This document presents the results of a study comparing the plasma osmolarity and levels of various biochemical parameters in healthy breastfed infants versus non-breastfed infants. The study found significantly higher levels of urea, sodium, potassium, total proteins and calculated plasma osmolarity in infants fed cow's milk or formula compared to breastfed infants. These differences indicate that non-breastfed infants have a higher renal solute load from alternative milks compared to the optimized nutrient balance provided by human breastmilk.

2. DIFFERENCES OF PLASMA OSMOLARITY
IN HEALTHY BREASTFED AND NON BREASTFED INFANTS
THESIS FOR
M.Phil BIOCHEMISTRY
BY
DR. MUHAMMAD MUSTANSAR
SUPERVISOR
DR. ZAMIR AHMED
PROFESSOR OF BIOCHEMISTRY
SERVICES INSTITUTE OF MEDICAL SCIENCES, LAHROE
INTRODUCTION
Breastfeeding is the most natural and safest way to feed an infant.
Breastfeeding provides a unique combination of proteins, lipids,
carbohydrates, and minerals in quite appropriate amount. Human milk
is a complex species-specific biological fluid for an infant with all
nutritional needs for growth, development and biochemical
equilibrium. The total protein content of human milk is the lowest
among all mammals due to relatively slow growth of baby in human
beings as compared to others. The concentration of minerals in
human milk is about 1/3rd lower than in cow’s milk. This fact together
with reduced protein content leads to a lower solute load, which is
adequate for the infants developing kidney. (Lawrence 1994)

3. Devies and Saunders (1973) described that the renal solute load of cow‟s milk is
considerably higher than that of breast milk. This is shown by high urea level in non-breast
fed infants. The blood urea level in breast fed infants was 22mg/dl and that of
non-breast infants was 47mg/dl calculated by „‟t‟‟ test for a sample of 61 infants.
The sodium level in cow‟s milk is 3.6 times more than in human milk. For cows milk it is
22meq/L and for human milk is 7meq/L. Hyper natermia is associated with cows milk
feeding. Experiments with newborn rats on high salt in takes have shown that
hypertension can develop. (Ruth 1989)
SOLUTE LOAD
Composition of Human, Cow
and Buffalo milk (100ml)
NUTRIENT HUMAN COW BUFFALO
Water g 85.7 88.0 84.0
Energy Kcal 70.0 61.0 97.0
Protein g 1.0 3.2 3.7
Fat g 4.4 3.4 6.9
Lactose g 6.9 4.7 5.2
Minerals g 0.20 0.72 0.79

4. Human milk provides optimal nutrition to infants and the appropriate balance of
nutrients. The relatively low contents of minerals and protein in breast milk are
adequate and present comfortable load to immature infant kidney. Human milk has
relatively low sodium content allowing the fluid requirement of the infant to be met
while not over taxing the kidneys.
The osmolarity of human milk approximates to that of human serum and is 286m
osmol/L, where as that for cows milk is 350m osmol/L. (Dale 1975)
OPTIMAL NUTRITION
Lonnerdal and Chen (1990) reported that formula-fed infants
experience much higher plasma levels of most amino acids and
consistently elevated blood urea levels. This implies a decreased
efficiency of utilization of the protein in the formula. Different
formulas with similar nitrogen concentration may contain
dissimilar levels of true protein and non-protein nitrogen
depending on their method of preparation.
FORMULA FEEDING

5. AIMS AND OBJECTIVES
The aim of the present study is to highlight the importance of mother’s milk
and to discourage the usage of infant formula or cows/buffalos milk to an
infant.
The objectives are
1) To find out Serum Sodium, Potassium, Urea, Glucose, Total
Serum proteins, Albumin, Globulin and A/G ratio
(Albumin/Globulin) in breastfed and non-breastfed infants.
2) To determine plasma osmolarity and its comparison in breastfed
and non-breastfed infants.
In our society due to some social and cultural believes many mothers
deprive their infants from the benefits of breastfeeding. The infants are
fed by cow milk/buffalos milk purchased from the market. The infant
formula milk is very costly and it is not possible for an average family
to afford formula milk for an infant. The milk of cows and buffalos is
having three times more proteins and 3-4 times more minerals as
compared to the human milk. By feeding the milk of cow or
buffalo/infant formula the infant is being loaded with more proteins
and minerals, which can lead to high osmolarity, sodium, potassium,
urea and serum proteins. The effects of these may not be visible
immediately but may have consequences in later life.
IMPORTANCE OF STUDY

6. MATERIALS AND METHODS
STUDY DESIGN
This was a descriptive cross-sectional study, which conducted on infants up to the
age of 6 months being fed either mother’s milk, infant formula or cow/buffalo milk.
SAMPLE SIZE
A total sample of one hundred and eighty infants up to the age of 6 months were
taken included. The sample were divided into three groups of sixty infants in each
group.
Group I:- Infants on mother’s milk.
Group II:- Infants on formula milk.
Group III:- Infants on cow/buffalo milk.
SELECTION CRITERIA
Infants up to the age of 06 months
Either sex
The Infants being fed milk. No weaning diets.
Taking the particular milk for at least one month.
Appropriate mile stone
EXCLUSION CRITERIA
Infants of less than one month or more than six months age
Low birth weight infants
Premature delivered infants
Infants having gross congenital anomaly of GIT, Urinary tract and Cardiovascular
System
The infants having history of recurrent chest infection
Infants on weaning diets
Infants on mixed feeding

7. SAMPLE COLLECTION
LABORATORY ANALYSIS
Serum Sodium Level (Gowenlock, 1988)
Serum Potassium Level (Gowenlock, 1988)
Serum Urea Level (Marsh et al, 1965)
Serum Glucose Level (Trinder, 1969)
Total Serum proteins Level (Reinhold, 1953)
Serum Albumin Level (Doumas et al, 1971)
Serum Globulin Level (Total proteins – serum albumin level)
A/G ratio Serum Albumin Level (Sood, 1994)
Serum Globulin Level
Plasma osmolarity (estimated) (Vapro Osmometer)
Plasma osmolarity (calculated) (Robert, 1999)
CALCULATION OF OSMOLARITY
Osmolarity mosm/l = 2{Na+meq/l+K+ meq/l}+ (glucose) mg/dl + (BUN) mg/d
18 2.8

8. Appendix I
PROFORMA
Name of Child:Name of Father:Age:Address:
Feeding Practice for the last one month:
Only on Mother’s Milk
On Buffalos Milk
On Cows Milk
On infant formula
Name: ________________ Preparation: ___________Dilution or without DilutionDilution or
without DilutionEXAMINATION OF INFANT
LABORATORY REPORT
Serum Sodium Level _______________meq/l
Serum Potassium Level _______________meq/l
Serum Urea Level _______________mg/dl
Serum Glucose Level _______________mg/dl
Total Serum proteins Level
_______________g/dl
Serum Albumin Level _______________g/dl
Serum Globulin Level _______________g/dl
A/G ratio _______________
Plasma osmolarity (estimated) ___________m osml/l
Plasma osmolarity (calculated) __________m osml/l
STATISTICAL METHODS
STATISTICAL METHODS (Danial WW 2005)
For the purpose of statistical analysis the
information recorded on the proforma was stored
in spreadsheet of SPSS software (Statistical
Program for Scientific Studies) version 13.
The data were analyzed as follows.
1. Description
2. Analysis ANOVA One Way.
3. Inference Post HOC Test.

9. AGE DISTRIBUTION OF BRESTFED AND NON BREASTFED INFANTS
Mode of Feeding No. of Cases (n)
Mean Age in
Months
Standard Deviation
(SD)
Formula Milk 60 4.03 1.657
Mother Milk 60 3.54 1.650
Fresh Milk 60 4.55 1.489
WEIGHT DISTRIBUTION OF BREASTFED AND
NON BREAST FED INFANTS
Mode of Feeding No. of Cases (n)
Mean Weight
in Kg
Standard Deviation
(SD)
Formula Milk 60 4.58 0.86
Mother Milk 60 4.86 1.16
Fresh Milk 60 4.84 0.80

10. BLOOD GLUCOSE LEVEL IN BREASTFED AND
NON-BREAST FED INFANTS
Mode of Feeding No. of Cases (n)
Mean Glucose
Level in
mg/dl
Standard Deviation
(SD)
Formula Milk 60 90.47 7.0
Mother Milk 60 95.30 9.2
Fresh Milk 60 95.43 9.3
BUN AND BLOOD UREA LEVEL IN BREASTFED AND NON BREAST FED INFANTS
Mode of
Feeding
No. of Cases
(n)
Urea mg/dl BUN mg/dl
Mean SD Mean SD
Formula Milk 60 22.81 4.02 10.66 1.88
Mother Milk 60 10.45 3.84 6.14 1.40
Fresh Milk 60 15.35 7.22 7.14 3.36

11. SERUM SODIUM AND POTASIUM IN
BREASTFED AND NON-BREAST FED INFANTS
Mode of
Feeding
No. of
Cases (n)
Na+ m mol/L K+ m mol/L
Mean SD Mean SD
Formula Milk 60 147.43 3.87 5.15 0.49
Mother Milk 60 138.43 4.20 4.83 0.34
Fresh Milk 60 146.5 5.17 5.27 0.55
COMPARISION OF CALCULATED AND ESTIMATED PLASMA OSMOLARITY IN
BREASTFED AND NON BREAST FED INFANTS
T TEST DISTRIBUTION
Mode of
Feeding
T d.f
Sig
(2Tailed)
Mean
Difference
95 % CI of the
differences
Lower
Bound
Upper Bound
Formula Milk 5.137 118 0.000 7.43 4.568 10.299
Mother Milk 5.158 118 0.000 5.23 3.214 7.220
Fresh Milk 4.296 118 0.000 6.90 3.764 10.202

12. CALCULATED AND ESTIMATED PLASMA OSMOLARITY IN BREASTFED AND
NON BREAST FED INFANTS
Mode of
Feeding
No. of Cases (n)
Calculated Osmolarity
m osm/L
Estimated Osmolarity m osm/L
Mean SD Mean SD
Formula Milk 60 314.05 8.48 306.62 7.32
Mother Milk 60 291.92 6.31 286.70 4.63
Fresh Milk 60 310.30 9.09 303.32 8.71
COMPARISION OF CALCULATED AND ESTIMATED PLASMA OSMOLARITY IN BREASTFED
AND NON BREASTFED INFANTS
(ANOVA)
Variable
s
Differen
ce
Sum of
Squares
d.f Mean
Squares
VR P Value
Calculate
d
Osmolarit
y
Between
Groups
16837.87
8
2 8418.939 129.872 0.000
Within
Groups
11474.03
3
177 64.825
Total 28311.91
1
179
Estimate
d
Osmolarit
y
Between
Groups
13673.54
4
2 135.788 13.847 0.000
Within
Groups
8911.767 177 50.349
Total
22585.31
1
179

13. COMPARISION OF CALCULATED AND ESTIMATED PLASMA OSMOLARITY IN BREASTFED
AND NON BREASTFED INFANTS
POST HOC TEST
Variable
Group
Code
Group
Code
Mean
Difference
*
P Value
95 % Confidence Interval
Lower
Bound
Upper
Bound
Calculated
Osmolarity
1
2 22.1333* 0.000 19.2324 25.0343
3 3.7500* 0.012 0.8491 6.6509
2
1 - 22.1333* 0.000 - 25.0343 - 19.2324
3 - 18.3833* 0.000 - 21.2843 - 15.4824
3
1 - 3.7500* 0.012 - 6.6509 - 0.8491
2 18.3833* 0.000 15.4824 21.2843
Estimated
Osmolarity
1
2 19.9167* 0.000 17.3601 22.4733
3 3.3000* 0.012 0.7434 5. 8566
2
1 - 19.9167* 0.000 - 22.4733 - 17.3601
3 - 16.6167* 0.000 - 19.1733 - 14.0601
3
1 - 3.3000* 0.012 - 5.8566 - 037434
2 16.6167* 0.000 14.0601 19.1733
*Mean Difference is significant at the 0.05 level.
Code Group 1 Formula Milk
Code Group 2 Mother Milk
Code Group 3 Fresh Milk
CONCLUSION
The present study has clearly highlighted that “Mother‟s Milk is the best milk”. The serum levels of
BUN, urea, total proteins, albumin, sodium, potassium and plasma osmolarity are all significantly low
as compared to the infants on bottle feeding taking infant formula or fresh milk. The bottle feeding
either by fresh milk or by the cheaper partially modified formula predispose the infants developing
systems to high solute loads and osmolarity which may not have immediate consequences but may
have in later life like childhood obesity and raised blood pressure.