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Identification particulars
Name of the Investigator
House No. / Address
Type of Family
Name of the Interviewee
Size of the Family
Particulars of household members
Name of
the family
Relation to
Sex Age the head of
the family
Other sources of income, if any (specify)
Monthly expenditure pattern
Shelter-Rent / Repaying house loan
Medicine / Hospital
Cooking fuel and electricity
Utensils and furniture
Recreation (Movies, Park, TV)
Others (Social events, donations,
Rs. Spent /
% of total
Monthly food expenditure pattern
Green leafy vegetables
Roots and tubers
Other vegetables
Milk and milk products
Meat, fish and poultry
Fats and oils
Sugar and jaggery
Processed / readymade foods
Rs. Spent/ % of Food
Dietary pattern of the expectant woman
Meal pattern of pregnant adolescents
Number of meals / day
Number of snacks / day
Frequency of intake of various food by the pregnant adolescence
Rice Products
Wheat / Wheat products
Horse gram
Green gram
Red gram
Bengal gram
Red gram
Bengal gram dhal
Black gram
Green leafy vegetables
Drumstick leaves
Pumpkin leaves
Colacasia leaves
Coriander leaves
Roots and tubers
Other vegetables
Papaya green
Bottle gourd
Ash gourd
Bitter gourd
Snake gourd
Tender jackfruit
Plantain green
Ladies finger
Tomato ripe
Milk and milk products
Fleshy foods
Nut and oil seeds
Fats and oil
Coconut oil
Palm oil
Sunflower oil
Gingelly oil
Sugar and Jaggery
Prepared / processed food
D-Daily, TRW – Thrice in a Week, TW – Twice in a week, OW – Once in a week, OFN – Once in fortnight,
OM – Once in a month, R - Rarely
Risk scoring schedule (Paul and Vijayalakshmi, 1994)
Risk factors
Age less than 18
Maternal height less than 145cm
Weight less than 45 kg more than 90 kg
Primi / multi 5+
Previous obstetric losses
Rh –ve
Previous low birth weight infant
Antepartum haemorrhage
Anaemia (Hb less than 8g/dl)
Pregnancy induced hypertension
Febrile ailment during pregnancy
Previous premature rupture of membranes
Foetal distress
Previous prolonged labour > 20
Pre-pregnant weight
Date of last menstrual cycle :
Para of pregnancy
Income of family
Anthropometric measurement of the expectant women
Month of Weight
Mid arm
pregnancy in kg circumference
if any
Foods included during pregnancy
Special foods included
Foods avoided during pregnancy
Foods avoided
Are you taking any nutrient supplementation as prescribed by the
physician ?
If yes, details
Nutrient composition
Iron and folate
Iron and calcium
Iron, folate and calcium
Iron, folate and zinc
Iron, folate, calcium with vitamin B3
Iron, folate and B Complex
Iron, vitamins with amino acids and minerals
Interval between previous and present pregnancy
Details regarding present delivery
Complications, if any
Obstetric loss
Foetal distress
Rupture of amniotic membrane before delivery
Antepartum haemorrhage
Change in position of the foetus
Labour pain
1st delivery
2nd delivery
3rd delivery
Type of delivery
Anthropometric measurements of the new born of the present pregnancy
Crown heel length
Head circumference
Chest circumference
Mid upper arm circumference
Block Name
Name of the interviewee
House No. / Address
1. What is the desirable age of pregnancy ?
2. What is the desirable weight gain during pregnancy ?
3. Mention the importance of nutrient supplementation
4. What is the importance of health checkups in pregnancy ?
5. What are the foods to be avoided during nausea ?
6. List out the foods to be included in liberal amounts during pregnancy.
7. Should the requirements of iron and calcium be increased during
8. Mention the foods to be included to overcome anaemia
9. Should you increase the intake of green leafy vegetables during pregnancy ?
10. What is the effect of malnutrition during pregnancy ?
11. What is the best exercise during pregnancy ?
12. What is the average birth weight of the new born baby ?
(Cyanmeth haemoglobin method)
(Raghuramulu et al., 2003)
In solution the ferrous ions (Fe2+) of the hemoglobins (Hb) are oxidized
to the ferric state (Fe3+) by potassium ferric cyanide to form methemoglobin. In
turn, methemoglobin reacts with the cyanide ions (CN-) provided by potassium
cyanide to form cyanmethemoglobin, which has the absorbance at 540nm.
Cyanmethemoglobin solution (Drabkin’s solution) : Dissolve 0.05g
potassium cyanide, 0.200g potassium ferric cyanide and 0,140g
dihydrogen potassium phosphate in 1L of distilled water . Add 1ml
of Triton X-100 and mix. Stable for atleat six months.
Haemoglobin standard : Lyophilized human methemoglobin
(supplied by Sigma USA). Each vial is equivalent to haemoglobin
concentration of 18g/dl whole blood when reconstituted in 50ml of
Drabkin’s solution. Stable for six months when refrigerated at 2-6 C.
Transfer 0.02ml of blood using a calibrated haemoglobin pipette, into a
tube containing 0.5ml of Drabkin’s reagent. Rinse the pipette several times
with the reagent. Allow diluted heamoglobin solution to stand for atleast five
minutes to achieve full colour development. Measure the absorbance at 530550nm of the unknown sample (Aunk) and that of a standard of known
heamoglobin content (Astd) against a reagent blank.
Haemoglobin unknown (g/dl)=
Aunk x Con. of Hb S tan dard (g / dl)
(Raghuramulu et al., 2003)
Protein forms a purple colour complex with copper ions in alkaline
solution. The biuret reaction takes its name from compound, which react in the
same way. The purple color developed is read colorimetrically at 500 m .
Sodium chloride diluent – Dissolved 9g of sodium chloride in one
litre of water.
Stock biuret reagent – Dissolved 45g of potassium sodium titrate
(Rochelle’s salt) in 400ml of 0.2N sodium hydroxide. Added 15g of
cupric sulphate. Stirred until it is dissolves. Added five grams of
potassium iodide and diluted it to one litre with 0.2N sodium
0.2N sodium hydroxide : 50g of sodium hydroxide dissolved in
1000ml of water.
Dilute biuret reagent - .Diluted 250ml of stock biuret and reagent to
one litre with 0.2N sodium hydroxide and added four grams of
potassium iodide.
Standard protein solution – Weighed 400mg of albumin and
dissolved in 0.9 per cent saline solution. Made up the volume to
100ml with saline.
22.5 per cent sodium sulphate.
Into a series of test tubes 0.5, 1.0, 1.5, 2.0, 2.5ml of standard protein
solution was pipetted out and then the volume was made upto 3ml with
distilled water. Into another test tube, pipetted out 0.2ml of serum and diluted
to 0.9 per cent saline upto five ml. From this, 2ml of the solution was taken,
made upto 3ml with distilled water and treated as unknown. Now added 3ml
dilute biuret reagent to all the test tubes. Along with this a blank was prepared.
The colour developed was read colorimetrically at 500nm after 30 minutes. The
amount of protein in the serum was calculated. This gives the value of total
Mixing 0.21ml of serum with 4.8ml of 22.5 per cent sodium sulphate
solution precipitates globulin. Stoppered the tube, inverted several times and
kept in the incubator at 40 C overnight. Filtered the solution the next day using
Whatman No. 42 filter paper. Took 2ml of the filtrate and carried out the
experiment as for total protein. The concentration in gram per cent of albumin
present in the globulin free filtrate is determined from the standard graph.
Globulin value can be obtained by deducting albumin value from total protein.
Total protein g/1000ml =
Amount of globulin
O.D of test
x concentration of standard
O.D of s tan dard
= Total protein - Serum albumin
(Raghuramulu et al., 2003)
The vitamin A is extracted with a suitable organic solvent and an aliquot
of the organic phase is injected onto a normal or reversed phase HPLC column,
followed by an eluting solvent of suitable polarity. Retinol, which is eluted as a
sharp peak within 1-6 min is detected by a sensitive UV detector set at 325328nm. Retinol is quantitated by use of peak height rations or peak area ratios
relative to an internal standard (retinyl acetate or other appropriate analogues).
Solvents of HPLC graph must be degassed and be free of particles.
Standards : A stock standard solution of retinyl acetate in ethanol (50
retinol/ml) is prepared by dissolving about one mg of retinyl acetate in 10ml of
ethanol, determining the concentration in a 1/30 dilution of an aliquot in
ethanol by use of the E11cm
at 325nm as 1795 and then diluting the stock
standard appropriately with ethanol. Then 10ml of this solution is diluted to
19.4ml to yield the stock standard with 50 g retinol/ml.
Normal phase
Transfer 100 l of serum (or plasma), 15 l of internal standard solution
(4 g/ml of retinyl acetate or propionate) and 100 l of methanol to a conical
centrifuge tube. Mix the contents of the tube with a vortex mixer. Add 200 l of
extraction solution (petroleum ether 80, dichloromethane 19.3, isoporpanol 0.7
by volume) and cap the tube. Extract by interrupted mixing on the vertex mixer
for 60s. After centrifugation (3000 rpm, 2 min) inject 100 l of the supernatant
into the column by the use of a Hamilton syringe. Elute with the same solvent
as used for extraction.
Chromatographic conditions
Procedure 1
15 x 0.2c i.. Micro Pak Si-10
Mobile phase
Petroleum ether :
Dichloromethane : isopropanol (80:19.3:0.7)
Flow rate
0.5 ml/min
10 kg/cm2
Detector wavelength 328 nm
Detection sensitivity 0.04 AUFS* on recorder
Recorder Speed
Not specified
Retention time (min)
Retinyl acetate
By use of an internal standard, losses due to incomplete extraction,
inaccurate aliquots, oxidation, etc. are automatically corrected. The internal
standard should have physical and chemical properties sufficiently similar to
retinol, is suitably separated from retinol on HPLC, should not coincide with
other 325nm absorbing materials in serum and is not converted to retinol under
the assay conditions. A precisely known amount of the internal standard is
added to the aliquot of plasma to be analyzed. By determining the relative
extraction efficiency and detector response of retinol and the internal standard,
a standard curve is fashioned in which the ratio of peak heights (or areas) is
plotted against the retinol concentration in plasma. In experimental samples,
the peak height (or area) ratio is determined and the appropriate plasma retinol
concentration determined from the standard curve.
A standard curve is prepared by adding varying amounts of retinol (i.e.,
10-120ng) to a fixed amount (i.e. 50ng) of internal standard in a final volume
of 100 l of eluting solvent, injecting the solution of HPLC under assay
conditions, measuring the peak heights and calculating the peak height ratio.
The peak height ratio is then plotted as the abscissa with the standard retinol
concentration (for a 100 l plasma aliquot) as the ordinate.
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