Chronic stress, as a result of economic deprivation, inadequate nutrition and/or poor health status, experienced by children in low-income countries, can have detrimental consequences on their wellbeing, physical and mental health later in life [1
]. Although unclear, the mechanism underlying the relationship between chronic stress and adverse health outcomes is hypothesized to be an increased activation of the hypothalamic–pituitary–adrenal (HPA) axis [4
], which may trigger an elevation in circulating cortisol levels [5
Traditional biological specimens for cortisol measurement include saliva, blood or urine samples, each with its advantages and disadvantages [6
]. Serum and saliva are subject to major physiological daily fluctuations and thus reflect acute stress [7
], limiting their usefulness as indicators of stress levels over long periods of time [8
]. In addition, blood collection for assessment of serum cortisol is invasive and stressful for children and may increase circulating cortisol levels, while collection of saliva, although less-invasive, presents other challenges related to protocol compliance [9
] and collection methods [10
]. In contrast to serum and saliva samples, a 24-h urine collection is non-invasive and is used for measuring cortisol secretion over a prolonged period [11
]. However, a 24-h urine collection is time-consuming, laborious and presents some difficulties in regards to the analytical method and protocol compliance [8
There is growing evidence that hair offers an alternative suitable matrix for monitoring long-term cortisol profiles by capturing systemic cortisol exposure over longer periods of time [8
]. Hair is easy to collect, non-invasive, less stressful and unlike blood, saliva and urine which require frozen storage, can be stored at room temperature in a sealed envelope. Only a small amount of hair is required to assess information prior to hair collection [13
] and analysis of sections along the hair shaft enables differentiation between several time periods in the past [6
]. Furthermore, it has been reported that hair cortisol is stable over time [14
], has a high overall validity and test-retest reliability [15
] and is not found to be influenced by acute stress [16
While hair cortisol concentration (HCC) has been widely used as a biomarker of chronic stress in studies among adults [17
], it is only emerging now as a promising marker of childhood chronic stress. To date there is no consensus on the best approach to assess chronic stress in young children [19
]. The main focus of the existing literature on HCC as a biomarker of chronic stress in young children has been limited to exploring the main determinants of HCC in terms of chronic stress exposure, such as low socio-economic status and maternal distress [20
]. Here, we investigate the utility of HCC as a stress biomarker in the context of a randomized controlled trial of zinc supplementation among 6 to 23 months old children.
Adequate zinc nutrition is essential for human health because zinc is involved in numerous metabolic processes as a catalyst, a regulatory ion or structural element of proteins [21
]. Because zinc participates in so many metabolic pathways, zinc deficiency affects multiple physiological systems, children’s physical growth, the risk and severity of a variety of infections and pregnancy outcomes. Preventive zinc supplementation provided daily in form of tablets or syrup has been shown to reduce the incidence of diarrhea and acute lower respiratory infection among young children [22
] which in turn may reduce chronic stress. Zinc is also commonly included in multiple micronutrient powders (MNP), which have consistently been found to reduce the risk of anemia and increase iron status [26
]. However, a recent meta-analysis of MNPs found a significant association between MNP and an increase in diarrhea incidence [27
], which was assumed to be due to potentially adverse effects of iron. Lastly, therapeutic zinc supplementation, as recommended by the World Health Organization (WHO) and UNICEF [28
], shortens the duration of diarrhea and reduces the number of children whose diarrhea persists for 7 days [29
Hair zinc, a biological marker of long-term zinc nutrition, has been reported to be negatively associated with HCC among 4–6 years old children in Vancouver [30
], and the authors speculated that the inverse relationship between zinc and cortisol concentrations in hair may suggest some chronic stress in the study population. Whether the above-mentioned various regimens of zinc supplementation with or without other micronutrients have an impact on HCC is unknown. Therefore, in the present study, we aimed to: (1) assess the impact of different strategies for delivering supplementary zinc on HCC in young Laotian children, (2) examine the risk factors associated with baseline HCC and, (3) assess the association between endline HCC and the diarrhea and fever burden in the previous 3 months.
Growing evidence indicates that HCC may be an objective biomarker of chronic stress, and given that zinc supplementation has been reported to reduce the morbidity burden, we hypothesized that zinc supplementation may reduce chronic stress by affecting the immune function and the morbidity burden. Thus, we examined the impact of different strategies for delivering supplementary zinc on HCC among young Laotian children. In our study population of 512 young children aged 6–23 months at enrolment, we found no significant impact of the different zinc supplementation strategies on HCC. However, there was a marginal difference in change in HCC between the preventive zinc and therapeutic zinc groups after statistical adjustment. This difference was too small to be considered of health significance.
To explore whether HCC may be a useful indicator of nutritional and health status during early childhood, we examined the association between HCC and potential risk factors and found that children’s CRP, AGP concentrations, high sTfR levels and lack of consumption of iron rich foods were risk factors for elevated HCC at baseline. We are uncertain why or how breastfeeding would be inversely associated with HCC and assume that this may be a chance finding. In contrast, the child’s stunting and wasting status were not associated with baseline HCC. We also found no associations between maternal characteristics (age, education, BMI), household SES status and baseline HCC. Furthermore, we evaluated the association between HCC and longitudinal morbidity and found that diarrheal and febrile morbidity in the previous 3–4 months did not translate into an increased HCC at endline.
It is unclear whether our intervention had no impact on HCC because (1) the tested interventions had no impact on chronic stress, or (2) the study population was not under chronic stress or (3) HCC is not a sensitive marker of chronic stress in this population. We will address each of these points below.
As previously reported, the present study found no overall impact of PZ, MNP and TZ on growth and morbidity outcomes, such as diarrhea, fever and respiratory distress [41
], although TZ reduced the incidence and duration of diarrhea episodes in older children (>18 mo), but not in younger ones. This beneficial impact on diarrhea outcomes did not result in lower HCC in the TZ group, nor did age have a modifying effect on HCC. Although MNP had no overall impact on growth and morbidity, we found that the provision of MNP was associated with a small adverse effect on linear growth among non-anemic children and on diarrhea among children with inherited hemoglobin disorders [43
]. Neither the beneficial nor the adverse effects observed in the Lao Zinc Study were reflected in the HCC, which was possibly due to the small magnitude of the effect or the limited sample size of children with HCC results in the respective subgroups. Another reason for the lack of impact of our interventions could be the relatively short duration of the intervention. We followed our study participants for ~36 weeks. Although this duration is consistent with previous studies of preventive zinc supplementation that found an impact on growth and morbidity outcomes [26
], the duration may have been inadequate to affect an indicator of chronic stress such as HCC. To the best of our knowledge, this study is the first intervention study to assess the impact of different strategies for delivering supplementary zinc or other micronutrients on HCC, making any comparison with the existing literature difficult. More evidence from supplementation trials is needed to understand whether zinc and MNP supplementation has an impact on chronic stress as reflected in HCC.
Another reason for the apparent lack of impact of our interventions on HCC may be that our study population was not under chronic stress, and thus HCC would not respond to supplementation. There are currently no cut-offs for HCC to define chronic stress. Mean baseline HCC in our study population was 28.8pg/mg which is similar to mean HCC (27.33 pg/mg) in 12 month old infants in Boston [46
] but higher than the mean HCC of the majority of the previous studies [20
], although there is a wide range in HCC reported in the literature, ranging from 5.0 pg/mg in 4–5 years old Dutch children [47
] to 40.9 pg/mg in 1-9 years old German children [48
] and 535.3 pg/mg in 3–18 years old subjects from Central African Republic and Ethiopia [49
]. Unfortunately, there are no published studies on HCC in 0–2 years old infants in low- and lower-middle-income countries. There was a high prevalence of stunting (38.2%), underweight (27.2%), zinc deficiency (75.4%) and iron deficiency (26.1%) in the study population, suggesting that many of these children may experience other types of chronic stress.
Even though studies have used direct validation and different strategies to indirectly validate hair cortisol as a biomarker of chronic stress [50
], only weak correlations have been reported between psychological tests (perceived stress for example) and HCC [51
], raising some concerns regarding its utility and applicability as a sensitive marker of childhood chronic stress. The risk factors associated with HCC in children have been previously examined [20
] and mixed findings have been reported in regards to household socio-economic status [30
], ethnicity [30
], maternal education [30
], child age [47
] and gender [48
]. It is worth mentioning that studies which reported socio-economic status, maternal education and child age to be risk factors of HCC [30
] included older children than those in the present study. Child age was not an effect modifier in our study. However, the age range in the present study was limited compared to some of the prior investigations in other populations mentioned above. Although the present study was implemented in rural communities of central Laos, children included in our study were young (6–23 mo) and may have either not had enough cumulative exposure to the effects of low socioeconomic status, or may use effective buffering mechanisms such that these associations do not emerge.
The association between HCC and child nutritional and health status has not previously been examined. We found that HCC was not significantly associated with child anthropometric measurements such as length, weight, MUAC, LAZ, WAZ, WLZ, stunting, wasting and underweight. In addition, no previous study has examined the associations between HCC and morbidity and we found no association between HCC and morbidity in the 3–4 months prior to endline hair collection. Moreover, HCC was neither associated with factors such as food security, adequate dietary diversity and minimum meal frequency. This lack of association between HCC and child nutritional and health status questions and challenges the usefulness of HCC as a sensitive marker of child nutritional and health status.
Previous studies found a high variability in HCC among different age groups [20
]. Hair sample collection method is unlikely to be a source of variability between our findings and other studies. As in other studies [58
], we cut hair in accordance with the guidelines published by the Society of Hair Testing [60
] from above the nape of the neck as close to the scalp as possible. Hair from this region has been reported to be the standard for hair analysis and to have higher cortisol levels compared with hair from other regions [61
]. However, cortisol in hair is usually quantified using enzyme linked immunosorbent assay (ELISA) and high performance liquid chromatography-mass spectrometry (HPLC/MS) [62
] and given the multitude of commercially available cortisol kits, the HCC detection method is more likely to be a source of the variability found. Other potential biases of the study could include the color of hair, but considering that the study was implemented in Laos, there was very little diversity in hair color. Data on hair care characteristics in terms of washing frequency, and use of hair products or type of hair products were not collected mainly because of the age-range of our study participants (6–32 months old). Previous studies that collected data on hair care characteristics were done in adults [63
] or in older children (4–14 years old) [47
] and findings from these studies have reported that HCC were not affected by hair color, hair washing frequency or use of hair products.
A notable strength of this study is its randomized placebo-controlled trial design, the high participation rate and the rigorous data collection, which included both regular training of data collectors and frequent supervision. The vast amount of collected data allowed the investigation of potential risk factors associated with HCC. This study also has some limitations. Adherence to the supplementation was based on weekly caregiver reports, which has been shown to be unreliable [64
]. However, plasma zinc concentrations increased in the PZ and the MNP groups and ferritin concentrations increased in MNP group as expected and thus suggest that adherence was adequate [41
]. In addition, hair samples were collected from a convenience sample for logistical reasons. However, hair collection was implemented among all four intervention groups simultaneously until the respective target sample size was achieved [31