Studies of serum lipoprotein concentrations in children: A preliminary report

65

Clinica Chimica Acta, 56 (1974) 65-74 @ Elsevier Scientific Publishing Company, Amsterdam -

Printed in The Netherlands

CCA 6607

STUDIES OF SERUM LIPOPROTEIN A PRELIMINARY REPORT

G.S. BERENSON, P.S. PARGAONKAR, RADHAKRISHNAMURTHY Departments (U.S.A.)

of Medicine

and Biochemistry,

CONCENTRATIONS

S.R. SRINIVASAN, L.S.U.

School

IN CHILDREN:

E.R. DALFERES, JR and B.

of Medicine,

New

Orleans, La.

(Received May 6, 1974)

Summary Serum lipid studies were performed on 333 children, aged 3-16 years. These studies measured total cholesterol, triglycerides, cr-lipoprotein cholesterol, /3- plus pre-/3-lipoprotein cholesterol, and the serum concentrations of p-, pre-0, and cr-lipoproteins. Mean serum cholesterol (170 mg/lOO ml) for children of 3-4 years was already as high as for young male adults 23-25 years (172 mg/lOO ml), but cholesterol derived from a-lipoprotein constituted 48% of the total cholesterol in children and only 28% in young adults. From childhood through maturation and adulthood, a transition appears to occur in the patterns of the different classes of serum lipoproteins. Serum triglycerides in children (77 mg/lOO ml) were slightly lower than in medical students (86 mg/ 100 ml), and their pre-fl-lipoprotein concentrations (47 mg/lOO ml) were about half those of the students (97 mg/lOO ml). These studies emphasize that evaluation of lipid abnormalities in children requires specific study of serum lipoproteins in addition to observations of integral components, such as cholesterol, common to each of the lipoproteins.

Introduction Observations concerning the chronological changes of serum lipids in individuals from infancy to adulthood need to be compared to information now available on adults. Although a number of factors have been shown to increase the risk for coronary disease, one of the most important factors is a high level of certain serum lipid fractions [l-4]. A limited number of studies on serum lipids in young children have been performed, but up to the present these lipids have been measured mostly in terms of cholesterol and triglycerides [ 5-91. Since it is now possible to detect abnormalities of lipoproteins in families, interest has been extended to the study of lipids even in newborns [lo-121 ;

66

however, there is little information on the levels of specific serum lipoproteins in young children and adolescents although the importance of lipid screening in [13] . Such observations are important because children has been emphasized the earlier abnormalities can be evaluated, the sooner treatment can be instituted with greater potential benefits. Materials

and Methods

Serum Serum was obtained from 333 children between the ages of 3-16 years seen in the Collaborative Child Development Program Clinic of Charity Hospital of Louisiana at New Orleans, in the out-patient clinics of the same hospital and of Touro Infirmary, and in the offices of private pediatricians. A large majority (75%) of the children were Negroes attending the Charity Hospital clinics. Blood was collected in tubes containing Thimerosal, an antibacterial agent (Aldrich Chemical Co., Milwaukee, Wis.) and was allowed to clot [14]. Approximately one-third of the sera were from non-fasting children. For comparison, studies performed earlier on male medical students 22-25 years old were used [15-181.

Total serum cholesterol

and triglyceride

Cholesterol was determined triglycerides by using a Technicon

Estimation

analyses

by the method of Pearson autoanalyzer [ 201.

et al. [19],

and

of serum p- plus pre-fl-lipoproteins

The turbidity obtained by the addition of Ca*+ and heparin, following a procedure previously described [15,18,21,22], was equivalent to the serum /3plus pre-&lipoprotein concentrations. These values are expressed as a /I- plus pre-P-lipoprotein index [16]. The difference between total cholesterol and the /3-plus pre-&lipoprotein cholesterol was considered as e-lipoprotein cholesterol. The measurement of fl- plus pre-fl-lipoproteins consisted of mixing serum (0.2 ml), distilled water (3.2 ml), heparin (140 units/mg, 0.25%, 0.1 ml), and CaC12 (0.5 M 0.5 ml), in the order given, and of measuring the turbidity after 15 min at 600 nm against a blank containing a similar mixture but omitting heparin. The presence of chylomicrons does not interfere with turbidimetric assay because the absorption due to chylomicrons is eliminated by measuring absorbance against a blank.

Agar-agarose gel electrophoresis Electrophoresis of serum lipoproteins was performed according to the method of Noble [23] and Cawley [24] with some modifications [ 181. The lipoprotein bands were scanned with a densitometer, and the curves were integrated to estimate the proportion of p- to pre-P-lipoprotein.

Determination

of p- and pre-P-lipoprotein

concentrations

Serum /3- and pre-/3-lipoproteins were calculated as previously described [21] . The calculations were based on the densitometric ratios of /3-to pre-fllipoprotein, /I- plus pre-&lipoprotein cholesterol content, and reported average

67

values of cholesterol present in P-lipoprotein (46.9%) and pre-fl-lipoprotein (22.2%) [25]. The validity of this method was established by comparing the results with those obtained by the analytical ultracentrifuge; the results were found to be in agreement, as well as more reproducible [15,16]. Estimation of a-lipoprotein Based on the serum cu-lipoprotein cholesterol content and on the reported average value of cholesterol present in e-lipoprotein (19%) [25], the approximate e-lipoprotein concentration was calculated by multiplying a-lipoprotein cholesterol by 5.15. Experimental

Results

Fig. 1 shows distributions of total serum cholesterol, triglycerides, and the p- plus pre-P-1’rpoprotein index (turbidity produced by heparin and Ca”) from the sera of children. The clear bars represent the number of children falling around the mean values (the actual mean values are given below), whereas the

Cholesterol

B+preP-Lipoprotein

8+ pro 8- Lipoprotein

Index

Fig. 1. In these graphs of distribution of serum cholesterol, triglycerides, and&- plus pre-&lipoprotein in a group of 333 children, the differently shaded areas represent arbitrary lhn@ for “normal”, borderline, and high values. The borderline area was assigned around the mean value for the sample.

TABLE

I

SERUM

LIPID

CONCENTRATIONS Age

group

IN CHILDREN

Index** Serum

Cholesterol*

Female

Male

Female

Male

Female

(11)

(40)

(29)

(54)

(44)

0.194

0.183 -?- 0.039

174

f * *

a-Lipoproteins*

**

P-Lipoproteins*

* *

f

***

* Number

of

Absorbance

28

165 +

35

+

86 80

k

22

i

27

29

*

72 k

89

26

181

169 zk

59

85 f

31

31

f

25

39 88

82 +

45 90

f

21

376

413

457

423

451

+ 172

f 145

? 132

+ 137

+ 131

? 110

196

193

186

169

174

62

f

53 * *

mg/lOO

i

0.187 * 0.074

379

Pre-P-Lipo. proteins*

48

0.172 * 0.051

173 +

73

33

f

in p- plus

t

74

Cholesterol*

20 72

66

a-lipoprotein

**

t

0.155 + 0.036

0.187 f 0.07

164

31 93

*

7-8

(21)*

Triglyce-

rides**

SEX

Male

f Serum

AND

5-6

f 0.065 * *

AGE

(fears)

3-4

/_-Pre-P-Lipoprotein

BY

+ subjects

32

k

42

40

z!

45

67

f

2

28

53 f

42

73

+

16

53

189 *

46 f

30

85 50

?

32

in parentheses.

Units/O.2

ml, Mean

2 S.D.

(absorbance

X 500

= approximate

concentration

Of cholesterol

pre-@-lipoproteins). ml,

Mean

f S.D.

different shaded areas represent the number of children falling above and below mean levels. The /3- plus pre-&lipoprotein index quantitatively represents the concentrations of these two lipoprotein classes in the serum [16]. It is interesting to note that unlike serum cholesterol, the /3-plus pre-@-lipoprotein index distribution is skewed to the left in the histogram. More children seem to fall into the high lipid category according to the total cholesterol assays than with the determination of the index. The difference is due to the specificity of individual laboratory methods that reflect different serum lipoproteins. Unlike total cholesterol and triglycerides which are derived from all classes of lipoproteins, the p- plus pre-P-lipoprotein index specifically reflects these two classes of lipoproteins. The present data indicate that in children, relative to values obtained on adults, higher proportions of the total cholesterol is derived from (Y-lipoprotein. (This is discussed below .) Table I and Fig. 2 show the serum lipid profile with respect to total serum cholesterol, triglycerides, fl- plus pre-/I-lipoprotein index, /3-plus pre-fi-lipoprotein cholesterol, fl-lipoprotein, pre-P-lipoprotein, and a-lipoprotein among the different age groups of children. Interestingly, total serum cholesterol at age 3-4 years is already at the levels noted for 15-16 years of age and for male medical students approximately 25 years of age [ 171. There were no significant changes in values among the various age groups, although girls showed a slight increase in the mean total cholesterol value over the period of 11-16 years. Although mean serum lipid levels varied little among age and sex groups, the individual levels varied considerably within the given age and sex group as seen from the high standard deviation (S.D.) values.

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9-10 Male (22) 0.175 f 0.055 164 * 31 77 * 40 76 * 22 392 +114 186 * 67 38 * 27

11-12 Female (4) 0.171 * 0.041 161 k 10 63 -t 27 75 t 19 386 f 98 185 t 53 34 f 11

Male (20) 0.163 + 0.052 159 + 26 72 f 30 78 A 21 403 *lo9 158 * 49 49 + 36

13-14 Female (19) 0.179 * 0.068 176 t 47 71 + 28 86 k 32 442 Ir165 174 * 68 55 + 34

Male (23) 0.147 F 0.048 153 i- 31 63 k 49 79 * 20 408 +104 145 f 52 41 + 29

16-16 Female (14) 0.189 * 0.079 171 + 34 74 t 29 77 + 23 395 + 120 188 f 85 54 t 43

Male (20) 0.193 + 0.110 174 * 66 87 * 51 78 ? 22 400 t115 189 *105 58 k 51

Female (12) 0.190 k 0.127 185 * 85 86 * 45 90 + 28 463 +144 195 r129 48 i- 43

Table II gives the serum lipids and lipoprotein levels in children as compared to young adults (medical students). It is evident from the P values that, except for serum cholesterol, all parameters studied in children differ significantly from those of the medical students. For instance, a considerable difference in cw-lipoprotein cholesterol can be seen between children and young adults, 81 mg/lOO ml compared to 48 mg/lOO ml, respectively. These values reflect the concentration of the cw-lipoprotein macromolecule in the serum, and significantly higher values are found in children. In contrast, the levels of pre-&lipoproteins for children (47 mg/lOO ml) were about half those of young adults (97 mg/lOO ml). These observations are clearly reflected in the marked differences in the (Y- to /3- plus pre-&lipoprotein ratio. The alterations in the relative proportion of these classes of lipoproteins in adulthood are further exemplified by the four-fold increase in cY/pre-P-lipoprotein ratios in children. In our studies the difference of a/pre-/I-lipoprotein ratios becomes even greater between normal individuals and individuals with diabetes mellitus (unpublished observations). These observations suggest that an inverse relationship or a transition occurs between CY-and pre-fl-lipoproteins observed in children with low pre-o-lipoproteins and commonly observed mixed hyperlipoproteinemia (combined elevated cholesterol and triglyceride) in adults. The comparison of triglyceride and pre-p-lipoprotein values reveals the usefulness of the present turbidity method. Our earlier studies (including the studies on medical students) indicated a correlation coefficient of 0.96 between serum triglyceride levels and pre-fl-lipoprotein concentrations [15] . The triglyceride levels due to chylomicrons increased in samples from non-fasting chil-

T

I T

ME GRWPS (YEARS)

PRE-P -LlPOP~T~l~

TRIGLYCERIDE

401

6oc

6oc

lcnc

34

T

3-6

T

T

T

7-6 9-KI 1112 ~314 B-16

I .

T

kc-LWWROTEIN

B+ PRE-~LIPoPROTEIN CHOLESTEROL

Fig. 2. Serum lipid profile in chit&en of different age groups is illustrated with respect to serum cholesterol, triglycerides, @- plus pre-&lipoprotein cholesterol, @-lipoprotein, pre-plipoprotein. and a-lipoprotein. The means and ranges are shown for both males and females. Note the extremely wide ranges observed in each category.

T

CHOLESTEROL

I FSllWb

71 TABLE

II

A COMPARISON Values are expressed

OF

SERUM

LIPID

CONCENTRATIONS

IN CHILDREN

AND

YOUNG

as Mean ? S.D. Children

Medical students (n = 67)

(n = 333) fl-Pre-fi-lipoprotein index* (absorbance units/O.2 ml) Serum cholesterol* * * (mg/100 ml) Serum triglycerides* * (mg/lOO ml) a-Lipoprotein cholesterol* (mg/lOO ml) a-Lipoproteins* (mg/lOO ml) P-Lipoproteins* (mg/lOO ml) Pm-P-Lipoproteins* (mg/lOO ml) a/Pre-P-Lipoproteins*

+ * * ? + * * f

or/P-Lipoproteins* * o/Pm-P

ADULTS

plus P-Lipoproteins* *

0.177 0.067 170 40 77 48 81 25 419 130 179 70 47 41 16 15 2.6 1.2 2.1 0.9

+ + * + * f * f * *

0.247 0.068 172 32 86 39 48 18 248 92 218 58 97 56 4 5 1.3 0.7 0.9 0.5

* P value 0.0001. ** P value 0.03. *** No significant difference.

dren while the pre-/3-lipoprotein values remained low. One of the problems of screening for lipids in a pediatric population is obtaining reliable information with fasting. Since the present method measures pre-P-lipoprotein but not the chylomicrons, one can get a good estimation of lipoprotein profiles. Fig. 3 shows two typical agar-agarose gel electrophoretic runs of multiple sera for lipoproteins. As for adults, marked variability is observed among chil-

Fig. 3. Two representative glass plates of agar-agarose gel electrophoretic runs for serum lipoproteins in several children are shown. p-. pre-(3-. and a-lipoproteins are well separated. Occasionally, multiple bands are observed; note. e.g.. a double pre-p band for No. 123. Chylomicrons at the origin in No. 127 were observed in some of the sera. See text for details.

72

205

i

Mole N=200

I

150.

.

Y

.

I ..I

% -I + P Q : i

200

I I

% I.50 I

Female N- 133

. .

. j

fl-Lipoprotein

mg%

Fig. 4. Scattergrams correlating @- and pre-(Mipoproteins in male and female children are shown. Divisions were made for p- and pre-~-tipoprote~ns based on mean values for the groups studied. Children with both p- and pm-&lipoproteins below the mean values are represented in the lower left quadrant. with high values for pre-@lipoprotein in the upper left quadrant, with high P-lipoprotein values in the lower right quadrant, and with mixed elevations of both lipoproteins in the upper right quadrant. The nature of the lipoprotein abnormality can easily be recognized from these scattergrams. The relationship of the lipoproteins and their distribution on the scattergrams have therapeutic implications.

dren. Compared to adults, most children have an intense band of ~-lipoprotein, and the average densitometric proportion of p- to pre-/&lipoprotein of 80/20 in children compared to 69/31 for adults was consistent with observations stated above. Chylomicrons were observed at the origin in 12% of “reportedly” fasting and 34% of non-fasting children, and these observations again indicate the difficulty of verifying fasting in children. Fig. 4 shows a scatter~am correlating p- and pre-~-hpoprote~s for both male and female children. In order to evaluate the nature of lipoprotein abnormalities graphically, divisions were made for /3-and pre$-lipoproteins based on mean values of the group studied. Children with both /_I-and pre$-lipoproteins below the mean values are represented in the lower left quadrant; with high values of pre-~-lipoprotein in the upper left quadrant; with high ~~lipoproteins in the lower right quadrant; and with mixed elevations of lipoproteins in the upper right quadrant. Even when the distributions were compared to a population of “normal” adults, about 15% of the children had levels above the means calculated for adults. Obviously, a much greater number appear elevated when the children’s mean levels are applied as “normal” levels. (It would be premature to use’such data as reference standards until further information becomes available on other groups of children.) It is interesting to note that the distribution of the serum lipoprotein concentrations in children among the four quadrants mimics the pattern observed in our earlier studies with adults [15,17], except that there is a quantitative shift towards lower values in children. Such findings cannot be reflected in studies of total cholesterol alone when the ~-~poproteins are high.

73

Discussion The conventional determinations for the assessment of lipid abnormalities include serum cholesterol and, more recently, triglycerides and lipoprotein electrophoresis. The serum cholesterol levels of the young children in these studies are in agreement with previous observations on juvenile populations in the United States [5,8,9] . However, the serum lipoprotein profile and the distribution of cholesterol in different classes of lipoproteins in children reveal some interesting observations which are not generally recognized. Since very low levels of cholesterol are reported in umbilical cord blood [10,12], the current findings show a striking change in children by 3 years of age. This has now been shown to occur at an even earlier age [26]. Another important point which will need further evaluation is that cholesterol derived from cr-lipoprotein in children constitutes a relatively greater part of the total cholesterol. Very low levels of total cholesterol are reported in umbilical cord serum, and a-lipoprotein cholesterol constitutes more than 50% of the total cholesterol [lO,ll, 13,271. The (Y-lipoprotein cholesterol levels observed in the present studies are higher than earlier observations [28] ; however, no data are available on the number of individuals tested in the specific age groups between 3-16 years, so it is difficult to compare these two sets of observations. Furthermore, the use of Ca*+ or Mg*+ in the polyanion precipitation method is more specific and quantitative than Mn 2+ which is known to coprecipitate other serum proteins including high density lipoprotein [ 22,291. Also, previous studies on a/3-lipoproteinemia indicated that heparin-Mn*+ treated serum yielded a precipitate containing albumin, y-globulin, and e-lipoprotein as well as trace amounts of other plasma proteins [30]. Since the precipitation method is generally followed to differentiate cr-lipoprotein cholesterol from p- plus pre-/3-lipoprotein cholesterol, it is important to recognize the specificities of the individual methods that are currently being developed for application in clinical studies. The high a/pre-&lipoprotein ratio observed in children as compared to young adults makes the understanding of the function and inter-relationships of different classes of lipoproteins all the more important. The high cY/pre-/3 ratio observed in children seems to reflect an effective lipid clearing mechanism in children. a-Lipoprotein is known to play a key role in lipoprotein metabolism by activating lipoprotein lipase and 1ecithin:cholesterol acyltransferase enzymes [31]. Furthermore, marked differences have been observed in a//3plus pre-P-lipoprotein between “normal” adult populations with low and high incidence of coronary artery disease [32]. Low levels of a-lipoprotein have been implicated in susceptibility to atherosclerosis [33]. In view of these facts, it will be important to elucidate the role of maturation in the gradation of changes of lipoproteins. Recent studies [7] on serum cholesterol levels of Mexican and Wisconsin children of age 14-15 indicated that the Wisconsin group was approximately twice as high as the Mexican. These observations make it apparent that some determinants are influencing levels of serum lipids in infancy and early childhood; therefore, the assessment of the exact type of lipoprotein abnormality at an early stage of life becomes essential for the development of preventive

measures. But to make such evaluations, the determination of total serum cholesterol and/or triglycerides alone has become inadequate. This is especially so in children. It is also apparent from these studies that, because of the extremely wide individual variability, larger samples of lipoprotein determinations will be needed in order to detect subtle differences reported for cholesterol which might be related to sex, race, and early age. The present studies illustrate the importance of lipoprotein determinations which can now be performed using simple methodology. Although the children studied were largely from outpatient clinics, further studies are in progress involving large populations of free-living children in a community setting. Acknowledgements Research was supported by funds from NHLI of the USPHS (HL 02942), Specialized Center of Research - Arteriosclerosis (HL 15103), the Louisiana Heart Association and the American Heart Association. The authors wish to thank Mr Charles Nicholas for technical assistance and Mrs Sarah Miravalle for statistical analyses. References 1

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