Treatment for Iron Deficiency Anaemia with a Combined Supplementation

Home , Iron deficiency, Zinc gluconate

Treatment for iron de®ciency anaemia with a combined supplementation of iron, vitamin A and zinc in women of Dinajpur, Bangladesh

P Kolsteren1*, SR Rahman2, K Hilderbrand2 and A Diniz3

1Institute of Tropical Medicine, Antwerp, Belgium; 2Dinajpur Medical College, Dinajpur, Bangladesh; and 3 Universidade Federal da Paraiba, Brazil

Objective: The study was set up to determine to what extent the addition of a supplement of vitamin A alone or in combination with zinc would improve standard iron treatment and correction of iron de®ciency anaemia. Design: 216 non-pregnant anaemic women of 15 ± 45 years of age with haemoglobin levels 100 g=l were randomly assigned to three treatment groups. One group (A) received iron alone, a second group (B) received iron and vitamin A, and a third group (C) received iron, vitamin A and zinc. Every woman was given one iron capsule per day for 60 days as FeSO4 containing 60 mg of elemental iron. In addition, groups B and C received 200 000 IU of vitamin A, given as a supervised dose, on the ®rst day of the treatment after collection of the blood sample. Group C received one zinc tablet per day for 60 days as zinc gluconate containing 15 mg of elemental zinc. Setting: The north-western part of Bangladesh in the urban slums of Dinajpur district between February and August 1995. Subjects: To select women with a haemoglobin level of 100 g=l, all the women of four randomly selected municipal slums of the district in the targeted age group (328) were invited to take part in the study. Blood samples were analysed for haemoglobin, serum iron, total iron binding capacity (TIBC), ferritin, retinol and zinc. Results: Out of the 328 women screened, 254 (77.5%) had a haemoglobin level 100 g=l and 322 (98%) 120 g=l. The three treatment schedules signi®cantly increased haemoglobin levels and improved iron parameters, except for serum iron in the group who received iron alone. The group who received iron, vitamin A and zinc responded best with an increase in haemoglobin of 17.9 g=l as compared to the group receiving iron alone (13.4 g=l). Iron and vitamin A treatment gave an intermediate response of 15.9 g=l. However, these differences are only statistically signi®cant only for the group who received iron, vitamin A and zinc and only for the increase in haemoglobin, P 0.03. Conclusion: The results are suggestive that the addition of vitamin A and zinc to the treatment for anaemia can increase haemoglobin levels more than with iron alone. Sponsorship: The present study was ®nanced by grant FWO 1.5.092.96 and Nutrition Tiers Monde. Descriptors: Bangladesh; iron de®ciency; vitamin A; women; zinc

Introduction factors such as poor compliance, the long duration of the treatment, side-effects and, in certain cases, insuf®cient Iron de®ciency, and in particular iron de®ciency anaemia, health system coverage (ACC=SCN1991). The correction remains a nutritional disorder of major importance. So of the de®ciency can also be hampered by the effect of much so that the Montreal meeting on micronutrients of concomitant nutrient de®ciencies (West, 1996). October 1991 and the international conference on nutrition Strong correlations between vitamin A status and hae- held in Rome in 1992, called for more efforts to control moglobin levels have been demonstrated (Suharno et al, vitamin A de®ciency and reduce iron de®ciency before the 1993). Iron de®ciency is more easily corrected when there end of the decade. It is estimated that more than 2 billion is no vitamin A de®ciency (Hodges et al, 1978; Mejia & people suffer from iron de®ciency. Women in particular are Chew, 1998; Suharno et al, 1993). Prolonged vitamin A affected and, although the prevalence ¯uctuates widely de®ciency in animals negatively affects haematopoiesis, between countries, it is estimated that 40 ± 50% of adult with a gradual replacement of the bone marrow by ®brous women in developing countries suffer from iron de®ciency tissue (Hodges et al, 1978). A depression of iron uptake by anaemia (WHO=FAO, 1992). the bone marrow (Beyen et al, 1992; Sijtsma et al, 1993) Notwithstanding the fact that the diagnosis of anaemia is was postulated to be one of the mechanisms in these fairly simple and the treatment cheap, the prevalence of studies. A food forti®cation trial in Guatemala with vitamin anaemia remains high. This is due to a combination of A and a supplementation study with vitamin A alone in Indonesia reported a bene®cial effect on haematopoiesis and haemoglobin status (Mejia & Arroyave, 1982; Bloem *Correspondence: P. Kolsteren, Nationalestraat 155, 2000 Antwerpen, Belgium et al, 1990). Haemoglobin levels increased through mobi- Received 20 November 1997; revised 11 August 1998; accepted 25 August lisation of iron stores. Vitamin A=zinc supplementation and anaemia P Kolsteren et al 103 Zinc also interacts with vitamin A. Zinc de®ciency examined further. Women were excluded from the study on decreases levels of serum albumin, pre-albumin and trans- the basis of the following criteria: suffering from acute or ferrin. Pre-albumin levels increase rapidly with a zinc chronic diseases or in¯ammatory conditions; acute or supplementation of 10 ± 15 days in zinc-de®cient subjects chronic blood loss or diseases such as aplastic anaemia, (Bates & McClain, 1981). This effect is probably mediated thalassaemia or haemolytic anaemia; intake of a megadose through the effect of zinc on protein synthesis, most notice- of vitamin A within the last 3 months; presently under iron able in the effect of zinc de®ciency on the synthesis of tablet supplementation; any drug consumption other than retinol binding protein (Smith et al, 1973). Zinc was found paracetamol and=or antihistamines for minor ailments; to be necessary for normal mobilisation of vitamin A from blood transfusion or blood donation within the last 4 the liver and for maintaining normal serum levels. A study months; and pregnancy. from India among malnourished children supports this concept. The children showed a signi®cant increase of Enrolment plasma vitamin A and retinol-binding protein in response Out of 328 women screened, the ®rst 216 meeting the to zinc supplementation (Shingwekar et al, 1979). In a criteria participated in the study. All the women were study performed in Thailand, no effect on retinol binding informed about the objectives and operational processes protein or serum retinol was noted when zinc was added to of the study in group meetings. Verbal consent was the vitamin A supplementation as compared to a group of obtained individually and all transport costs were reim- children who received vitamin A alone (Udomkesmalee et bursed. The date of last menstruation was obtained to al, 1992). In this last study, however, it is not clear whether ensure non-pregnancy, which was rechecked in the subse- the children were signi®cantly zinc de®cient or to what quent months of drug supplementation. degree. It can therefore be proposed that zinc, through its The ethical committee of the Institute of Tropical effect on retinol mobilisation, can affect utilisation of iron Medicine of Antwerp approved the study. stores and so affect iron de®ciency anaemia. Considering that iron de®ciency anaemia is a major Anthropometric measurements problem and that the treatment is often unsuccessful, the Body weight (Wt) was measured with women wearing present study was set up to determine to what extent the usual light clothing, to the nearest 0.1 kg, using an ordinary addition of a supplement of vitamin A alone or in combina- bathroom scale. Height (Ht) was measured to the nearest tion with zinc would improve standard iron treatment and 0.1 cm by ®xing a measuring tape to the wall and having correction of iron de®ciency anaemia. women stand barefoot on a concrete ¯oor. Body Mass The study took place in Bangladesh where iron de®- Index (BMI) is calculated as Wt=Ht2. Measurements of ciency anaemia is very prevalent and where both vitamin A height were taken in duplicate by the same person, and the and zinc de®ciency have been documented (Simmer et al, average was calculated. 1990; Zeitlin et al, 1992; Baqui et al, 1994). Drug supplementation Each woman was given one iron capsule per day for 60 Subjects and Methods days as FeSO4 containing 60 mg of elemental iron (Bengal Non-pregnant women (n 328) between 15 and 45 years of Tech Pharmaceuticals, Bangladesh). A plastic container age were screened for anaemia. The ®rst 216 women with a was provided with 30 capsules during the blood sample haemoglobin level 100 g=l were randomly assigned to collection sessions at the start and after one month. In three treatment groups. One group received iron alone (A; addition, groups B and C received 200 000 IU of vitamin A n 72), a second group received iron and vitamin A (B; (Hoffman-La Roche) given as a supervised dose, on the n 72) and a third group received iron, vitamin A and zinc ®rst day of the treatment after collection of the blood (C; n 72). It was not considered ethical to include a sample. Group C received one zinc tablet per day for 60 placebo group as control. To perform randomisation, num- days as zinc gluconate containing 15 mg of elemental zinc bers from 1 to 216 were drawn out of a bag and allocated (Laboratory of Pharmaceutical Technology, University of alternately to treatment groups to end up with groups of Gent, Belgium). The tablets were packed in strips of 30 equal size. The sample size was calculated to demonstrate a tablets and were given together with the iron tablet dis- difference of 5 g=l haemoglobin between two groups with tribution. Instructions were given to take the zinc and iron an alpha error of 5% and a beta error of 10%. The standard tablets with separate meals. Drug compliance was super- deviation was estimated from the literature as being 10 g=l. vised by twice-weekly household visits by two female slum The calculated sample size was in¯ated to allow 30% health workers by asking whether the tablets had been defaulters. Blood samples were taken at the start of the taken the days before (iron and zinc where applicable). The study, and at the end of 60 days treatment. The study was activities of the health workers were also directly super- conducted in the north-western part of Bangladesh in the vised by household visits by one of the authors. urban slums of Dinajpur district between February and August 1995. Serum sample collection Blood collections were carried out in a clinical laboratory Screening on the ®rst day of treatment before the start of medication To select women with a haemoglobin level of 100 g=l, and at the end of 60 days of treatment. Women still all the women of four randomly selected municipal slums anaemic at the end of the study continued to receive iron areas of the district in the targeted age group (328) were treatment for one more month. In each round, an average of invited to take part in the study. Blood was collected by 5 ml of venous blood was drawn by vacutainer syringes ®nger prick for haemoglobin estimation by the cyano- under cover of light protective aluminium foil, from which methaemoglobin method with 2 ± 4 h (Lynch et al, 1969). 20 ml was transferred immediately for the determination of Every woman with a haemoglobin value below 100 g=l was blood haemoglobin levels by the cyanomethaemoglobin Vitamin A=zinc supplementation and anaemia P Kolsteren et al 104 method. Serum was separated after centrifugations, from same reason with the Kruskal ± Wallis test. Where the which 0.5 ml serum was transferred to an aluminium foil- parameters were normally distributed and more than two covered test tube for determination of the retinol, which groups needed to be compared, one-way analysis of var- was then deep frozen below 20C. To the rest of the iance was used. Serum ferritin differences between begin- serum, 3 ± 5 minute crystals of sodium azide were added ning and end are calculated and tested after natural and also frozen ( 4C to 12C). These were used for all logarithmic transformation given their non-normal distribu- other determinations in the study. All the plastic test tion. Comparisons between the group who received iron tubes, tips, syringes and needles used were warranted alone and the two others were made using the paired t-test. (Sarsted Ltd, Germany) to contain minerals in such a negligible amount that they would not in¯uence the analysis. Results Out of the 328 women screened, 254 (77.5%) had a Biochemical analysis haemoglobin level 100 g=l and 322 (98%) 120 g=l. Transferrin was analysed by an immunochemical reaction The turnout for screening was more than 90% of the target with the Behring Nephelometer 100 (Germany) using population. protein standard, control and reagent from Behring in Of the 216 women who participated in the trial 171 are accordance with the New International Standardisation included in the analysis. The defaulter rates are comparable IFCC=BCR=CRM 470 (International Committee for Stan- in the three groups: A 22%, B 21%, C 20%. The reasons for dardization in Haematology, 1967). Estimation of serum exclusion were illness during the treatment period (n 9), iron was done by an automated determination technique pregnancy (n 2), haemolysis of blood sample (n 1), using clinical chemistry slides in Ektachem 700XR from refusal to participate further in the study (n 13) and Johnson & Johnson clinical diagnostics. Ferritin was deter- poor compliance, i.e. no treatment taken more than 10% mined quantitatively in an immunoenzymatic assay (Sorin of the time (n 20). ETI Lab, Italy). Total iron binding capacity (TIBC) was Table 1 shows the composition of the three participating determined by saturating the transferrin with iron, remov- groups at the start of the treatment. The three groups were ing the excess from serum and estimating iron with an comparable in initial haemoglobin, serum retinol, zinc, automated technique (method of Caraway, modi®ed by Zak serum ferritin, transferrin saturation, total iron binding & Epstien). Serum retinol was determined by high-pressure capacity and serum iron. The mean age (standard deviation) liquid chromatography (HPLC) (Arroyave et al, 1982), and was 28.8 years (7.2) for group A, 28.0 years (7.9) for group serum zinc by atomic absorption spectrophotometry. B and 28.3 years (7.7) for group C. The mean BMI (s.d.) for Iron indices were analysed in the clinical laboratory of group A was 19.1 kg=m2 (2.6), for group B 19.3 kg=m2 the Institute of Tropical Medicine, Antwerp, Belgium. (3.1), and for group C 18.9 kg=m2 (3.0). Comparison of the Retinol and zinc estimations were done in the laboratory three groups by a one-way analysis of variance showed that of the biochemistry and nutrition department of the Inter- they were not signi®cantly different (F probability for Age national Centre for Diarrhoeal Disease Research, Bangla- was 0.86 and for BMI was 0.75). The body mass index in desh (ICDDR, B). all groups was rather low and overall 44.4% of the women had a BMI below 18.5 and are to be considered under- Statistical analysis weight (James et al, 1988). The prevalence of thinness in Differences between biochemical parameters at the begin- all three groups is comparable; in group A it was 46%, in ning and at the end within the treatment groups were tested group B 37% and in group C 50% (w2 2.15, P 0.43). by the Wilcoxon signed rank test given that we are dealing The serum retinol values are well within the normal with an anaemic population and the distributions of the range, with only one value in the de®ciency range (below biochemical parameters are not Gaussian. The biological 0.35 mmol=l) and 6% having low values ( < 0.70 mmol=l) baseline data between the three groups were tested for the (Arroyave et al, 1989).

Table 1 Effect of supplementation with vitamin A, iron and zinca

Total iron Haemoglobin Retinol Zinc Ferritin Transferrin binding capacity Serum iron (g=l) (mmol=l) (mmol=l) (mg=l) saturation (%) (mmol=l) (mmol=l)

Iron (n 56) Baseline 92.9 (9.8) 1.45 (0.6) 14.4 (3.3) 34.1 (23.6) 18.3 (9.5) 65.8 (12.5) 11.5 (5.7) 8 weeks 106.2 (9.6) 1.42 (0.6) 13.5 (2.9) 54.1 (40.9)22.0 (9.7) 60.5 (11.7) 12.9 (5.4) Pb < 0.001 0.69 0.097 < 0.001 0.002 < 0.001 0.024 Iron vitamin A (n 57) Baseline 89.5 (11.9) 1.30 (0.5) 14.2 (2.6) 33.9 (41.7) 15.2 (9.1) 62.3 (12.6) 9.08 (5.8) 8 weeks 105.4 (8.0) 1.39 (0.4) 14.2 (2.7) 47.7 (40.9)20.9 (11.6) 59.7 (12.0) 12.0 (5.7) Pb < 0.001 0.16 0.86 < 0.001 < 0.001 0.04 0.001 Iron vitamin A zinc (n 58) Baseline 88.5 (11.4) 1.44 (0.6) 14.1 (0.3) 31.5 (30.6) 14.3 (8.3) 64.5 (13.3) 9.2 (5.5) 8 weeks 106.5 (9.9) 1.47 (0.5) 14.3 (0.3) 47.5 (41.4)20.3 (10.7) 60.3 (11.7) 11.8 (5.8) Pb < 0.001 0.17 0.68 < 0.001 < 0.001 0.004 0.001 Comparison of baseline P 0.13 P 0.35 P 0.87 P 0.47 P 0.054 P 0.32 P 0.02 data between the three groups, Kruskall ± Wallis

aValues are means with s.d. in parentheses. bWilcoxon signed rank test. Vitamin A=zinc supplementation and anaemia P Kolsteren et al 105 Table 2 Mean (s.d.) differences in biochemical parameters between baseline and post-intervention values

Iron Iron and vitamin A Iron, vitamin and A and zinc (n 57) (n 57) z (n 58) f

Haemoglobin (g=l) 13.4 (9.6) 15.9 (8.5) P 0.14 17.9 (13) P 0.03 Retinol (mol=l) 0.03 (0.4) 0.09 (0.4) P 0.14 0.04 (0.5) P 0.53 Zinc (mmol=l) 0.09 (4.4) 0.02 (3.9) P 0.26 0.02 (4.3) P 0.19 Ferritin (mg=l) 19.9 (27.6) 13.3 (24.5) P 0.95 15.73 (24.8) P 0.69 Transferrin saturation (%) 3.61 (10.4) 5.7 (10.9) P 0.32 6.08 (10.0) P 0.21 Total iron binding 4.96 (9.0) 2.57 (10.5) P 0.20 4.27 (11.5) P 0.73 capacity (mmol=l) Serum iron (mmol=l) 1.38 (6.5) 2.87 (6.2) P 0.23 2.84 (5.6) P 0.30

z, Probability of paired t-test on the difference compared to the group who received iron alone. f, Probability of paired t-test on the difference compared to the group who received iron alone.

Serum ferritin levels were below 12 mg=l for 23% of the et al, 1996) owing to their different sensitivity and speci- women. Transferrin saturation was found to be abnormal ®city. Punnonen and coworkers (Punnonen et al, 1997) ( < 16%) in 53% of the analyses and 63% had low serum estimated that in anaemic patients a cutoff limit of 41 mg=l iron levels ( < 11.6 mmol=l) (Guyatt et al, 1992). for ferritin provides the optimal diagnostic ef®ciency for The three treatment schedules signi®cantly increased iron de®ciency. In our population, 77% of the women can haemoglobin levels and improved iron parameters, except be considered iron de®cient on the basis of that criterion. for serum iron in the group who received iron alone (Table Vitamin A de®ciency does not appear to be a very 2). Retinol levels did not alter over the 8-week period even serious problem in the studied populations, with 6% of in the groups who received a supplement. Zinc values are the women having low serum retinol values. The expected within normal range and again show no increase in the bene®t of a vitamin A supplement could therefore have zinc-supplemented group. Whereas all women had Hb been less than in a more de®cient population. This also values below 100 g=l at the beginning of the study, 74% explains the observation that retinol values did not change. had Hb values above 100 g=l at the end. In 36% it had In mild de®ciency states, the supplements are mobilised to increased above 110 g=l and only 9% were in the normal replenish the reserves with little change in serum retinol range. No difference in prevalence could be found between values (McLaren & Frigg, 1997). the three groups. The studies that found an effect of a vitamin A supplement on haemoglobin values gave supplements on a daily basis, whereas in the present study only one megadose was Discussion administered. Single massive doses were indeed found to Nutritional anaemia is a very serious problem worldwide, be less effective in improving haemoglobin levels in particularly for women in developing countries. In the children (Bloem et al, 1989, 1990). slums of Dinajpur the present study documented a surpris- An additional intervening factor might be related to the ingly high prevalence of anaemia, with 98% of the screened study group itself. Our study concerned adult non-pregnant women having haemoglobin values below 120 g=L, and women, whereas other supplementation studies gave vita- 77.5% below 100 g=l. min A to pregnant women. In Indonesia (Suharno et al, Vitamin A de®ciency is a cause of nutritional anaemia 1993) the increase in haemoglobin was found to be 50% (Shingwekar et al, 1979). Hodges found in 1971 that higher when vitamin A was supplied with iron and suf®- volunteers on a prolonged vitamin A-de®cient diet anaemia cient to eliminate anaemia in 97% of those anaemic that responded to vitamin A but not to iron (Hodges & pregnant women who received both vitamin A and iron. Kolerd, 1971). Subsequent studies demonstrated correla- The needs for iron during pregnancy are, however, con- tions between serum vitamin A and haemoglobin (Meija et siderably higher. The effect of vitamin A might thus have al, 1977; Suharno et al, 1992). Vitamin A forti®cation of induced higher haemoglobin changes owing to the higher sugar of Guatemala (Meija & Arroyave, 1982) and mono- demands and the need to mobilise iron stores. Iron is in sodium glutamate in Indonesia (Muhilal et al, 1988) also particular sequestrated in liver and bone marrow in a improved haemoglobin status in adults and children. Daily vitamin A de®ciency state (Bloem, 1995). Women of vitamin A supplements given either to children or to Dinajpur are also very thin; 44% of them have a pregnant women signi®cantly increased haemoglobin BMI < 18.5 kg=m2 and should be considered malnourished. levels in the observed populations. This general degree of undernutrition might have decreased The present study demonstrated the bene®cial effect of the possible response to treatment. Indeed, an increase of adding vitamin A and zinc to iron treatment for anaemia. 1.5 g=l of Hb over 2 months is well below the 20 g=l Vitamin A added to the iron treatment did not signi®cantly increase over 3 weeks that is the normal response to increase the response in terms of haemoglobin increase, treatment in iron de®ciency (Frewin et al, 1997). It although an increase could be seen. There are several should be emphasised that in the present population 2 confounding factors that could have in¯uenced the results. months of treatment only corrected anaemia in 6% of the A multitude of factors, nutritional and others, cause anae- women who received treatment. mia. Iron de®ciency is only one of them. The Dinajpur area A ®nal explanation is related to the sample size, which is malaria free (Dr S Rahman personal communication), was calculated to demonstrate signi®cant differences with a however. To identify iron de®ciency, biochemical para- difference of 5 g=l for a standard deviation of 10 g=l. In the meters are accepted as useful indicators, but these indica- present study the standard deviation in the group receiving tors separately identify different population groups (Hastka all three supplements was 13 g=l and the differences Vitamin A=zinc supplementation and anaemia P Kolsteren et al 106 between the groups were smaller than ®rst expected. This Fairweather-Tait SJ, Whart SG, Fox TE (1995): Zinc absorption in infants might explain the fact that an inter-group difference of 3 g=l fed iron-forti®ed weaning food. Am. J. Clin. Nutr. 62, 785 ± 789. Frewin A, Henson A, Provan D (1997): ABC of clinical haematology. Iron was not found to be signi®cantly different. de®ciency anaemia. Br. Med. J. 314, 360 ± 363. The women who received zinc as part of the treatment Golden MHN, Golden BE (1981): Trace elements. Potential importance in showed no increase in serum values. It is very probable that human nutrition with particular reference to zinc and vanadium. Br. the women took all medication at the same time, even when Med. Bull. 37, 31 ± 36. requested to take it with separate meals. The most likely Guyatt GH, Oxman AD, Ali M, Willan A, Mcillroy W, Patterson C (1992): Laboratory diagnosis of iron-de®ciency anemia. J. Gen. Intern. Med. 7, explanation would then be that the therapeutic dosage of 145 ± 153. iron inhibited most of the zinc uptake, as it is known that Hastka J, Lasserre JJ, Schwarzbeck A, Reiter A, Hehlmann R (1996): these elements compete for the absorption receptors (Fair- Laboratory tests of iron status: correlation or common sense? Clin. weather-Tait et al, 1995). In Nigeria, however, no effect of Chem. 52, 718 ± 724. iron supplementation could be demonstrated on zinc meta- Hodges RE, Kolerd H (1971): Experimental vitamin A de®ciency in human volunteers. In Summary of proceedings. Workshop on Biochem- bolism (Arnaud et al, 1993). The authors concluded that ical and Clinical Criteria for Determining Human Vitamin A Nutriture, iron supplementation did not interfere with normal zinc 28 ± 29 January, 1971, pp. 10 ± 16. Washington DC: Food and Nurition absorption. 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