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A pilot study on the effects of curd (dahi) & leaf protein concentrate in children with protein energy malnutrition (PEM).

Background & objectives: In protein-energy malnutrition (PEM) there is a significant impairment of immunity, both cell-mediated and humoral, which may be reversed with nutritional rehabilitation. With the use of probiotics like curd (dahi) and micronutrient-rich leaf protein concentrate (LPC), this immune recovery may be hastened. This study was conducted to assess the impact of supplementation of curd and LPC on nutritional status, and immunity as assessed by anthropometry, haemoglobin, ferritin levels, T-cell subpopulation and C-reactive protein (CRP), in children suffering from PEM.

Methods: Eighty moderate to severely malnourished children (1-5 yr) were randomized to receive either curd or LPC in addition to WHO recommended two-step diet over 15 days. Nutritional, immunological and haematological parameters were measured before and after supplementation and compared within the groups.

Results: The change in weight, haemoglobin level and CD4:CD8 T-cell subpopulation was significant in both the groups after supplementation. Response of CRP was blunted in PEM. Serum ferritin decreased significantly after supplementation in both groups.

Interpretation & conclusion: Curd and LPC when added to diet of malnourished children, may have therapeutic value by accelerating immune recovery. More studies need to be done on a larger sample to confirm these findings.

Key words CRP--curd (dahi)--leaf protein concentrate--protein-energy malnutrition--serum ferritin--T-lymphocytes

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Protein energy malnutrition (PEM) is the most frequent cause of secondary immune deficiency in children especially in developing countries (1). In PEM there is a significant impairment of cell-mediated and humoral immune responses (2). Following nutritional rehabilitation the immunological impairments take longer to recover than the anthropometric and some biochemical measurements (3). Therefore dietary supplements are needed to enhance immune recovery. Recently probiotics like curd (dahi) and various micronutrients have been recognized for their immunomodulating effects. Incorporating them in the diet of malnourished children may play an important role in the recovery of the immunological impairments as well as diarrhoea (4). Though curd is an integral part of the Indian diet, micronutrient-rich leaf protein concentrate (LPC) may serve as a cheap alternative. Plant leaves are valuable source of protein, vitamins and minerals and the protein has been rated as good as egg protein (5). Composition of many common leaves has been studied (5-7). Aloe Vera (8), Olive leaf (Olea europeal) (9) and tulsi (Ocimum sanctum) (10) are known to have immunogenic properties. Berseem is rich in protein, [beta]-carotene, B complex vitamins and minerals on feeding to young women, and showed significant improvement in haemoglobin (11). Information on immunogenic effects of feeding curd and LPC to children suffering from PEM is not avalilable. We conducted this study in children suffering from PEM to study the impact of supplementation of curd and LPC on the nutritional status as measured by weight, haemoglobin levels and serum ferritin levels; and the effects on the immune status as measured by the T-cell subpopulation and C-reactive protein (CRP) levels.

Material and Methods

Eighty moderately to severely malnourished children (grade II and III, respectively in Gomez's classification (12), using National Child Health Survey (13), 50th centile for weight as standard) were selected. Children between 1 to 5 yr (mean age 24.91 [+ or -] 11.13 months) requiring in-patient management of PEM were enrolled in a tertiary care hospital in east Delhi during September 2003 to August 2004. It was planned to have minimum of 30 patients in each group. The children were admitted for a 15-day follow up study, with informed parental consent and hospital ethical committee approval. Children with a history of allergy to milk and dairy products, those suffering from severe infections, those on immunosuppressive medication and those with known immunodeficiency states like HIV infections, were excluded.

Of the 80 children, 68 could complete the study. These children were randomized into two groups using a computer-generated random number table. The children in group A (n=32) received curd as a supplement while children in group B (n=36) received LPC. The main reason for not having a control group was due to ethical reason. The follow up was restricted to a minimum of 15 days due to socio-economic reasons, as most patients' attendants could not afford a prolonged loss of working days. Also, the bed occupancy in the paediatric ward of the hospital is 96 per cent and thus the turn over of patients is very high.

At admission a detailed history was taken and clinical examination, anthropometry and relevant biochemical investigations were done for each child. All children were given oral albendazole at admission to render them parasite free and oral chloroquine (25 mg/kg). Children received parenteral Ampicillin (100 mg/kg/day) and Gentamycin (5 mg/kg/day). Clinical non-improvement or deterioration, after 48 h of treatment warranted change to ceftriaxone (75 mg/kg/ day) and amikacin (15 mg/kg/day). All children received a two-step mixed vegetarian diet from the hospital kitchen over 15 days, as per the recommended WHO guidelines (14).

Initial treatment phase (Day 1 to 7): Involved treatment of dehydration, correction of electrolyte imbalance, institution of appropriate antibiotics for associated acute infections and initiation of feeding using feeds providing about 80-100 kcal/kg/day.

Rehabilitation phase (Day 7 to 15): Involved increasing the calorie intake to 150-200 kcal/kg/day.

Of the total protein intake for each subject, 6 g were provided using either curd or LPC. The later was provided in the form of dried leaf powder in sachets such that three such sachets provided 6 g of protein. The subjects were required to consume three sachets of LPC per day by incorporating it in the dough used to make chapattis (bread) or in khichdi (a preparation of rice and pulse) or mixed with honey in subjects who could not consume chapattis.

LPC was prepared using leaves of Berseem by ultrafiltration and acid thermo coagulation (100g contains--344 calories; fat 22.5g, CHO 12.5g, fibre 1g, [beta]-carotene 86700 [micro]g, vitamin B complex- [B.sub.1] 0.5mg, [B.sub.2] 0.5mg, [B.sub.5] 24.2mg, [B.sub.6] 1mg, [B.sub.9] 330mg, Pantothenic acid 4.3mg, vitamin C 2.2mg, vitamin K 1mg, [Ca.sup.2+] 187mg, [P.sup.3+] 604mg, [Fe.sup.2+] 99mg, [Zn.sup.2+] 9mg, [Mg.sup.2+] 384mg, [Cu.sup.2+] 2.1 mg and [K.sup.+] 713mg). Curd was set in the hospital kitchen using the starter provided by National Dairy Research Institute, Karnal. 1g of curd contained [10.sup.8] colony forming units (cfu), each of Lactobacillus bulgaricus and Streptococcus thermophilus.

Curd (100g contains- energy 60cal, protein 3.1g, fat 4g, CHO 3g, [beta]-carotene 102 [micro]g, [B.sub.1] 0.05mg, [B.sub.2] 0.16mg, [B.sub.5] 0.1mg, [B.sub.9] 12.5 [micro]g, vitamin C 1mg, [Ca.sup.2+] 149mg, [P.sup.3+] 93mg, [Fe.sup.2+] 0.2mg, [K.sup.+] 130mg, [Na.sup.+] 32mg) was supplied to the subjects under refrigerated conditions. 200 g of curd was provided in two containers of 100 g each, which were consumed by the subjects with meals. Parents of the enrolled children were advised to refrain from giving any other fermented product or any additional micronutrient supplements to their children during the period of the study.

All selected parameters were recorded before intervention and after 15 days of treatment. Anthropometric measurements were done by one of us (PD) to avoid observer bias and measurements were repeated three times and the mean of two closest values was recorded. Weight was recorded to the nearest 25 g using a beam balance scale. Haemoglobin was estimated using freshly collected venous blood on a coulter in the hospital laboratory, which was, standardized regularly. The patient's serum were separated by centrifugation and stored at -20[degrees]C. Serum ferritin was estimated on stored sera using a kit (Spectro Ferritin Kit, Ramco Lab Inc., Huston, TX, USA) based on solid phase ELISA. The reference range of serum ferritin levels in children 6 months-15 yr is 7-140 ng/ml (15). The reference range of serum ferritin level below 12 ng/ml indicates depletion of body's iron stores. In patients with chronic infection, inflammation or malignancy serum ferritin level below 50ng/ml is associated with reduced or absent iron stores (15). CRP was measured on stored sera using a kit (AVITEX-CRP Latex Test, Omega Diagnostic Ltd., Scotland, UK) based on slide agglutination. The upper level for normality was taken as 6 mg/dl. The T cell subpopulations were estimated on freshly collected venous blood using the fluorescent automated cell sorter technique, at the AIDS Reference Centre, National Institute of Communicable Diseases, New Delhi. Fluorescent-tagged antibodies directed against T cell subpopulations were used and estimation was done using a flow cytometer (Becton Dickinson, Netherlands).

The study groups were compared with regards to nutritional parameters, haematological parameters and immunological parameters before and after intervention. The statistical analysis was done using Pearson's Chi-square test and student's unpaired and paired t-test. The level of significance was at P<0.05 and 95 per cent confidence interval.

Results

Of the 80 children enrolled in the study, 12 were excluded (7 in group A and 5 in group B). Six (3 in each group) were lost to follow up as they either absconded or left against medical advice. Four (3 in group A and 1 in group B) were excluded as they refused to consume the food supplement and two patients suffering from bronchopneumonia (1 in each group) died during the study. Thus, 32 patients in group A and 36 in group B completed the study. Both the groups were comparable with respect to age, sex and malnutrition characteristics. At the time of enrollment 60 per cent had bronchopneumonia, 37 per cent had acute gastroenteritis and 3 per cent had pyoderma.

The changes in nutritional parameters viz. weight, haemoglobin and serum ferritin following nutritional supplementation at the end of the study are shown in Table I. While a significant average increase in weight (P<0.001) was seen in both the groups, a mean fall in weight was seen in patients suffering from kwashiorkor. One patient in group A and 3 in group B were suffering from kwashiorkor. The mean fall in weight of these patients was -0.250 kg and -0.950 kg, respectively.

The haemoglobin rose significantly in both the groups after supplementation. There was a significant fall in serum ferritin levels in both the groups at the end of the study period (P<0.001) (Table 1).

Though all patients had clinical or laboratory evidence of infection, only 22 (32%) patients were CRP positive, initially. Of these, 8 were in group A and 14 in group B. After supplementation, One in group A and 5 in group B remained CRP positive. Of these, 4 patients were not positive for CRP at the beginning of the study.

The mean T cell subpopulations in children in both the groups were within the normal reference range for age as depicted in Table II (16). The changes in mean CD3, CD4 and CD8 counts in both the groups after treatment were not significant, after supplementation. However, there was a significant increase in CD4:CD8 ratio in both the groups after treatment (Table II).

Discussion

After 15 days of intervention there was increase in weight, and haemoglobin, with decrease in serum ferritin in both the groups. Similar results have been reported earlier (3,17,18). CRP was positive in one third of the patients only; CRP has been reported to have a sensitivity of 85 per cent and a specificity of 80 per cent as an acute phase protein in the presence of infection. Our findings suggest a possibility of blunting of the acute phase response in PEM, supporting earlier reports (19,20).

Though, PEM has been known to be associated with a decrease in absolute T lymphocyte count and a reduction in CD4:CD8 (21,22), these values were in normal limit in the present study. The rise in the CD4:CD8 ratio after nutrition intervention supported findings of Chandra (23) . In the present study such changes were observed in a much shorter span of treatment in both the groups. This may be attributed to the immunoenhancing effects of curd and the micronutrient-rich LPC. Improvement in the total, CD4 and CD25 T lymphocyte counts have been reported by Gill et al (24) in elderly volunteers following consumption of Bifidobacterium lactis HN019 for 3 wk. Devi et al (25) also showed that lactobacillus supplementation along with a protein rich diet for fifteen days improved the immune status of malnourished preschool children indicating its therapeutic role in PEM. LPC is inexpensive, thus more studies are needed to explore full potential of this low cost vegetable source.

In conclusion, our results showed that supplementation of diet with curd or leaf protein concentrate for about two weeks to children with moderate to severe malnutrition produced significant improvement in weight and haemoglobin levels. Ferritin was not found to be a reliable marker of iron status in the malnourished children. Also the response of CRP to acute infection was blunted in PEM. Immune recovery as assessed by CD4:CD8 can be accelerated in PEM by supplementing the diet with curd or leaf protein concentrate indicating that curd or leaf protein concentrate could be an inexpensive and effective food therapy in malnourished children.

Acknowledgment

Authors thank National Dairy Research Institute, Karnal for providing the starter for setting curd and Shrimati Jasbir Kaur, Senior dietician and Shrimati Kamlesh Sethi, Dietician, Department of Dietetics, Guru Teg Bahadur Hospital for providing curd and adding LPC in dietary items for our patients. Authors also acknowledge help of Dr C.K. Katiyar, Dabur Research Foundation Ltd, Sahibabad, UP for providing leaf protein concentrate. Thanks are due to the India National Science Academy, New Delhi for financial support.

Received May 31, 2006

References

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(2.) Chandra RK. Nutrition and the immune system: an introduction. Am J Clin Nutr 1997; 66 : 460s-3s.

(3.) Chevalier P, Sevila R, Sejas E, Zalles L, Belmonte G, Parent G. Immune recovery of malnourished children takes longer than nutritional recovery: implications for treatment and discharge. J Trop Pediatr 1998; 44:304-7.

(4.) Agarwal K N, Bhasin S K. Feasibility studies to control acute diarrhea in children by feeding fermented milk preparations Actimel and Indian Dahi. Eur J Clin Nutr 2002; 56 (Suppl 4) : 556-9.

(5.) Nagy S Telek L, Hall N T Berry R E. Potential food uses for protein from tropical & subtropical plant leaves. J Agric Food Chem 1978; 26 : 1016-8.

(6.) Ejot R, Mbiapo T, Fokou E. Nutrient composition of the leaves and flowers of Colocasia esculenta and fruits of Solanum melongera. Plant Foods Hum Nutr 1996; 49 : 107-12.

(7.) Oluwasola A J. Characterization of the leaf meals, protein concentrates and residues from some leguminous plants. J Sci Agr 2006; 86 : 1292-7.

(8.) Opii WO, Joshi G, Head E, Milgram NW, Muggenberg BA, Kleen JB, et al. Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: Relevance to Alzheimer's disease. Neurobiol Aging 2006; 19 : 1.

(9.) J Cinatl, Morgenstern B, Bauer G, Chandra P, H Rabenau, Doerr HW. Glycerrhizin, an active components of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361 : 2045-6.

(10.) Mehndiratta PK, Dewan V, Bhattacharya SK, Gupta VS, Maiti PC, Sen E Effect of Ocimum sanctum on humoral immune response. Indian J Med Res 1988; 87 : 384-8.

(11.) Kumawat N, Mathur R, Kohli GK. Impact of green leafy vegetable powder (alone/in combination with ascorbic acid) in prevention of anemia in young women. In: Abstracts: Nutrition goals for Asia- Vision 2020 IX Asian Congress of Nutrition; 2003 February 23-27, New Delhi, India. New Delhi: Nutrition Foundation of India, 2003 p. 220.

(12.) Gomez F, Galvan RR, Frenk S, Munoz JC, Chavez R, Vasgnez J. Mortality in second and third degree malnutrition. J Trop Pediatr 1956; 2 : 77-83.

(13.) Hamill PVV, Johnston FF, Grams W. Height and weight of children: United States, Vital Health Statistics Series 11, No. 104, Washington DC: US Government Printing Office; 1970.

(14.) World Health Organization. Manual of Severe Malnutrition: A Manual for Physicians and Other Senior Health Workers. Geneva: WHO; 1999.

(15.) Centers for Disease Control and Prevention. Recommendations to prevent and control iron deficiency in the United States. MMWR Morb Mortal Wkly Rep 1998; 47 (RR-3) : 1-29.

(16.) Coman-Bitter WM, de Groot R, van den Beemd R, Neijens HJ, Hop WC, Groenveld K, et al. Immunophenotyping of blood lymphocytes in childhood: reference values for lymphocyte subpopulations. J Pediatr 1996; 130 : 388-93.

(17.) Wickramsinghe SN, Gill DS, Broom GN, Akinyanju OO, Grange A. Limited value of serum ferritin in evaluating iron status in children with protein-energy malnutrition. Scand J Hematol 1985; 35 : 292-8.

(18.) Subotzky EF, Heese HD, Sive AA, Dempster WS, Sachs R, Malan H. Plasma zinc, copper, selenium, ferritin and whole blood manganese concentrations in children with kwashiorkor in the acute stage and during refeeding. Ann Trop Pediatr 1992; 12 : 13-22.

(19.) Doherty JF, Golden MH, Raynes JG, Griffin GE, McAdam KP. Acute-phase response is impaired in severely malnourished children. Clin Sci (Lond) 1993; 84 : 169-75.

(20.) Reid M, Badaloo A, Forrester T, Morlese JF, Heird WC, Jahoor E The acute-phase protein response to infection in edematous and nonedematous protein-energy malnutrition. Am J Clin Nutr 2002; 76 : 1409-15.

(21.) Parent G, Chevailier P, Zalles L, Sevilla R, Bustos M, Dhenin JM, et al. In vitro lymphocyte-differentiating effects of Thymulin on lymphocyte subpopulations of severely malnourished children. Am J Clin Nutr 1994; 60 : 274-8.

(22.) Fakhir S, Ahmad P, Faridi MMA, Rattan A. Cell-mediated immune responses in malnourished host. J Trop Pediatr 1989; 35 : 27-30.

(23.) Chandra RK. Numerical and functional deficiency in T helper cells in PEM. Clin Exp Immunol 1983; 51 : 126-32.

(24.) Gill HS, Rutherford KJ, Cross ML, Gopal PK. Enhancement of immunity in the elderly by dietary supplementation with the probiotic Bifidobacterium lactis HN019. Am J Clin Nutr 2001; 74 : 833-9.

(25.) Devi S, Devi PY, Siva Prakash M. Effect of Lactobacillus supplementation on immune status of malnourished pre-school children. Indian J Pediatr 1999; 66 : 663-8.

Reprint requests: Dr K.N. Agarwal, D-115, Sector 36, Noida, Utta radish 201301, India email: adolcare@hotmail.com/kna_ped@yahoo.com

Pooja Dewan, Iqbal Kaur *, D. Chattopadhya ([dagger]), M.M.A. Faridi & K.N. Agarwal ([double dagger])

Departments of Paediatrics & * Microbiology, University College of Medical Sciences, ([dagger]) AIDS Division, National Institute of Communicable Diseases, Delhi, & ([double dagger]) Indian National Science Academy, New Delhi, India
Table I. Weight, haemoglobin and serum ferritin before and after
supplementation

 Weight (kg)
 Curd group LPC group

Before supplementation 7.46 [+ or -] 2.15 6.42 [+ or -] 1.74
After supplementation 7.96 [+ or -] 2.43 6.84 [+ or -] 1.75
Change +0.50 +0.42
 P<0.001 P<0.001

 Hemoglobin (g/dL)
 Curd group LPC group

Before supplementation 9.33 [+ or -] 1.45 8.07 [+ or -] 1.73
After supplementation 9.63 [+ or -] 1.48 8.59 [+ or -] 1.36
Change +0.29 [+ or -] 0.68 +0.52 [+ or -] 0.92
 P<0.05 P<0.01

 Serum ferritin (ng/ml)
 Curd group LPC group

Before
 supplementation 195.56 [+ or -] 321.22 173.86 [+ or -] 288.05
After
 supplementation 50.87 [+ or -] 82.88 71.69 [+ or -] 154.94
Change -144.69 [+ or -] 292.74 -102.17 [+ or -] 288.29
 P<0.001 P<0.001

Values are mean [+ or -] SD.

n=32 in curd group

n=36 in LPC group

Table II. T cell subpopulations before and after supplementation

 CD3 (cells/[micro]l)
 Curd group LPC group

Before
 supplementation 2217.17 [+ or -] 938.28 2137.93 [+ or -] 959.15
After
 supplementation 2171.07 [+ or -] 956.69 2146.56 [+ or -] 657.57
Change -46.10 [+ or -] 608.489 +17.79 [+ or -] 871.07

 CD4 (cells/[micro]l)
 Curd group LPC group

Before
 supplementation 1166.10 [+ or -] 454.07 1078.44 [+ or -] 402.45
After
 supplementation 1220.17 [+ or -] 490.43 1209.97 [+ or -] 422.08
Change +54.07 [+ or -] 418.5 +82.16 [+ or -] 390.5

 CD8 (cells/[micro]l)
 Curd group LPC group

Before
 supplementation 913.14 [+ or -] 462.06 946.41 [+ or -] 440.62
After
 supplementation 859.55 [+ or -] 521.11 1027.12 [+ or -] 764.69
Change -53.59 [+ or -] 296.92 82.16 [+ or -] 390.50

 CD4/CD8 (cells/[micro]l)
 Curd group LPC group

Before
 supplementation 1.42 [+ or -] 0.54 1.29 [+ or -] 0.58
After
 supplementation 1.79 [+ or -] 0.92 1.55 [+ or -] 0.8
Change 0.37 [+ or -] 0.60 +0.33 [+ or -] 0.51
 P=0.001 P=0.001

Values are mean [+ or -] SD.

n=32 in curd group and n=36 in LPC group
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Author:Dewan, Pooja; Kaur, Iqbal; Chattopadhya, D.; Faridi, M.M.A.; Agarwal, K.N.
Publication:Indian Journal of Medical Research
Article Type:Clinical report
Date:Sep 1, 2007
Words:3451
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