Within a nutritional study of the Family Health Project in Laos in 2000/2001, a total 1514 Laotian preschool children of the Province of Bolikhamxay were examined with regard to their nutrition, health and socio-economic situation in order to determine different causal factors between infants having a stunted growth and those with a normal growth. All children aged 0 to 59 months were eligible for entry in this study. The regional ethic committee approved the study. Participation was voluntary and informed consent was obtained from all mothers of participating children.

Laotian field workers collected information by questionnaire that included social-economic-education status, nutritional habits and queries to diseases.

Anthropometric measurements comprising height and weight, were measured by standardized equipment.

The daily food intake by quantity and amount was investigated over three consecutive using an estimated food record. The results were analysed with a computer programme called INMUCAL, which were developed by the Institute of Nutrition of the Mahidol University in Bangkok. The food frequency questionnaire (FFQ) determined the regular nutrition habits of the sample children over a period of time. Afterwards the daily consumption were calculated. The mothers were asked in an interview to estimate how often (daily, weekly, monthly, yearly) the child eats given food items from a list. The amount of a consumed food item the mother had to specify by using the cup and spoon set from Thailand. The food item from the given list were scaled with a digital scale according to the different sizes of the cups and spoon (SOEHNLE attache 8020, max. 2 kg, division 2 g, Germany).

Blood samples were obtained from a total of 81 children of different sex aged between 35 and 59 months. Data for the European control group (n = 101) has been published previously 13 and are included in this paper for reasons of comparison. Capillary blood was drawn into heparinized microcapillaries (60 μl) by puncturing the pad of the finger. To obtain plasma samples the microcapillaries were immediately centrifuged at the site for 4 minutes at 11500 g in a battery-driven miniature centrifuge (M 1101, Bayer Diagnostik GmbH, Germany). The plasma was transferred into opaque Eppendorf cups and stored in an ice-filled box. Within four hours they were transferred to the nearest hospital to be frozen at -15°C. From there they were shipped by air on dry ice to Germany where they were stored at -80°C. Analysis was performed within six months.

In order to compare the carotenoid and vitamin concentrations between capillary and venous blood, blood samples were obtained simultaneously from 14 individuals of different sex and age recruited from the institutional staff who gave informal consent to this sampling. Venous blood samples were taken from the antibrachial vein, capillary blood was drawn into heparinized microcapillaries (60 μl) by puncturing the pad of the finger. Samples were centrifuged and treated in a comparable way.

Carotenoids (lutein, zeaxanthin, α-carotene, β-carotene, β-cryptoxanthin, lycopene), α-tocopherol and retinol and retinyl esters were separated and quantified by reversed-phased HPLC 14. Due to the limited amount of sample available the method was modified. Briefly, 200 μl of ethanol were added to 20 μl plasma diluted with 180 μl H₂O. After vortexing for 30 sec, the samples were extracted twice with n-hexane (1 ml each time stabilized with 0.05 % butylated hydroxytoluene (BHT)) and vortexed for 3 min. The supernatants were removed, pooled, and evaporated under nitrogen and reconstituted in 50 μl isopropanol and injected (40 μl) into the HPLC-system (Waters, Eschborn, Germany). Accuracy and precision of the analyses were verified using a standard reference material (SMR 968a fat-soluble vitamins in human serum; National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA). Coefficient of variability over time using control plasma was less than 4 % for carotenoids, retinol, retinyl esters and α-tocopherol. The recovery rate was above 95% for all components.

Normally distributed data are expressed as means and standard deviation (SD), and non-normally distributed data are expressed as medians and ranges. Statistical analysis was performed with a paired t test. Probability values below 0.05 were considered significant.

Table 1 shows differences in plasma retinol, individual carotenoids (lutein, zeaxanthin, α-carotene, β-carotene, β-cryptoxanthin, lycopene) and α-tocopherol between plasma obtained from the same individuals either by puncturing of a vein or the capillaries of the fingertip. In general, all components were higher in plasma obtained from capillary blood. The most obvious differences were observed for α- and β-carotene. This observation is in accordance with a previous study in which, however, only β-carotene and α-tocopherol were investigated 21. The increased levels have been attributed to the reduced fluid in capillaries due to differences in hydrostatic pressure and colloid osmotic pressure in the capillaries. In this case, however, one would expect that all components measured would show similar quantitative changes. Nevertheless, the differences between plasma obtained from capillaries or veins are in the same order of magnitude. Thus, capillary blood sampling is a valid procedure to determine micronutrients such as retinol, α-tocopherol and carotenoids under field condition not only in developing countries but also in neonatal and pediatric medicine. With regard to the situation in developing countries, the procedure not only eliminates invasive blood sampling but also greatly reduces the equipment needed. Plasma can be obtained using a battery powered centrifuge and the small sample volumes can be stored and transported in well isolated storage containers even under less favourable conditions.

Table 2 summarizes the results on plasma concentrations of retinol, carotenoids and α-tocopherol and shows obvious differences between groups of children sampled in Europe and in Laos as well as sex differences in Laotian children. Studies that determined the levels of these components in plasma are limited and mostly restricted to developed countries. One reason for this, as has been pointed out earlier, are the difficulties to obtain appropriate samples. As reported for adults and children in Nepal and Africa the levels observed for carotenoids are substantially lower in children from Laos compared to slightly older children from Austria 13 or Germany 22 or from much older ones in the USA 23. Similarly low levels were found in children from Nigeria and Pakistan 2425. The differences in dietary carotenoid pattern are further strengthened by the striking differences in the carotenoid pattern. While in European infants the dominant carotenoid was β-carotene representing 52% of total carotenoids, in infants from Laos the dominant carotenoid was lutein with 62 % (P < 0.001) (Table 3). Both lower carotenoid plasma levels as well as differences in the plasma carotenoid pattern clearly reflects differences in the quantitiative and qualitative availability of carotenoids. High lutein levels are indicative of a consumption of dark green leafy vegetable and are frequently found as dominant carotenoid in plasma samples obtained from populations in developing countries 24. The relative contribution of individual carotenoids to total carotenoids in Laotian children was independent of origin or sex (data not presented).

The numerically smaller difference between the two groups was observed for plasma retinol, which can be explained by the homeostatic regulation of its plasma levels 26. In none of the children plasma levels were indicative of deficiency. Despite low vitamin A intakes, plasma retinol concentrations of an average of 0.91 – 1.07 μmol / l (95% C.I.) could be measured. Based on our protocol stunted children got 39 % and non-stunted children got 43 % of the recommended daily requirements. Other studies, however, show strong relationship between low vitamin A intake and low serum retinol concentrations 27. The cause of this contradiction might be the homoeostatic regulation of the retinol and possible sufficient amounts present in the main storage organ for vitamin A, the liver. As shown in Table 4 the effect of growth retardation and the area in which the children lived was of less importance for the level of retinol, carotenoids and α-tocopherol in plasma.

Table 2 shows that in children from Laos substantial sex differences exist with regard to the levels of retinol, carotenoids and α-tocopherol. In boys from Laos all components were significantly lower (P < 0.01). This is in contrast to our results from Europe and studies from Pakistan in children and from Algeria in adults, in which no such sex differences were observed 13222428. In the study conducted in Pakistan however, levels of lycopene and β-cryptoxanthine were below detection limit and β-carotene was in traces in only 8% of the children and in the study from Algeria only β-carotene and α-tocopherol were measured. The gender-specifically higher plasma concentrations of carotenoids in girls, however, have been reported by one study 29. Another study found slightly higher levels of carotenoids only significant for lycopene in boys 23. Based on the nutritional questionnaires taken in this study, this phenomenon cannot be explained by differences in nutrition. Additionally, the age of the children would exclude known effects of sex hormones on plasma levels of the investigated components.