A Community Based Study of Vitamin a and Vitamin E Status of Adolescent Girls Living in the Shire Valley, Southern Malawi

Home , Vitamin E

A community based study of vitamin A and vitamin E status of adolescent girls living in the Shire valley, Southern Malawi

G Fazio-Tirrozzo1, L Brabin1, B Brabin1, O Agbaje1, G Harper1 and R Broadhead2

1Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA, Liverpool, UK and 2University of Malawi, College of Medicine, Private Bag 360, Chichiri, Blantyre 3, Malawi

Objective: To assess vitamin A and E status and anaemia in non-pregnant Malawian adolescent girls. Design: A cross-sectional study in rural village communities in the Shire Valley, Southern Malawi. Subjects: Adolescent girls, n 118, aged between 10 and 19 y, 112 of whom were unmarried. Methods: Socio-demographic information was collected by questionnaire, heights and weights were measured. Vitamin A was assessed by the Modi®ed Relative Dose Response (MRDR) test, in addition to serum retinol values. Blood samples were collected 4±5 h after administration of 3,4-didehydroretinyl acetate. Retinol and a- tocopherol levels were measured by HPLC. Serum retinol results for non-pregnant girls were compared with values for 43 adolescent pregnant girls which were available from a previous study. Results: 26.6% of non-pregnant girls had serum retinol values < 0.70 mmol/L; 40.2% had an MRDR ratio > 0.060. In 59.3%, serum tocopherol levels were < 11.5 mmol/L and the tocopherol/cholesterol ratio was < 2.2 in 23.9%. 11.3% had a haemoglobin 12 g/dl. Vitamin A levels were signi®cantly related to age, and younger girls were more likely to be de®cient. Signi®cant correlations were found between serum retinol, MRDR values and serum tocopherol. Girls with a low body mass index for age had tocopherol cholesterol ratios < 2.2. Low serum retinol values occurred signi®cantly more often in stunted girls (P 0.01). Serum retinol values of adolescent pregnant girls were signi®cantly lower than those of non-pregnant adolescents (P 0.002). Conclusions: Vitamin A and E de®ciency and anaemia were common in adolescent non-pregnant girls, and thought to partly result from increased growth requirements. Girls who become pregnant at an early age are at risk of depletion of their nutritional reserves. Measures to reduce nutritional de®ciencies before the ®rst pregnancy are needed. Sponsorship: Shell Fellowship (Liverpool), European Union and Department for International Development. Descriptors: adolescent girls; serum retinol; MRDR; vitamin E; anaemia; growth

Introduction essential for pubertal growth and sexual maturation (Brabin & Brabin, 1992). There have been few studies of vitamin A or vitamin E in Vitamin A de®ciency is common in Malawi (Tielsch et adolescents, especially from developing countries. The al, 1986; Chirambo et al, 1986; Semba et al, 1994) where authors of a recent study of serum retinol and iron status studies have been conducted in children and adults but not among adolescent school girls in urban Bangladesh in adolescents. Vitamin A supplementation for children and (Ahmed et al, 1996) noted that they were unaware of any for women following delivery became government policy other similar information. Studies from developed countries in Malawi in 1992 (M'manga, 1994). Both vitamin A have focused on the pattern of various nutritional para- de®ciency and anaemia are common amongst pregnant meters during adolescence and the need for age-speci®c women living in the Shire Valley (Chimsuku, 1997; reference values (MichaeÈlsson et al, 1976; Widhalm et al, Brabin et al, 1998). There is no information on vitamin E 1985; Garry et al, 1987; Lockitch et al, 1988). Some of (tocopherol) status in this area. This paper reports a com- these studies have demonstrated that levels of plasma munity based pilot study to assess the nutritional status of retinol or its transport protein are higher during adolescence pre-pregnant rural adolescent girls. The objectives were to (Vahlquist et al, 1975). There is a correlation between measure vitamin A and E status, body mass index, stunting vitamin A utilisation and growth (Glover, 1983) and plasma and anaemia prior to the ®rst pregnancy. The hypothesis retinol concentrations are positively related to stage of was that girls are likely to become pregnant before their sexual maturation (Herbeth et al, 1991). Retinol and growth process is complete, when nutrient stores are low tocopherol are absorbed and transported by different and requirements will be high. mechanisms but are apparently correlated (Lockitch et al, 1988; Herbeth et al, 1991). It is likely that hormonal factors in¯uence the levels of both vitamins, especially during Methods adolescence and pregnancy (Yeung & Chan, 1975; Glover, 1983; Garry et al, 1987. These observations Study population In 1996, 118 girls were contacted in villages within the would suggest that vitamin A and perhaps vitamin E, are catchment area of Chikwawa District Hospital and Mon- tfort rural hospital, Nchalo, the site of a previous pregnancy Correspondence: Dr L Brabin. study (Verhoeff et al, 1998). People in this area live by Received 4 December 1997; revised 14 April 1998; accepted 9 May 1998 subsistence farming, mainly maize, sorghum, millet and Vitamin A and vitamin E status of adolescent girls G Fazio-Tirrozzo et al 638 vegetables, with some cash crops such as cotton and method developed by Tanumihardjo et al (1990). This groundnuts. Some families own cows and goats. The involved the use of two HPLC UV detectors connected in village leadership structure is a pyramidical organisation series, one set to monitor retinol and the other set to of Traditional Authorities with the Paramount Chief Lundu monitor a-tocopherol. Cholesterol was measured using a on top and chiefs of single villages at the base. Access to commerical assay kit (Wako Chemicals, UK). communities must be made through this organisation. The clinical ®eld of®cer (GFT) visited village communities to explain to village committee members, elders and senior Data analysis women why the medical team wished to screen adolescent, Data were analysed with SPSS for Windows Release 7 unmarried girls, to seek their permission and to request (1995) and Minitab for Windows Release 11.12 (1996). them to send girls for screening. This process was lengthy Initial examination of the data was done to process the data as Malawian villages remain traditional (Helitzer-Allen et into a suitable form for analysis. The distributions of all al, 1994) and are generally reluctant to agree to having variables were checked for normality, using Kolmogorov- blood samples drawn. Smirnov test for normality. Natural logarithm and square Surveys were carried out in four villages where an elder root transformed values were used respectively to reduce would make available a large compound where girls could the skewness of the MRDR and tocopherol : cholesterol assemble and wait. Groups of girls from neighbouring ratios. Distributions and summary statistics (means and villages came accompanied by their chiefs or an older standard deviations) for indicators of vitamin A, vitamin lady who supervised all procedures. The Modi®ed Relative E and anaemia were calculated. Pearson Chi-square ana- Dose Response (MRDR) Test (Tanumihardjo et al, 1996) lyses were done to examine the difference in proportions of was used to assess marginal vitamin A de®ciency. adolescent girls strati®ed between cut-off points of indica- Although regarded as the best tool currently available, it tors of vitamin A, vitamin E and haemoglobin levels in presents logistical dif®culties when used in the ®eld due to relation to menarche. Trend tests were used to examine the the need to administer the 3,4-didehydroretinyl acetate strength and the direction of associations for all biochem- (DRA) some 4±6 h in advance of taking a blood sample. ical indicators and haemoglobin in relation to different age It is also given with an oily meal or snack that is low in groups. Relative odds and Con®dence Intervals for low vitamin A content. Senior women were asked to prepare height-for-age (stunted) and low body mass index in rela- breakfasts to accompany adminstration of the 3,4-dide- tion to various indicators of vitamin A, vitamin E and hydroretinyl acetate, as well as a cooked meal at the end haemoglobin levels at different cut-off points were calcu- of the surveyÐwhich was a way of thanking girls and lated. Pearson's correlation test was performed to examine elders for their participation. Non-pregnant girls aged 10± the strength of relationships among different indicators of 19 y were included but only if they were prepared to spend vitamin A, vitamin E and haemoglobin. a minimum of ®ve hours in the compound to complete the Two provisional cut-off values for MRDR exist namely survey. In the time they waited, they ate breakfast, com- 0.03 for the United States and 0.06 for Indonesia (Tanu- pleted a questionnaire and received health education from mihardjo et al, 1994). In this study the 0.06 cut-off was two trained nurses. On several occasions a village youth used. The serum retinol cut-off for de®ciency was taken as band was hired to provide entertainment. Girls who < 0.70 mmol/L (Gibson, 1990). The concentration of total attended the survey could not be randomly selected. tocopherol in serum is the most commonly used index of vitamin E status. In adults serum total tocopherol concen- Study procedures trations < 11.6 mmol/L have been associated with > 5% The questionnaire collected data on socio-demographic haemolysis in the peroxide haemolysis test in adults and factors such as age, schooling, literacy, household socio- with low vitamin E status. Children have lower lipid economic status and domestic duties, and medical history, concentrations without evidence of vitamin E de®ciency including age of menarche, menstrual problems, pregnancy and for them a cut-off of 8.8 mmol/L is used (Gibson, 1990). history and treatment seeking behaviour. Cut-offs for adolescents are not well de®ned, and in this A supply of DRA was obtained from the Dept. of paper, wherever possible, a range of values is shown. Biochemistry and Biophysics, Iowa State University and Otherwise the adult cut-off of < 11.6 mmol/L is applied. used as recommended by this group (Tanumihardjo et al, Tocopherol:lipid ratios are the preferred measure of vita- 1990). The acetate was kept frozen until required where- min E status. For haemoglobin values the mean of two upon it was transplanted in a cool box and protected from samples was calculated and anaemia was de®ned as light. A single oral dose of 8.8 mmol/L DRA was adminis- 12.0 g/dl. tered directly into the mouth with a disposable pipette. Reference data for the British 1990 Growth Charts (Cole Blood samples were drawn 4±5 h after dosing and kept in et al, 1995; Freeman et al, 1995) were obtained from the amber vials in a cool box until they were returned to the Child Growth Foundation and precise age cut-offs were laboratory, where they were spun, serum separated and calculated. Weight and height measurements were con- frozen, and protected from light. Haemoglobin was mea- verted to body mass index using the standard formula sured in the ®eld using a spectrophotometer and samples weight/height2 (kg/m2). The second centile was used to were measured in duplicate. Height and weight were taken indicate low body mass index for age. The second centile with girls wearing minimum clothing, using a Minimetre height for age was used to de®ne stunting. scale against a ¯at wall, and Seca electronic weight scales Permission and ethical approval for the study were which were regularly checked. granted by the Malawian Health Sciences Research Com- Serum samples were returned frozen to the Liverpool mittee. Girls found to be anaemic at the time of the survey School of Tropical Medicine for further assays. Retinol, were treated, as were other illnesses like malaria. If 3,4-didehydroretinol (DHR) and a-tocopherol levels were necessary, girls were referred to the doctor at Montfort measured simultaneously, using a modi®cation of an HPLC Hospital, Nchalo. Vitamin A and vitamin E status of adolescent girls G Fazio-Tirrozzo et al 639 Results height for age) and 13.9% (16) had a low mean body mass- for-age (< 2nd centile). No girl with a body mass index One hundred and eighteen non-pregnant girls were below the 2nd centile had a normal tocopherol-cholesterol screened; all but six were unmarried. Most were from the ratio (> 2.2). Low serum retinol values but not marginal two predominant ethnic groups: Manganja (57) and Sena de®ciency, occurred signi®cantly more often amongst (43). Forty-four girls were aged 10±13 y, 59 were 14±16 y stunted girls (P 0.01). and 14 were 17±19 y (one age was unknown). 37 had Comparative serum retinol values were available for 43 attained menarche and mean age of menarche for these adolescent pregnant girls who had been screened in a girls was 14.4 y (s.d. 1.5). For ®ve girls, menarcheal status previous study. Their mean serum retinol value was was unknown. Many girls were illiterate: 37% (43) could 0.63 mmol/l (Æ 0.6) which was lower than the mean of read easily in the local language while the remainder could 0.92 (Æ 0.4) for non-pregnant adolescents (P 0.002). only read with dif®culty or not at all. All of them reported Thirty-two (74.4%) of pregnant adolescents had serum responsibility for a range of domestic tasks such as house- retinol values below 0.7 mmol/L. Seven pregnant adoles- work (99.1%) and farmwork (81.9%). In the farming cents were in their second or third pregnancy. Although not season 67.2% were expected to do manual work for at signi®cantly lower, the mean serum retinol values for these least four hours a day. girls was 0.47 mmol/L (Æ 0.3). Table 1 shows the distribution of values for serum retinol (n 113), MRDR ratios (n 112), serum tocopherol (n 113), tocopherol:cholesterol ratios (n 113) Discussion and haemoglobin (n 114). 26.6% (30) of girls had serum retinol values below 0.70 mmol/L compared to A large number of non-pregnant girls seen in this study 40.2% (45) with an MRDR ratio > 0.060. 59.3% (67) were anaemic and de®cient in vitamins A and E. Although girls had serum tocopherol levels < 11.5 mmol/L of which this could re¯ect the fact that the sample was small and 40.7% (46) were equal to or fell below the paediatric cut- self-selected, this seems unlikely given the even higher off value of 8.8 mmol/L. Tocopherol:cholesterol ratios levels of anaemia and morbidity which were previously below 2.2 were found in 23.9% (27). In 14.8% (17) of reported for pregnant adolescents from the same locality girls haemoglobin values were < 8.0 g/dl and only 11.3% (Brabin et al, 1997). This suggests that nutritional de®cien- had a normal haemoglobin value ( 12 g/dl). Positive blood cies commonly occur before the ®rst pregnancy and that smears for malaria were found in 15.9% (18) and 20.0% during pregnancy, nutrient levels are further diminished. In (22) reported blood in the urine, indicative of urinary Malawi many girls become pregnant shortly after schistosomiasis which is endemic in the area. 9.7% (11) menarche, following peak growth and a period of high had recently taken antimalarials and 21.9% (15), paraceta- nutritional demand. At the stage when many become mol. 2.6% (3) also reported taking vitamin A and 4.3% (5) pregnant, the growth process is not yet complete. Older took iron tablets in the last two weeks. nulliparous girls were found to be less often vitamin A Vitamin A levels were related to age (Figure 1) and de®cient than younger girls (Figure 1), perhaps because younger girls were more likely to have low serum retinol menarche was several years behind them. It is possible that values (P 0.012) and high MRDR ratios (P 0.013) than when vitamin A intakes are adequate and girls become older girls. Neither vitamin E de®ciency or moderately pregnant several years after menarche (as in most devel- severe anaemia (< 10.0 g/dl) was related to age. Girls who oped countries), vitamin A stores have time to replenish had attained menarche were less likely to be vitamin A before pregnancy. This is not the situation in Malawi, de®cient or to be moderately anaemic but these differences where the diet is poor and early marriage prevails. Of 43 were not signi®cant (data not shown). adolescents in their ®rst pregnancy for whom serum retinol The correlation between different measures of vitamin values were available, 32 (74.4%) had values < 0.7 mmol/L A, vitamin E and haemoglobin status are shown in Table 2. at the time of their ®rst antenatal visit. According to the Both measures of vitamin E status were highly correlated, MRDR results, 40.2% of pre-pregnant girls had low stores, as were serum retinol and MRDR values. Both measures of re¯ecting the marginal situation of girls who will soon vitamin A correlated with serum tocopherol but not with become pregnant. the tocopherol:cholesterol ratio nor with mean haemo- Vitamin E values were also low in this population globin values. (Table 1) although the signi®cance of vitamin E de®ciency Table 3 shows the relationship between the various is as yet unclear. In this study tocopherol:cholesterol ratios nutritional parameters and anthropometric status. Height below 2.2 were found more often in girls with a low mean and weight measurements were unknown for three girls. body mass index-for-age, that is in girls who were thin or Overall 33.9% (39) of girls were stunted (< 2nd centile possibly, chronically energy de®cient (Brabin et al, 1997)

Table 1 Distribution of values of vitamin A and vitamin E status and anaemia in adolescent girls

Serum retinol MRDR ratio Serum tocopherol Tocopherol/cholesterol ratio Haemoglobin mmol/L % (no.) % (no.) mmol/L % (no.) % (no.) g/dl % (no.)

< 0.35 2.7 (3) > 0.06 40.2 (45) 8.8 40.7 (46) < 2.2 23.9 (27) < 8.0 14.8 (17) 0.35±0.70 23.9 (27) < 0.06 59.8 (67) 8.9±11.5 18.6 (21) > 2.2 76.1 (86) 8.0±9.9 32.2 (37) > 0.70 73.5 (83) 11.6 40.7 (46) 10.0±11.9 41.7 (48) 12.0 11.3 (13) MeanÆ s.d. 0.92Æ 0.34 0.061Æ 0.039 10.02Æ 5.6 4.5Æ 3.1 9.9Æ 1.8 Vitamin A and vitamin E status of adolescent girls G Fazio-Tirrozzo et al 640

Figure 1 Vitamin A and E de®ciency and anaemia in relation to age.

Table 2 Correlation coef®cients for vitamin A and vitamin E indicators and haemoglobin in adolescent girls

Variable Serum retinol MRDR ratioa Serum tocopherol Tocopherol/cholesterolb Haemoglobin

Serum retinol Ð 70.672 0.246 0.091 0.161 P value Ð 0.00 0.01 0.34 0.09 MRDR ratio Ð Ð 70.257 70.029 70.029 P value Ð Ð 0.00 0.76 0.76 Serum tocopherol Ð Ð Ð 0.768 70.036 P value Ð Ð Ð 0.00 0.71 Tocopherol/cholesterol ratio Ð Ð Ð Ð 70.114 P value Ð Ð Ð Ð 0.24

aNatural log transformed values. bSquare root transformed values. Vitamin A and vitamin E status of adolescent girls G Fazio-Tirrozzo et al 641 Table 3 Distribution of values for vitamin A, vitamin E and haemoglobin according to whether girls were stunted or had a low body mass index (BMI) for age

Stunted Low BMI for age

<2nd centile >2nd centile Odds Con®dence <2nd centile >2nd centile Odds Con®dence Nutritional indicator % (n) % (n) ratio interval P-value % (n) % (n) ratio interval P-value

Serum retinol (mmol/l) (n 110) < 0.7 53.3 (16) 46.7 (14) 3.01 1.26±7.19 0.01 13.3 (26) 86.7 (26) 0.87 0.26±2.95 0.82 0.7 27.5 (22) 72.5 (58) 1.00 15.0 (12) 85.0 (68) 1.00 MRDR ratio (%) (n 109) > 0.06 37.8 (17) 62.2 (28) 1.34 0.60±2.97 0.47 11.1 (5) 88.9 (40) 0.60 0.19±1.87 0.38 0.06 31.2 (20) 68.7 (44) 1.00 17.2 (11) 82.8 (53) 1.00 Serum tocopherol (mmol/l) n 110 < 11.6 35.4 (23) 64.6 (42) 1.10 0.49±2.44 0.82 12.3 (8) 87.8 (57) 0.65 0.22±0.188 0.42 11.6 33.3 (15) 66.7 (30) 1.00 17.8 (8) 82.2 (37) 1.00 Tocopherol/cholesterol (ratio) (n 110) < 2.2 19.2 (5) 80.8 (21) 0.37 0.13±1.10 0.06 0.0 (0) 100.0 (26) a a a 0.02 2.2 39.3 (33) 60.7 (51) 1.00 19.0 (16) 81.0 (68) 1.00 Haemoglobin (g/dl) (n 115) < 12 35.3 (36) 64.7 (66) 1.82 0.47±7.03 0.38 10.8 (11) 89.2 (91) 0.19 0.054±0.70 0.01 12 23.1 (3) 76.9 (10) 1.00 38.5 (5) 61.5 (8) 1.00 aZero frequency observed (odds ratio not de®ned).

(Table 3). Stunting was more likely to be associated with Although their nutritional needs are high before pregnancy, serum retinol levels < 0.7 mmol/L. In adolescents, as in adolescent girls are excluded from current policies for children (Tarwotjo et al, 1992), inadequate stores of vita- vitamin A, mainly because of fears of vitamin A toxicity min A and perhaps vitamin E, could result in faltering if girls are inadvertently given a massive dose of vitamin A growth. Since serum retinol values do not re¯ect liver when pregnant and especially in the ®rst trimester (Hath- stores some girls may have had suf®cient stores to catch cock et al, 1990). However, fears of giving vitamin A to up on growth. However, stunted girls showing evidence of girls of reproductive age must be weighed against the marginal de®ciency (MRDR > 0.06) may not `catch up', adverse consequences of the failure to prevent vitamin A especially if they become pregnant and have no chance to de®ciency before the ®rst pregnancy. Supplementation replenish vitamin A stores. either during or after pregnancy comes too late to bene®t Many non-pregnant girls were moderately anaemic and mother and fetus in the ®rst pregnancy. One solution would 4.4% were severely anaemic (< 7.0 g/dl) compared to be to extend childhood vitamin A supplementation 11.8% of girls in the pregnancy study (Brabin et al, (200 000 IU periodically) up to at least the age of about 1998). Since vitamin A de®cient subjects have been 12 y, but this might be dif®cult to sustain in Malawi since found to be less responsive when administered iron many children do not attend even primary school and are (Mejia & Arroyave, 1982), recovery during pregnancy not easily reached. An alternative (or complementary) may be hampered in spite of iron supplementation, espe- strategy which we have initiated in this area, is the devel- cially since vitamin A supplementation is not given during opment of a community based literacy programme for pre- pregnancy. Serum retinol values of the 47 pregnant ado- pregnant adolescent girls through which nutritional infor- lescents remained similar at delivery to values at booking mation is given, and supplementation could be made (data not shown), a result which would argue for dealing available. We believe that programmes are necessary for with vitamin A de®ciency prior to the ®rst pregnancy. young girls, to facilitate their growthÐphysical, social and Where interactions between iron and vitamin A have intellectualÐbefore the long years of pregnancy and been described, it has often been in areas where malaria motherhood, and if possible, to encourage girls to delay is less intensely transmitted, (for example Indonesia and pregnancy till they are older and growth is complete. Guatemala). A signi®cant degree of anaemia in this Mala- wian population is malaria attributable and 15.9% of girls AcknowledgementsÐDr Gabriella Fazio-Tirrozzo was awarded a Shell had a positive malaria blood smear. S. haematobium infec- Fellowship from the Liverpool School of Tropical Medicine to help tion is also common. That much anaemia was infection- support her during this project. Additional funds were made available by related may explain why anaemia was not more prevalent the Tropical Child Health Group who had completed a study of malaria in girls who were stunted or had a low body mass index and anaemia in pregnant women in the Shire Valley with EU support. The (Table 3), nutritional states which would be more likely to remaining project costs were contributed by the Overseas Development result from chronic iron de®ciency. Administration (now DFID) through its Work Programme grant to Dr L Brabin. The ODA, however, takes no responsibility for the opinions expressed on this paper. Our special thanks go to the four study nurses Conclusions working on the project, Mrs Berlina Makwiza, Mrs Stella Mlanga, Mrs Nakoma and Miss Gwaza, and to Mr Richard Fudzulani and Mr Gibson Adolescent girls living in the Shire Valley experience a Ching'ani, the Malawian laboratory technicians who read the malaria high level of morbidity before and during pregnancy. slides. The driver, Mr Harry Banda, acted as translator and cultural Vitamin A and vitamin E status of adolescent girls G Fazio-Tirrozzo et al 642 mediator. We thank the Chikwawa District Commissioner, Mr Chiguduli, Lockitch G, Halstead AC, Wadsworth L, Quigley G, Reston L & Jacobson and most of all the Paramount Chief Lundu, Chief Katunga, the village B (1988): Age and sex-speci®c pediatric reference intervals and communities themselves and the girls who took time to participate. We correlations for zinc, copper, silenium, iron, vitamins A and E and gratefully acknowledge the support of the College of Medicine, Blantyre, related proteins. Clin. Chem. 34, 1625±1628. M'Manga R (1994): Success and failure of vitamin A capsule distribution and SUCOMA for technical support. during drought: results for Chikwawa and Machinga. Malaw. Med. J. 10, 16±18. References Mejia LA & Arroyave G (1982): The effect of vitamin A forti®cation of sugar on iron metabolism in pre-school children in Guatemala. Am. J. Ahmed F, Khan MR, Karim R, Taj S, Hyderi T, Faruque MO, Margetts Clin. Nutr. 36, 87±93. BM & Jackson AA (1996): Serum retinol and biochemical measures of MichaeÈlsson G, Vahlquist L, Mellbin JT & Bratt L (1976): Zinc and iron status in adolescent schoolgirls in urban Bangladesh. Eur. J. Clin. vitamin A. serum concentrations of zinc and retinol-binding protein Nutr. 50, 346±351. (RBP) in healthy adolescents. Scand. J. Clin. Lab. Invest. 36, 827±832. Brabin L & Brabin B (1992): The cost of successful adolescent growth and Semba RD, Miotti P, Chiphangwi JD, Saah AJ, Cannon JK, Dallabetta GA development in girls in relation to iron and vitamin A status. Am. J. & Hoover DR (1994): Maternal vitamin A de®ciency and mother-to- Clin. Nutr. 55, 955±958. child transmission of HIV-1. Lancet 343, 1593±97. Brabin L, Ikimalo J, Dollimore N, Kemp J, Ikokwu-Wonodi C, Babatunde Tanumihardjo SA, Koelner PG & Olson JA (1990): The Modi®ed Relative S, Obunge O & Briggs ND (1997): How do they grow: A study of Dose Reponse (MRDR) assay as an indication of vitamin A status in a south-eastern Nigerian adolescent girls. Acta Paediatr. 86, 1114±1120. population of well nourished American children. Am. J. Clin. Nutr. 52, Brabin L, Verhoeff FH, Kazembe P, Brabin BJ, Chimsuku L & Broadhead 1068±1072. R (1998): Improving antenatal care for pregnant adolescents in southern Tanumihardjo SA, Permaesih D, Dahro AM, Rustan E, Muhilal, Karyadi D Malawi. Acta Obstet. Gynecolog. Scand. 77, 402±409. & Olson JA (1994): Comparison of vitamin A status assessment Chirambo MC, Tielsch JM, West KP, Katz J, Tizazu T, Schwab L, techniques in children from two Indonesian villages. Am. J. Clin. Johnson G, Swartwood J, Taylor HR & Sommer A (1986): Blindness Nutr. 60, 136±141. and visual impairment in southern Malawi. Bull. WHO 64, 567±572. Tanumihardjo SA, Cheng J-C, Permaesih D, Muherdiyantiningsih, Rustan Chimsuku LHE (1997): The Pattern and Determinants of Anaemia in E, Muhilal, Karyadi D & Olsen JA (1996): Re®nement of the modi®ed- Pregnant Women and Their Infants in a Malarious Area. PhD thesis, relative-dose-response test as a method for assessing vitamin A status in University of Liverpool. a ®eld setting: experience with Indonesian children. Am. J. Clin. Nutr. Cole TJ, Freeman JV & Preece MA (1995): Body mass index reference 64, 966±971 curves for the UK 1990. Arch. Dis. Child. 73, 25±29. Tarwotjo I, Katz, J, West KP, Tielsch JM & Sommer A (1992): Xerop- Freeman JV, Cole TJ, Chinn S, Jones PRM, White EM & Preece MA thalmia and growth in pre-school Indonesian children. Am. J. Clin. Nutr. (1995): Cross sectional stature and weight reference curves for the UK, 1990. Arch. Dis. Child. 73, 17±24. 55, 1142±1146. Tielsch JM, West KP, Katz J, Chirambo MC, Schwab L, Johnson GJ, Garry PJ, Hunt, WC, Bandrofchak JL, Vanderjagt D & Goodwin JS (1987): Vitamin A intake and plasma retinol levels in healthy elderly Tizazu T, Swartwood J & Sommer A (1986): Prevalence and severity of men and women. Am. J. Clin. Nutr. 46, 989±994. xeropthalmia in southern Malawi. Am. J. Epidemiol. 124, 561±68. Gibson RS (1990): Principles of Nutritional Assessment. New York: Valhquist L, Rask P, Petersen A & Berg T (1975): The concentration of Oxford University Press. retinol-binding protein, prealbumin, and transferrin in the sera of newly Glover J (1983): Factors affecting vitamin A transport in animals and man. delivered mothers and children of various ages. Scand. J. Clin. Lab. Proc. Nutr. Soc. 42, 19±30. Invest. 35, 569±575. Hathcock JN, Hattan DG, Jenkins MY, McDonald JT, Sundaresan R & Verhoeff FM, Brabin BJ, Chimsuku L, Kazembe P, Russell WB & Wilkening V (1990): Evaluation of vitamin A toxicity. Am. J. Clin. Broadhead R (1998): An evaluation of the effects of intermittent Nutr. 52, 183±202. sulphadoxine-pyrimethamine treatment in pregnancy on parasite clear- Helitzer-Allen D (Ed) (1994): An investigation of community-based ance and risk of low birthweight in rural Malawi. Ann. Trop. Med. communication networks of adolescent girls in rural Malawi for HIV/ Parasitol. 92, 141±150. STD prevention messages. International Center for Research on Widhalm K, HoÈlzl M & Brubacher G (1985): Lipids, lipoproteins and Women, Women and AIDS Research Programme, Report Series No. alpha-tocopherol relationship and changes during adolescence. Ann. 4. Washington. Nutr. Metab. 29, 12±18. Herbeth B, Spyckerelle Y & Deschamps JP (1991): Determinants of Yeung DL & Chan PL (1975): Effect of a progestogen and a sequential plasma retinol, b-carotene and a-tocopherol during adolescence. Am. type oral contraceptive on plasma vitamin A, vitamin E, cholesterol and J. Clin. Nutr. 54, 884±889. triglycerides. Am. J. Clin. Nutr. 28, 686±691.