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 ::  Abstract
 ::  Introduction
 ::  Material and Methods
 ::  Results
 ::  Discussion
 ::  Acknowledgements
 ::  References
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ARTICLE
Year : 1976  |  Volume : 22  |  Issue : 2  |  Page : 88-93

Serum lipoproteins, phospholipid and cholesterol levels in normal children


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:
S T Babar
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
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Source of Support: None, Conflict of Interest: None


PMID: 1032681

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 :: Abstract 

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%.



How to cite this article:
Babar S T, Modi P J, Gharpure S V. Serum lipoproteins, phospholipid and cholesterol levels in normal children. J Postgrad Med 1976;22:88-93

How to cite this URL:
Babar S T, Modi P J, Gharpure S V. Serum lipoproteins, phospholipid and cholesterol levels in normal children. J Postgrad Med [serial online] 1976 [cited 2019 Jul 16];22:88-93. Available from: http://www.jpgmonline.com/text.asp?1976/22/2/88/42838



 :: Introduction Top


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, [11] Salt and Wolff, [13] Chatterjee et al, [2] and Choremis et al, [3] 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.


 :: Material and Methods Top


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. [7]

The determinations of total, free and ester cholesterol levels, and serum phos­pholipids were done as per methods given by Varley [15] and Youngburg and Young burg. [16] Lipoprotein electrophoresis on paper was carried out using modifications suggested by Lloyd [7] to the method by Jenks and Durrum. [6] 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.


 :: Results Top


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.


 :: Discussion Top


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. [4] 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. [4] 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. [4] This has played an im­portant part in the classification of hyper­lipoproteinemia by Fredrickson. [4] So it is really important to know the level of Omega in normals.

Rafstedt [11] 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 [11] and Chatterjee et al. [2] The Beta-lipoprotein levels are higher and Alpha-lipoprotein levels within agreeable range as compar­ed to the levels found by Chatterjee et al, [2] The higher level of Omega fraction re­ported by Chatterjee et al, [2] may be due to pre-stained serum used for electropho­retic separation which is not very satis­factory. [8]

Salt and Wolff [13] 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, [3] 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. [1],[3],[4],[5]

Values obtained by Boyd [1] 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 [5] has reported a value of 176.00 -± 32.00 mgm% for total cholesterol. Rad­win et al , [10] 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 [11] 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 [12] 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 [9] found serum total cholesterol 181.9 ± 17.1 mgm% in their 35 normal children group. Chatterjee et al, [2] 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 , [4] reported serum total cholesterol 170.00 ± 34.00 mgm%; Cho­remis et al, [3] 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 [14] 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, [10] had reported mean value of 170.00 mgm% which is slightly on the lower side. Rafstedt [11] had reported 235.00 ± 5.3 mgm% which is higher than our values.

Choremis et al, [3] reported mean serum phospholipid levels of 157.00 mgm%. Nath and Chatterjee [9] 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.


 :: Acknowledgements Top


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.

 
 :: References Top

1.Boyd, E. M.: Lipid composition of blood in newborn infants, Am. J. Dis. Child., 52: 1319-1324, 1936.  Back to cited text no. 1    
2.Chatterjee, K. and Chaudhuri, J. Nag.. Serum lipids in malnutrition of children, Indian J. Pediat., 28: 195-202, 1961.  Back to cited text no. 2    
3.Choremis, C., Kyriakides, V. and Papa­dakis, E.: Studies on the blood lipids and lipoproteins in thalassaemia and sickle cell anemia, J. Clin. Path., 14: 361-364, 1961.  Back to cited text no. 3    
4.Fredrickson, D. S., Levy, R. I. and Lees, R. S.: Fat transport in lipoproteins-an integrated approach to mechanisms and disorders, New England J. Med., 276: 34. 94, 148, 215, 1967.  Back to cited text no. 4    
5.Hansen, A. E.: Serum lipids in eczema and in other pathological conditions, Am. J. Dis. Child., 53: 923-S46, 1937.  Back to cited text no. 5    
6.Jenks, W. P. and Durrum, E. L.: Paper electrophoresis as a quantitative method -the staining of serum lipoproteins, J. Clin. Invest., 34: 1437-1448, 1955.  Back to cited text no. 6    
7.Lloyd, J. K.: Disorders of serum lipo­proteins-.. Lipoprotein deficiency states, Arch. Dis. Childh., 43: 393-433, 1968.  Back to cited text no. 7    
8.Mukherjee, K. L.: Mechanism of liver in Kwashiorkor-A review, Indian Pediat., 2: 1-9, 1965.  Back to cited text no. 8    
9.Nath, R. L. and Chatterji, A.: Studies on blood lipids in red cell and plasma, Bull. Calcutta, School Trop. Med., 10: 4-6, 1962.  Back to cited text no. 9    
10.Radwin, L. S., Melnick, J . , Michelson, J. P. and Gottfried, S.: Blood lipid pat­tern in hypothyroidism of childhood, Am. J. Dis. Child., 60: 1120-1136, 1940.  Back to cited text no. 10    
11.Rafstedt, S.: Studies on serum lipids and lipoproteins in infancy and childhood, Acta Paediat., 44: (Suppl 162): 1-109, 1955.  Back to cited text no. 11    
12.Ramanathan, M. K.: Biochemical changes in the serum nutritional oedema syndrome, Indian J. Med. Res., 43: 517-520, 1955.  Back to cited text no. 12    
13.Salt, H. B. and Wolff, O. H.: The appli­cations of serum lipoprotein electropho­resis in pediatric practice, Arch. Dis. Childh., 32: 404-412, 1957.  Back to cited text no. 13    
14.Thomas, E. M.: Total and fractional blood lipid levels in disease of childhood, Am. J. Dis. Child., 74: 563-575, 1547.  Back to cited text no. 14    
15.Varley, Harold: Lipids, "Practical Clinical Biochemistry", 4th Edition, William Heinemann Medical Books, Ltd., and Interscience Books Inc., New York, pp. 311-313, 1967.  Back to cited text no. 15    
16.Youngburg, G. E. and Youngburg, M. V.: Determination of serum phospho­lipids, lipid phosphorous, J. Lab. Clin. Med., 16: 158, 1.930. (Cited by Varley,H. 15 ).  Back to cited text no. 16    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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