integration tracings of electrophoretograms from a normal neonate and neonates with increased lipoproteins. The vertical lines delineate the lipoprotein zones. The number of integration units within each zone is expressed in dye units.
13.72 9.07 16.27
15-85 5-60 5-85
6.90 3.09 11.78
4.10 2.63 6.54
33.04 13.63 53.35
• S.D. 14.1 9.2 15,4
lipoprotein made up 53 per cent of the total lipoproteins. Although alpha lipoprotein was usually found separated into alpha and pre-alpha lipoprotein, 21 boys and 24 girls did not have detectable amounts of pre-alpha lipoprorein. Beta lipoprotein represented 33 per cent of total lipoproteins, and a pre-beta lipoprotein content of 13 per cent of total lipoprotein was found in both male and female infants. However, no detectable pre-beta lipoprotein was seen in 12 boys and 8 girls. Two of the male neonates demonstrated two pre-beta lipoprotein bands. Cord blood lipoprotein levels greater than cut-off limits (derived experimentally but selected arbitrarily as m e a n cord blood lipoprotein + 2 S.D.) were identified as elevated. Seven male neonates and four female neonates exceeded the cut-off limits for beta lipoprotein, and five boys and six girls exceeded the cut-olTlimits for pre-beta lipoprotein. Two boys and two girls exceeded the cut-off limits for both beta and pre-beta lipoprotein. A normal pattern of low amounts of lipoproteins, compared with densitometer scans showing increased beta lipoprotein, or increased pre-beta lipoprotein, or a pattern in which both beta and pre-beta lipoprotein were increased, is shown in Fig. 1. A correlation was not observed between values for lipoproteins and birth weight. Five of 110 neonatal sera had detectable amounts o f lgA antibody, and one of these had an increased amount of betalipoprotein. DISCUSSION
and dye uptake of individual fractions was expressed as per cent of total dye uptake of each serum sample. The results of lipoprotein determinations are given in Table I, A statistically significant difference between boys and girls in mean concentrations of each lipoprotein was not found. Amounts of lipoproteins were low and in somewhat different distribution than found in adults. Alpha
The preliminary results indicate that, although cord blood lipoproteins were low in the majority of neonates, some had elevated values that could be detected by agarose gel electrophoresis. The finding of a lipoprotein pattern of (1) increased beta-lipoprotein, (2) increased pre-beta lipoprotein, or (3) both beta and pre-betalipoprotein increased in umbilical cord serum raises the question as to whether or not this is an early expression
Volume 84 Number 4
o f type II or IV hyperlipoproteinemia. H o w e v e r , it remains to be established if the elevated lipoproteins r e p r e s e n t a transient or genetic h y p e r l i p o p r o t e i n e m i a u n t i l f o l l o w - u p s t u d i e s i n c l u d i n g c h o l e s t e r o l and triglyceride determinations of these neonates and their relatives h a v e been performed. The authors thank Mr. John Ryan for assistance with the statistical computations. REFERENCES 1. Levy, R. I., and Rifkind, B. M.: Diagnosis and management of hyperlipoproteinemia in infants and children, Am. J. Cardiol. 31: 547, 1973.
Lipoproteins in neonates
587
2. Beaumont, J. L., Carlson, L. A., Cooper, G. R., et al.: Classification of hyperlipidaemias, Bull. W. H. O. 43: 891, 1970. 3. Kwiterovich, P. O., Levy, R. I., and Fredrickson, D. S.: Neonatal diagnosis of familial type II hyperlipoproteinemia, Lancet 1: 118, 1973. 4. Glueck, C. J., Heckman, F., Schoenfeld, M., Steiner, P., and Pearce, W.: Neonatal familial type I1 hyperlipoproteinemia: Cord blood cholesterol in 1800 births, Metabolism 20: 597, 1971. 5. Darmady, J. M., Fosbrooke, A. S., and Lloyd, J. K.: Prospective study of serum cholesterol levels during first year of life, Br. Med. J. 2: 685, 1972. 6. Noble, R. P.: Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res. 9: 693, 1968.