Effects of Probiotics and Calcium Supplementation on Growth, Development, and Metabolic Parameters

Stunting is still a major problem in developing countries, including Indonesia, and has been associated with impaired development. Stunted children have also a higher risk of metabolic syndrome in adulthood. The gut microbiota, as a part of intestinal integrity, may promote intake of nutrient during childhood. Probiotics supplementation may optimize the balance of gut microbiota and further improve child growth during the window period. Furthermore, calcium could also improve child growth by increasing the resistance to intestinal infection. However, the long-term effects of gut microbiota optimization during childhood using probiotics and calcium on growth, development, and the metabolic condition has not widely studied.

This study was a 10-year follow-up study of randomized controlled trials of 6-month probiotics and calcium supplementation of Lactobacillus reuteri DSM 17938 or Lactobacillus casei CRL 431 in children aged 1-6 years (n=494) living in urban low socioeconomic communities of East Jakarta in 2007-2008. We re-enrolled the subjects to assess the long-term effects of probiotics and calcium supplementation on growth, development, and metabolic parameters at the age of 11-17 years.

Healthy children (n = 238) were included in this follow-up study. Each subject previously assigned to one intervention group: low-lactose milk with a low calcium content (LC = 53), a regular calcium content (RC = 70), regular calcium with L. reuteri DSM 17938 (n = 55), and regular calcium with L. casei CRL 431 (n = 60).

Anthropometric measurements were performed by measuring the weight and height of the subjects, and further combining the results to report BMI-for-age z-score. Height was also plotted to the chart of the World Health Organization (WHO) Child Growth Standards. Gut integrity was assessed with the lactulose-mannitol ratio using high-performance liquid chromatography (HPLC) method. Cognitive function, symptoms related to depression, behaviour, and serum brain-derived neurotrophic factors (BDNF) of the adolescents were evaluated used to investigate the effects of supplementation on development. We quantified lipid profile and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) as metabolic parameters using the vein blood sample. The metabolic parameters were measured in at least three groups of intervention.

Eligibility Criteria:
Inclusion Criteria at the previous clinical trial:

* Apparently healthy children aged 1-6 years, with emphasize on aged 2-5 years, living permanently in low socio-economic urban areas of East Jakarta for at least six months
* Parents are willing to sign the informed consent and give the supplements to the children for six months
* Capable and willing to drink liquid milk with a straw (acceptance to be tested at the screening by providing a sample drink and placebo straw to be consumed under supervision for two days)

Exclusion Criteria at the previous clinical trial:

* Calcium intake exceeding 75% of the Recommended Dietary Allowance (RDA) for calcium (\<375 mg/d) based on a Food Frequency Questionnaire
* Currently breastfed children
* Siblings of already included children that are living in the same household, except if it is a twin sibling
* Severely malnourished with or without edema (weight-for-height Z-score of \<-3.00 SD)
* Symptoms of chronic/congenital diseases and disabilities, suspected Tuberculosis by clinical examination, and/or history of allergic disease.
* Taking (any) antibiotics during 2 weeks prior to the start of the study (children will be included after 3 weeks of last antibiotic intake
* Participation in another clinical trial at the same time or 2 months prior to the start of this study
* Both mothers and other caregivers present in the family are illiterate

Inclusion criteria of follow-up study:

* Healthy adolescent participated in the previous trial study willing to take part in the follow-up study

Exclusion criteria of follow-up study:

* History of type 1 diabetes
* Taking the cholesterol-lowering drug, an anti-diabetic drug, or oral corticosteroid for more than 2 consecutive weeks in the last 3 months
* Pregnant