Serum lipoproteins, phospholipid and cholesterol levels in normal childrenST Babar, PJ Modi, SV Gharpure
Department of Pathology, B. J. Wadia Hospital for Children and Institute of Child Health, Parel, Bombay and Department of Biochemistry, Seth G. S. Medical College, Parel, Bombay 400 012., India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 1032681
Source of Support: None, Conflict of Interest: None
Serum lipid phosphorus, phospholipids, serum total cholesterol, free cholesterol and serum lipoproteins i.e. omega (Chylomicron), beta and alpha-lipoproteins were estimated in 60 healthy children below the age o f 12 years to assess the normal levels. Electrophoretic separation of serum proteins on absorbent paper was used for the separation of three fractions of serum lipoproteins. The pre-beta lipoprotein-band was not seen in all the 60 normal children under this study. The levels of serum lipoproteins found in these children were as follows: Omega-12.99 ± 2.27%; Beta-61.59 ± 2.90% and Alpha-25.37 ± 3.08%. Similarly, the serum total phospholipids was 189.45 ± 27.73 mgm.% and serum total cholesterol level 170.73 ± 23.47 mgm%.
Estimation of the serum lipids and lipoproteins is of prime importance in the study of metabolic disorders during childhood. Analysis of serum proteins by electrophoresis has become an every day test in most laboratories. Lipoprotein electrophoresis has not yet attained such acceptance though its research value is becoming recognised.
The literature contains a number of studies concerned with abnormalities of the blood lipids in various diseases in children. Rafstedt,  Salt and Wolff,  Chatterjee et al,  and Choremis et al,  have studied the serum lipoproteins by paper electrophoresis in normal children. However, the work done in India on lipids and lipoproteins in children is very scanty, and our knowledge about these in normal children is still incomplete. In order to appreciate the changes in serum lipids, total cholesterol, cholesterol esters, phospholipids in diseases, their levels were estimated in healthy children. An attempt was made to establish the normal levels in healthy children from general population to assist evaluation of the levels in abnormal cases, e.g. in hyperlipoproteinemia. The children under study were males and females from middle class group and were on normal mixed diet. Many cases with lipoprotein abnormalities have been reported in Indian literature but levels of lipoproteins in normal Indian children have not been established. We, therefore, decided to establish the normal pattern of lipoproteins in Indian children.
Sixty healthy children of either sex, under the age of 12 .years, from population at large, were selected for study. The blood was collected after overnight fast and serum was used for the analysis rather than plasma, as the addition of anti-coagulants (especially heparin) distorted the electrophoretic pattern. 
The determinations of total, free and ester cholesterol levels, and serum phospholipids were done as per methods given by Varley  and Youngburg and Young burg.  Lipoprotein electrophoresis on paper was carried out using modifications suggested by Lloyd  to the method by Jenks and Durrum.  For better understanding, the procedure is shortly discussed here.
The separation of different fractions of proteins by electrophoresis is based upon their mobilities in an electric field at o particular pH. Since proteins are in combination with lipids, separation of proteins permits the separation of serum lipoproteins. Mainly three lipoprotein zones have been recognised, i.e. omega at the point of application, beta in the beta-globulin zone and alpha in alphaglobulin region. The following instruments and material were used for electrophoresis.
Shandon hanging-strip vertical apparatus.
Paper strips (5 x 36 cms) were cut from Whatman No. 3 mm thick filter paper.
Buffer: Barbitone buffer of ionic strength 0.075, pH 8.6.
Voltage: 250 volts (constant). Time: Time for run was 6 hours.
Staining solution: Oil red 0: 0.4 g dissolved in 1000 ml of 60% ethanol.
Elution solution: This was prepared freshly by adding 75 ml of ethanol and 25 ml of glacial acetic acid.
Paper electrophoresis was performed at room temperature using Durrum hanging strip method. The buffer was placed in all compartments except the central one. The filter paper strips were marked, folded at the centre and suspended over a tensioned nylon string and the buffer was allowed to ascend to the apex of inverted V. A volume of serum 0.06 nil was applied with a micro-pipette to the apex of each filter paper strip. Then the apparatus was closed and a constant potential of 250 volts (about 20 mA) was applied to the 6 strips for 6 hours. After this, the current was switched off and the strips were spread flat on a blotting paper and dried in oven at 100-110°C for 30 minutes (one of the strips was charged with 0.02 ml of the serum and protein fractions were stained with bromophenol blue). Then all the strips were immersed overnight in oil red O solution for staining lipoproteins. They were then washed in tap water at room temperature and rinsed with constant agitation until alcohol was removed from the paper (about 5 minutes). The strips were allowed to dry at room temperature.
Elution of dye: The stained strips were marked into two 2 cm segments containing alpha-and beta-lipoprotein bands with the point of division of the two segments about 2 mm ahead of the sharply defined front of the beta-band. Then 2 cm segment was marked at the point of application and named as Omega. A 2 cm segment was marked off from protein-free Cathode side of the strip to serve as blank. The segments were cut and placed into 6 ml of acetic acid-ethanol mixture in stoppered tubes and kept at room temperature for 6 hours. After this the tubes were shaken, the supernatant was poured into cuvettes, and the readings were taken in a Leitz photometer using 520 millimicron green filter.
The mean values are calculated in each of the parameters and given in [Table 1]. There was no significant difference found in these parameters in between males and females in this study, which is easily seen from [Table 2] and [Table 3]. Serum lipoprotein electrophoretic pattern with protein electrophoretic pattern in normal and abnormal are shown in [Figure 1] and [Figure 2].
The values obtained in the present study are as follows: (a) Omega-lipoproteins 12.99 ± 2.27%; (b) Beta-lipoproteins 61.59± 2.90% and (c) Alpha-lipoproteins 25.37 ± 3.08% and (d) ratio Beta/Alpha 2.47 ± 0.39; and (e) serum phospholipid is 189.45 ± 27.73 mgm% ; (f) total cholesterol 170.73 ± 23.47 mgm% ; (g) free serum cholesterol 44.15 ± 7.41 mgm% and (h) ester cholesterol 126.01 ± 19.11 mgm% were obtained.
The pre-beta lipoprotein, as is well-known, is rather important in lipid profiles. It was not seen in any of the 60 normal children in this study. On the basis of electrophoresis on paper, one can see that pre-beta band is usually absent in normal children. It is possible that paper is not the optimal medium for this separation.  However, we did succeed in getting pre-beta separation on paper in some abnormal cases, which can be seen in [Figure 2].
Electrophoresis is less quantitative than ultra-centrifugation; but is much more convenient and economical. It is adaptable to the screening of large numbers of subjects at relatively low cost. Visual inspection of properly stained strips permits the immediate recognition of most normal patterns and certain abnormal ones of specific types.  The elution technique which is used in this study gives better quantitation in lipoproteins.
The so-called Omega band at the point of application is nothing but "neutral fat" or exogenous triglyceride in serum i.e. chylomicron particles, which is important in differentiating the various abnormal types.  This has played an important part in the classification of hyperlipoproteinemia by Fredrickson.  So it is really important to know the level of Omega in normals.
Rafstedt  had recorded Omega 21.8%, Beta 50.60% and Alpha 27.60%. The Omega-lipoprotein value in the present studies is less than that of Rafstedt  and Chatterjee et al.  The Beta-lipoprotein levels are higher and Alpha-lipoprotein levels within agreeable range as compared to the levels found by Chatterjee et al,  The higher level of Omega fraction reported by Chatterjee et al,  may be due to pre-stained serum used for electrophoretic separation which is not very satisfactory. 
Salt and Wolff  reported Omega 6.3 % , Beta 64.0% and Alpha 29.0% in 27 normal children (the values are taken by converting into per cent from mgm%). Their values for Beta and Alpha-lipoproteins are similar to ours.
Choremis et al,  obtained Omega 10.62%, Beta 59.55% and Alpha 30.70% in 17 normal children. Their values are in agreement with present studies.
The values in the present study for serum total, free and ester cholesterol are in agreement with the values found by some of the Western authors. ,,,
Values obtained by Boyd  for serum total cholesterol 162.0 ± 32.0 mgm%, free cholesterol 47.0 ± 7.0 mgm% and serum ester cholesterol 115 ± 27.0 mgm% are similar to our values.
Hansen  has reported a value of 176.00 -± 32.00 mgm% for total cholesterol. Radwin et al ,  studied 50 normal children and reported serum total cholesterol level to be 193.00 ± 28.00 mgm%, which is slightly higher; the free cholesterol level of 47.2 ± 10.1 mgm% is in agreement with that in the present study. The levels obtained by Rafstedt  for serum total cholesterol, (188.00 ± 5.00 mgm%), were nearer to those in the present study, but their levels of serum ester cholesterol were slightly higher.
Ramnathan  studied 12 normal Indian children and found the serum total cholesterol levels 189.50 ± 11.19 mgm%, serum free cholesterol 66.50 mgm% and ester cholesterol 123.0 ± 10.66 mgm%. The values of total and free cholesterol were on the higher side and the values of ester cholesterol on the lower side as compared to our values. This can be explained on the basis of different methods used in estimation.
Nath and Chatterjee  found serum total cholesterol 181.9 ± 17.1 mgm% in their 35 normal children group. Chatterjee et al,  found serum levels of total cholesterol 169.6 mgm%, serum free cholesterol 44.00 mgm% and ester cholesterol 130.00 mgm% in their normal group which is in agreement with levels obtained by us.
Fredrickson et al ,  reported serum total cholesterol 170.00 ± 34.00 mgm%; Choremis et al,  reported 162.00 mgm% which agrees with the values obtained in the present study, but free cholesterol levels were slightly higher and ester cholesterol levels were on the lower side.
The mean serum total phospholipid obtained in the present study is 189.45 ± 27.73 mgm%. Thomas  had studied 24 normal persons upto 24 years and his mean values were 190.00 mgm% which is in agreement with values in the present study. Radwin et al,  had reported mean value of 170.00 mgm% which is slightly on the lower side. Rafstedt  had reported 235.00 ± 5.3 mgm% which is higher than our values.
Choremis et al,  reported mean serum phospholipid levels of 157.00 mgm%. Nath and Chatterjee  have reported a slightly lower value for serum phospholipids than those found by us. This lower value can be explained on the basis of different procedures followed for estimations.
We are thankful to Dr. R. A. Irani, M.S., Dean and Dr. G. M. Dhadphale, M.D., Pathologist, B. J. Wadia Hospital for Children and Institute of Child Health for facilities given for this work. We also thank Prof. K. G. Tank sale, M.Sc., Head of the Department of Biochemistry, Seth G. S. Medical College for all the facilities made available for this work, and his valuable advice in this work. We thank staff members of the Pathology Department, B. J. Wadia Hospital for Children for their kind co-operation during this study.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]