X VITAMIN A STATUS AND THE EFFECT OF ORAL SUPPLEMENTATION IN PREGNANT NIGERIAN WOMEN IYABODE OLUWAREMILEKUN ADEYEFA (MISS) B.Sc, M.Sc (Hum.Nutr) (lb). VITAMIN A STATUS AND THE EFFECT OF ORAL SUPPLEMENTATION IN PREGNANT NIGERIAN WOMEN BY IYABODE OLUWAREMILEKUN ADEYEFA S ? A THESIS IN THE DEPARTMENT OF HUMAN NUTRITION SUBMITTED TO THE FACULTY OF BASIC MEDICAL SCIENCES, COLLEGE OF MEDICINE IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF IBADAN 1991 i i i C E R T I F I C A T I O N I CERTIFY THAT THIS STMPX HAS"BEEN CARRIED 0UT BY IYABODE OEUWAREMILEKUN ADEYEJrA• ' P 1 .* ,’ * iIN THE DEPARTMENT OF j NUTRITION, FACULTY OF BASIC MEDICAL SCIENCES, COLLEGE OF MEDICINE, UNIVERSITY OF IBADAN, NIGERIA < r SUPERVISOR Cw DR. A.O. KETIKU Ph.D (Ibadan) CO-SUPERVISOR PROFESSOR BABATUNDE OSOTIMEHIN MD(Birgm), FRCP(Lond). I V A B S T R A C T This study was designed to investigate vitamin A nutritional status of pregnant and non pregnant non lactating Nigerian women. The beneficial effects of oral vitamin A supplementation was also investigated in the pregnant women. The study was carried out in three pOhasyes: Phase one was the cross-sectional study carried out on 22, 88 and 61 pregnant women in the 1st, 2nd and 3rd trimesters. The controls were 35 non pregna^ty non lactating women in the proliferative phase of the menstrual cycle. Their ages ranged from 18 to 45yrs with a mean age of 27.8+/— 6.82yrs. The subjects were randomly selected from both the University teaching and Adeoyo hospitals, Ibadan and the study lasted for a period of nine months. The result of the study showed that 11% of the subjects had plasma vitamin A levels in the deficient range << 20ug/dl) while 60% had marginal values (20 - 29ug/dl). Plasma vitamin A levels was observed to decrease as pregnancy progressed vi i ACKNWOLEDGEMENT--- ____________ _______ vi i i TABLES OF CONTENTS x i i LIST OF FIGURES--- x i i i LIST OF TABLES---- xv LIST OF APPENDICES x vi i LIST OF ABBREVIATION x vi i i 3 LIST OF ACRONYMS x i x INTRODUCTION----A- i LITERATURE REVIEW---- 12 MATERIALS AND METHODS- 52 RESULfSfeJ-------------- 76 DISCUSSION------------ 109 SUMMARY--------------- 139 RECOMMENDATION-------- 140 BIBLIOGRAPHY---------- 142 APPENDIX-------------- 160 X 1 1 1 L I S T O F F I G U R E S FIGURES TITLE PAGE 2-1 Vitamin A alcohol 14 2 -2 Vitamin A aldehyde 14 2-3 Vitamin A acid 14 2-4 Isomers of Vitamin A 18 2— 5 B- carotene 18 2 -6 Isomers of B—carotei 18 3- 1 B-carotene standard curve 60 3-2 Vitamin A sta_ ndard tc urve 63 3-3 RBP and TTR czu lture plate 66 3-4 RBP standar curve 68 3- 5 TTR standard curve 69 4- 1 Histogram depicting the changes between the PCV values of the subjects in the three trimesters of pregnancy and the controls in cross sectional study. 82 4-2 Histogram showing the plasma B-C levels o-f the subjects and the controls in the cross sectional study. 84 4-3 Histogram showing plasma vitamin A levels in the subjects and the controls in the cross sectional study. 86 4-4 Histogram showing the plasma albumin levels o-f the subjects and the controls in the cross sectional study. 89 X I V 4-5 Graphical presentation of the changes over time in the PCV values and plasma albumin levels in both the subjects and the controls in the longitudinal study. 95 4-6 Changes in the vitamin A levels over time during the longitudinal study period. 97 4-7 Changes in the levels of pi a —carotene over time in both the con and the subjects during the ongitudinal study period. 98 4-8 Changes in the levels of plasma ALB levels in both the subjects and controls in the longitudinal study lOO 4-9 Changes in the l&evels of RBP levels in both the"< subjects and the controls in the longitudinal study 102 4-10 Changes in the levels of TTR levels in both the subjects and controls in the longitudinal study 104 X V L I S T O F T A B L E S TABLE TITLE PAGE 2-1 B—carotene and vitamin A contents of various foods consumed in Africa 1 Means +/— SD Age, pari wt of subjects and coi in the cross sections Age distribution jd^ the subjects and the controls in the cross sectional study 78 Class distribLumticon of the subjects and the controls in the cross sectional study 79 4 Various reasons given for missed meals by the pregnant subjects 80 A list of the B-carotene and vitamin A rich foods and their consumption pattern 80 PC^3 VIT A, B~C, and ALB levels of the subjects and the controls in the cross sectional study 83 Analysis of variance between and within the different parameters in the cross sectinal study 88 8 Mean age, WT, HT, basal plasma vitamin A and RDR values of pregnant subjects and NPNL controls 91 9 Mean-*-/—SD age, PT, WT, HT, of supplemented subjects and XVI placebo controls 93 10 Mean +/-SD PCV, VIT A, B-C, levels in the supplemented subjects and the placeo treated controls A 11 Mean +/- SD ALB, RBP, TTR levels in the supplemented and the placebo groups ^ 1 101 12 Mean +/- SD APSC, BW, HT, PCV levels of the neonates 106 13 Mean +/- SD VIT A, B-C, ALB, TTR, RBP of the neonates 106 14 Mean +/- SD and median CALS, PROT, FAT, B-C, and VIT A intake of pregnant women 108 X V I I L I S T O F A P P E N D I C E S Appendi x 1 Consent Appendix 11 Ethical Commitee Approval Appendix 111 Appendix IV < r X V I 1 1 L I S T O F A B B R E V I A T I O N S mm mi 11imetre cm centimetre ug microgram < Iess than > greater than 7. percentage Tig -figure a alpha B beta Y gamma xix L I S T O F A C R O N Y M S B-C Beta carotene a-C alpha carotene Y-C gamma carotene VIT. A Vitamin A ALB Albumin RBP Retinol Binding Protein TTR Transthyretin vV PCV Packed Cell Volume CSS Cross Sectionai i j p i ! LS Longitudinal Study RDRT Relative Dose Response Test SD Standard Deviation S Subjects c trol s I.U AC"ternational Unit. C H e r - o N n r> !. I C T 0 N Although the importance of Vitamin A m Nutrit ion has been known for almost a century its role m metabolism is poorly understood. A number of metabolic roles have been ascribed to the Vitamin - these include its on5 in vision, growth, reoroduction. maintenance of epithelial cells and immune properties. The specific role it olays in each case however has not been fully explained except for its well elucidated role in vision (Wald, i960). The role of Vitamin A in reproduction has been the focus of attention J^y various workers (Moore, 1957: Thompson et a1, 19£fr4: Howell et al. 1964: Bodansky et al, 1962: Pulliam et al, 1962). Severe deficiency of Vitamin A has been found to cause infertility in all vertebrate soecies studied. The conseauences of the deficiency ascrihable to Vitamin A range from a) disruption of the oestrous cycle and b) permanent keratinization of the vagina c) necrosis of the junctional zone of the placenta, and d) foetal mishaps such as resorption stillbirths and various congenital malformations. The failure of fertilization or imolantation are uncommon features of vitamin A deficiency 1(Bates, 1983). At the other extreme hyoervitammosis A is also associated with an increased risk of congenital malformation (Cohlan, .1953: Bates.1983). Earlier. the function of retinol could not be ascertained until evidence was adduced/\ to demonstrate that retinoic acid would correct th* non-Boecific effects of vitamin A deficiency except those of vision and reoroduction (Thompson et 1964; Howell et al, 1964)« Various mechanisms have been postulated to explain the observed reoroductiVe functions of vitamin A. These include effects in steroid hormonooenesis„ It has been demonstrated that vitamin A <1) improved the reproductive performance of female rabbits supported survival of pups and lactation in the dams (Ganduly et al, 197lab). Thus retinol deprivation probably can cause a disturbance of steroid normone production although the extent to which this leads to the overall impairment of reproductive capacity is not clear. It has been observed that when pregnancy is allowed to proceed to term in rabbits and pigs which are receiving marginal amounts of vitamin A just sufficient to prevent total resorption of fetuses a hig cidence of concern ta1 malformations is observed. The type of defects depends on the timing anrwdH duration of the deficiency (Wilson et al 1953; Palludah, 1966). O'Toole et al, (1974), in a study of the effect of hypovitaminosis A on eight Rhesus monkeys observed abortions, and Xerophthalmia at birth but not congenital mal formation. It -kN&s then suggested that primate embryoes may present with milder forms of vitamin A deficiency signs when compared to other mammals. A few instances of congenital malformations possibly attribute#'-to vitamin A deficiency have been reported in hum subjects (Bates, 1983). The evidence that uncomplicated vitamin A deficiency can be teratogenic in human is however inconclusive. Thus it is apparent that there is a critical amount of vitamin A required for successful reproduction. Hume and Krebs (1949) demonstrated that pregnancy imposed an increased demand for vitamin A. This observation has been confirmed by other groups (Howell et al, 1964; Thompson et al, 1964). However the exact amount of vitamin A required in order to meet optimal needs varies with the different physiological states. The adequacy of vitamin A intake during pregnancy will depend on the prevalent food cultures as dictated by the geographical zones of the world. MATERNAL STATUS OF VITAMIN A DURING PREGNANCY A number of studies have described night blindness and 1PPaiPPd PaP, adaPtat_ ^ . diet inadequate in its vitamin A content (Rodriguez and Irwin, 1972)- Also,, a decrease in plasma retinol levels during the course of pregnancy? and an increase post partum has been reported (Pulliam et al 1962? Vekatac ha 1 am et © 1962; McGanity et al, 1969, Edozien et. al 1976). The observed increase in plasma vitamin A levels Dost partum however is not sustained and values may fa deficient levels if intake is not increased (LunI d acnftd Kimble 1943)- Sr >3 ies have also revealed the inadequacy of the intakeof vitamin A and its precursors amongst women in developing countries (Rodriguez and Iwin, 1972; Bates, 1983; Le Francois et al, 1981). 4 & The control of olasma vitamin A levels during pregnancy clearly differs from that of the other lioid soluble components of the blood. While vitamin A level decreases in pregnancy that of vitamin £ and other lipid soluble substances increases . Takahashi et al, (1975) found that the concentration of vitamin A in the liver of neonates increased 1.4 fold when maternal concentrations increased 100 fold. 5 However at very iow maternal intakes., the liver A concentration in the neonates was observed to be substantia1ly reduced. Takahashi et al, 1977 also found that accumulation of vitamin A in the conceotion followed a complex pattern. During the early stages (day 7 - 9) the vitamin accumulated to a high concentration in the placenta- From day 9 - 11, the concentration fell abruptly to less than 20% of the initial peak and duriofl days 11 - 14- both vitamin A and R.8P accumulated lf% parallel. During days 1 6 - 2 0 the fetal liver started to synthesize RBP and accumulated vitamin A anciid, foe1tal stores increased. Gal and Parkinson (1974) showed that there was a reduction in the olasms /2u>tamin A levels in pregnancyin the early 5th to 9t h weeks and after the 36th week of gestation, Though they attributed this to the effect of circulating sex hormones, the trend they observed follows the oAbservat..:ion made by Takahashi et al (1971). It reveal|ed- > rt•hat the timing of the drop in plasma vitamin A levels in the mothers corresponded to the period of increased concentrations of vitamin A in the foetus. These studies are consistent with the observation that the supply of Vitamin A to the foetus is from the retinol RBP complex from maternal stores via the 6 maternal blood (Bates 1983), Human cord oiasma retinol levels have however been shown to be lower than the corresponding maternal levels (Lewis et al, 1947; Vekatachalam et al, 1962) except when maternal levels are very low in which case the relationship may be reversed (Mclaren and Ward, 1962. Rodriauez & Irwin, 1972). EFFECT OF VITAMIN A SUPPLEMENTATI ON IN PREGNANCY Several attempts have been made to solve the problem of vitamin A deficiency in pregnancy through oral suppiemention with vitamin A ranging from 650 - 9000ug dailv with varyinq decrees of successes- Lewis et a.1 , (1947) found that 3UOOug vitamin A or the equivalent amount of carotene given daily during the last trimester of pregnancy, had no effect on plasma retinol levels in the neonates but significantly increased the levels in the mothers. In Cows and pigs, large doses of vitamin A supplied to the mother increased the extent of placental transfer to a moderate iarn e d oses of carotene in contrast were entirely jout effect on foetal vitamin A stores and very little per se was transferred to the foetal plasma. Vekatachalam et al (1962), gave 9000ug vitamin A per day throughout that last trimester of pregnancy to 7 twelve rna 1 nourished Indian women who apparently had very- low dietary sources, and observed significant higher cord levels in them than in the unsupplemented controls. Lund and Kimble in 1943 also showed that the administration of 10?OOOiu/day of vitamin A brought about a maintenance of normal levels of the vitamin in the mother's blood throughout the course of pregnancy. They observed that an amount greater than this level was without benefit under normal conditions RATIONALE FOR THE STUDY Vitamin A is an essent:iiaal nutrienntt*v faor normal growth and development. Since pregnancy is a time of active growth for both the mother and the foetus this period therefore deserves special attention. Chronic vitamin A de$ficiency plagues many developing regions of the world with its tragic consequences such as increased morbidity and mortality,, different stages of eye affectations and blindness observed mostly in c h 1 1 d r e n S i .1 d e r f o r m s o f v i t a m m A d e f i c i e n c y h a v e a 1 s o beeno obserrved in pregnant women (Vekatacha1 am et ai. 1 962>). In the Nigerian situation, vitamin A deficiency been identified in the Northern part of the country containing a six-membered carboxylic ring, and an eleven carbon side chain. Vitamin A activity in mammals is not only found in the retinols but also provided by certain 14 carotenoids widely distributed in plants, particularly a, B - Y carotenes. These carotenes have no intrinsic vitamin A activity per se but are converted into vitamin A by enzymatic reactions in the intestinal mucosa and the liver. B-carotene5 a symmetrical molecule, is cleaved in its centre to yield two molecules of retinol. Retinol occurs in the tissues of mammals and is transported in the blood in the form of esters of lonq chain fatty acid Vitamin A, an organic compound foun alyr in the animal kingdom, is a very pale yellow (almost colourless) substance composed of carboy hydrogen and oxygen. The vitamin is soluble only in fat and organic solvents. The carotenoid pigments are composed of carbon and hydrogen. The c f r * Is of the pigments are a deep red colour but they are intensely yellow in solution (Steenboc k, 1919) Vitamin, O &p? ists naturally in several isomeric forms. A cis-trans isomerism resulting from configurational differences at. the double bonds in the side chain illustrated in fig. 2-4. The naturally occurring form of Vitamin A is the all- trans isomer. Neo-Vitamin A (13-cis) has about 85 per cent of the potency of the all-trans form, and the 11- 15 cis isomer (neo-b) has 75 per cent of the biological activity of the all-trans isomer (Goodhart and Shills, 1974). Dehydro-retinol has only about half the biological activity of retinol (Goodhart and Shills, 1974) also exists in various isomeric forms. Retinol, Re t i n a 1 and Retinoic Acid Retinol (vitamin A alcohol) is the most import.ant form of vitamin A. It performs all the known VTflumnctitons of vitamin A since it can he oxidised tc other forms nf the vitamin. Retinol is therefore used synonymously with vitamin A. Retinal (RCHO) is the aldehyde corresponding to retinol. It is the active form tamin A required for vision (Wald, 1960) and certain other functions of Vitamin A (Goodhart and Shills, 1974). The blindness prevented by vitamin A is specific in that the early stages of the blindness can only be treated by the vitamin. Ret m o Vv : l d a t i on & Potassium permanganate oxidation of retinol yields retinal (Marton, 1941). This has led to the use of manganese dioxide as an oxidant to convert allylie alcohols into the corresponding aldehydes (Ball et al- 1948%. A petroleum ether solution of retinol, left in the dark at room temperature in the presence nf manganese dioxide yields retinal. Reduction Lithium aluminium hydride reduces Vitamin A aldehydes. acids, and esters to the corresponding retinol analog (Robeson, 1955), sodium and potassium borohydride have the same effect (Brown and Wald, 1956). 1somerization Retinal is isomerized by exposure to light. Each isomer gives a steady state mixture of all possible isomers with the all-trans retinal always dominanl : ,7?'W own and Wald. 1956). Thermal isomerization of aqeous solutions also occurs (Wald et al. 1955). Instability to acids Vitamin A is extremely sensitive to acids; they can cause rearrangement of the double bonds and dehydratmn (Bentel et al. 1955). Co1our-reactions Acidic reagents gij/e transient blue colour reactions with Vitamin-A. These tests are useful for qualitative or comparative measurements. The purple color obtained with suJlphuri c aci d was one of the first methods used tn iden Vitamin A in liver oils. Later, arsenic trichloride and the Carr-Price reagent (antimony S r ichloride in chloroform) were used (Carr and Price, 1926). Other Lewis acid such as trifluoroacetic acid have been used for quantitative determinants of Vitamin A (NeeId and Pearson, 1963). 20 Provitamin A B-carotene melts at 181 to 182oc. In Petroleum ether, all-trans B-carotene has absorption maxima at 453, 481 and 273nm (Zechmeister and Polqar, 1949)- Pure synthetic? crystalline all-trans B—carotene, after drying in a high vacuum drying pistol« has absorption maxima in n—hexane at 468nm. (Goodhart and Shills, 1934), B-carotene is readily soluble in carboA^aisulphide, chloroform and benzene but partiallv soluble in- Carotene is rapidly oxidised in air giot absorb significant amounts of carotenoid pigments. However they can convert provitamin A to theV vitamin in the gut (Thompson et al. 1950). The tsmpall intestine is the mostimportant organ involved in the conversion of provitamin A to the Vitamin. The liver and the kidney are aisn capable of carrying out this process (Goodhart and Shi 1 Is,1974). The process of conversion involves two soluble enzymes, B-carotene 15-15' dioxygenase and retinaldehyivfdeeN rer? eduucct t.iase. The first enzyme catalyzes the cleavaqe <%>B—carotene at the central double bond by a dioxygenase mechanism, to yield two molecules of retinaldehyde (Goodman and Olson, 1969). Retinadehyde reductase reduces the retinaldehyde to retinol. <5e absorption of dietary carotenoid is significantly reduced when the diet is unusually low in fat (Reels ef al. 1958). The amount of carotene absorbed from raw carrots is highest in the presence if low molecular weight and short chain fatty acids hmuk et al . 1964), although a portion is converted to retinoic acid CFidqe et al. 1968). Transport of retinol Retinyl esters in chylomicrons formed in the intestinal mucosal colls travel through the lymphatic system, via the thoracic duct, to the blood stream, and are stored in the liver. The uptake of the chylomicron retinol esters hydrolysis and reesterification occur in the liver, where the resulting retinyl esters (mainly retmy 1 pa Imitate) are stored. Vitamin A is mobilized as the free alcohol, retinol, bound to a specific plasma transport protein, retinol binding protein cRBP). The retinol then travels via the blood stream to the tissues. Only 10 to 17 per cent of the Vitamin A content of the blood in normal human subjects in the fasting state is in the ester form. However ojq postprandial state after Vitamin A intake, the pe^^rtage of the ester in the circulating blood increases rapidly. This is as a result of the Vitamin A ester arriving in the blood stream from the gut via the lymphatic system (Hoch, 1959). The blood level of Vitamin A is independent of the liver reserves: as long as there are very small reserves of Vitamin A present in the liver, the blood level remains norm w As soon as the liver is depleted of its Vitamin A re serves to a certain level, the blood Vitamin A level fallIs rapidly (Goodhrt and shills, 1974). Retinol Binding Protein and Prea1bumin Retinol Binding Protein (RBP) is a single polypeptide chain with a molecular weight close to 20,000, and a 24 single binding site for one molecule of retinol- In plasma, most of RBP normally circulates as the retinol- RBF* complex (holo-RBP); the usual level of RBP in plasma is about 40—50ug/ml (Goodman? 1980). RBP interacts strongly with transthyretin and normally circulates as a 1:1 molar protein-protein complex. In addition to its role in i/itamin A transport,transthyretin plays a role in the binding and plasma of thyroid hormones. The formation of the RBP- transthyretin complex serv•ees to reduce trhe glomerular filtration and renal catabotlliism .of VRBP (Gotodman, 1980). Plasma RBP levels are low j€ rpa ti ents with liver disease but high in patients wit chronic renal disease. These findings reflect the fact that RBP is produced in the liver and mainly catabolised in the kidneys. The transthyretin molecule is a stable tetramer, composed of Vour identical subunits with a molecular weight of 54,980. Transthyretin appears to contain four binding sites for RBP (Goodman, 1980). Several studies have examined the retxnol transport- system in patients with protein-ca1orie malnutrition, who have been found to have decreased concentrations of plasma RBP, pre-albumin, and Vitamin A. Low intake of dietary protein and calories is frequently accompanied by an inadequate intake of Vitamin A. However, even in cases of malnutrition where there is adequate Vitamin A intake, the plasma RBP and Vitamin A levels are low reflecting a functional impairment in the hepatir release of Vitamin A because of defective production of RBP (Goodman. 1980). RBP is responsible for the delivery of retinol from the liver to the extra-hepatic sites of action of the vitamin. Evidenced is available that this delivery process may involve speci f i c cell surface receptors for RBP (Chen and Heller, 1977). The retinol thus carried is delivered to the specific sites where it enters the cell for subsequent metabolism and action. The apo RE*P returns to the circulation, where it shows a reduced affinity for transthyretin and is selectively filtered he renal glomeruli. Studies in the rat and in humans have suggested that Vitamin A toxicity occurs in vivo. This occurs when the level of Vitamin A in the body is such that retinol begins to circulate in olasma and to be presented to membranes, in a form Oo-threr than bound to RBP (Uoodman, 1980). It has been suggested that the nonspecific and unregulated delivery of Vitamin A to biological membranes, in <5ontrast. to the specific and regulated delivery via RBP, leads to Vitamin A toxicity. Vitamin A mobilization from the liver, and its delivery 26 to peripheral tissues, are highly regulated by factors that control the rates of RBP production and secretion by the liver- One factor that specifically regulates RBP secretion from the liver is the vitamin A nutritional status of the animal. Retinol deficiency specifically blocks the secretion of RBP from the liver, so that plasma RBP levels fall and liver RBP 1 eve1s rises Conversely, repletion of vitamin A deficient rats intravenously with retinol stimulates the rapid secretion of RBP from the expanded liver pool (in the deficient rat) into the plasma- This release of RBP is not blocked by inhibitors of protein synthesis indicating that it comes from the expanded liver pool of RBP, rather (Goodman, 1979) A depletion is highly specific for RBP. Thus neither Vitamin Of depletion and deficiency, nor retinol repletion of deficient rats significantly altered plasma levels of transthyretin. The secretion of RBP ctnd that of transthyretin appear to be independently regulated processes with formation of the RBP-transthyretin complex occurring in plasma after secretion of the two proteins from the liver cells (Goodman, 1979). RBP in the liver is mainly associated with the liver 27 fnicrosofnes, and is especially enriched in the rough microsomal fraction. The Golgi apparatus was found tn contain a maximum of 22/1 of RBF* in the liver in normal rats, and 97. in Vitamin A~deficient rats. The Golgi apparatus is therefore not a major sub-cellular locus for RBF* in either normal or deficient rats- Inconclusive evidence that the microtubules are involved in the secretion of RBF has been obtained in studies with the drug colchicine. Smith et al.<1978) found two lines of dif f erentiated rat hepatoma cells thz*t synthesize RBP during culture in vitro. When the cells were incubated in a Vitamin &rfree serum-less medium, . a relatively large proportion of the RBP synthesized was retained within the cells. Addition of retinol to the medium tr ansferred to the foetal 1i ver. These studies are in agreement with the idea that the supply to the foetus is mainly from the retinol—RBP complex in maternal stores, except when they fall to very low lGeve/ W r ls (Bates, 1983). Values for foetal liver Vitamin A levels at autopsy are "id! 4 < The early studies were put together by Moore (1957). Gal et al. (1972) observed a range of values from less than 10 to more than 150ug/g in livers of British infants near term. A wide range was also observed by Montreewasuwat & Olson <1972) in Thailand, although in this study, and that of Iyengar & Apte (1972) on foetuses from poorly nourished indian women, there were few values above 50ug/g, and mean values were of the order of 20ug/g. Although foetal levels are generally lower than those of adults, it is difficult to be certain about the quantitative relationship between maternal status and intake and foetal liver in humans,, and whether they are as tightly controlled as they are in the rat. Human maternal values for plasma ret]:-inolo usrlually tend to be higher than the corresponding c o r ^ plasma retinol levels (Lewis et al . 1947). Valhlquist et al. (1975) found that RBP and thyroxine binding transthyretin levels at birth, in a presumably well-nourished population are about half the adult levels. Preterm infants have been observed to have lower plasma retinol levels at birth than term infants (Brande et al. 1978; Shenai et al. >. However babies of low birth weight but not premature had plasma retinol levels similar to those of normal birth-weight (Baker et al. 1977). Lewis \e \1 e,tico )n, Vitamin A is released from the liver of the r-at in amounts much greater than required by the ani mal The actively growing tissues of the foetus may also utilise considerable amounts of Vitamin A. The basal metabolic rate increases during the latter half of pregnancy. There is general agreement that most of this increases is due to the growth of the foetus, although it may also be in part accounted for by a possible activator of the maternal endocrine glands (Du Bois, 1936). - Lund and Kimble (1943) observed that economic status and health education influenced the adequacy of the intake of Vitamin A in the pregnant population s-t udied. They also observed a sign ificant correl atVrfi between the intake of Vitamin A and the plasma values for the groups of subjects. A diet which was adequate non-pregnant women was also adequate for pregnant women during the first trimester except when such complications as hyperemesis gravidarum intervened. During the second trimester, only the best met the needs. By the third trimester there was ĝtheed for supplements of Vitamin A in addition to amount supplied by the diet. Gal and ■ Parkinson (1974) also observed a significant effect of Vitamin A supplementation with as slow as 2,500 i.u.daily when treated subjects were compared with the untreated control group. Optimum plasma Vitamin A levels could be maintained during the last trimester of pregnancy by the addition of 10,000 i.u. of Vitamin A. Larger supplements of 20,000 i.u. had no additional 41 effect, (Lund & Kimble 1943) Lund & Kimble (1943) gave some reasons for the pattern of plasma Vitamin A in pregnancy. Haemodilution which accompanies pregnancy might be considered as a possible cause of the progressive decline in vitamin A blood values. But since there was no decline in caroteAne> value of the subjects during the same oeriod thkiis does not hold true. _ The ingestion of large amounts of carotene, whether by diet or supplements, elevated carotene without affecting the plasma Vitami n » < \ , r oItene metabolism in pregnancy is however poorly understood. Vitamin A mobilization ^pito the blood stream postpartum was observed by Lun d & Kimble <1943). A single common -factor to every pat i eint is emptying of the uterus. Labour, Caesarean section, anaesthesia & analgesic had no effect on the mobilization of Vitamin A postpartum (Lund & Kimble, 1943; Clauses et al. 1942). Puerperal diuresis was also eliminated in the possible causes of Vitamin A mobilization postpartum. Vitamin C and carotene values did not change with Vitamin A. Mobilization of Vitamin A was noticed - as early as 6 hours postpartum and lasted up to 24 hours. Large doses of Vitamin A 6000,000 and 330,000 i.u. given to mothers 42 before delivery in an effort to produce very high plasma levels though succeeded but could not maintain the plasma levels in early puerperium. The puerperal values decreased unless the supplements were continued (Lund and Kimble, 1943). There was no correlation between the toxaem i a of pregnancy and plasma Vitamin A, even though low values might be anticipated because of the li amage common in some toxaemias. Plasma Vitamin A could also not be correlated with the duration o-f labour. Considering these various ob Lund and Kimble (1943) advocated Vitamin A supplements of 5,000 i.u. of Vitamin A daily in the 2nd trimester and 10,000 i.u. in the third trimester and they also observed that amounts greater than this are not necessary under usual conditions. They further suggested that since the provitami & cannot be efficiently converted and absorbed, a diet which supplies generous amounts of vitamin A itself rather than one which depends principally on conversion from carotene is preferable (Lund and Kimble, 1943). Gal and Parkinson in 1974 showed that there was no difference in the plasma levels in the three trimesters though there was a reduction in the early 5th to 9th 43 weeks and after 36th week of pregnancy. They attributed their findings to the effect of circulating sex hormones. Studies have shown that both plasma progesterone and estradiol increase during pregnancy (Bates, 1983). The initial decrease in the serum Vitamin A content in early preqnancy could therefore be related to the changing site of progesterone production from the corpus luteum to the placenta. Similarly the decrease in circulating levels of Vitamin A in the final stages of pregnancy could be related to a decrese in the c,rculti"9 pro9esterone ^ ced" — “ • VITAMIN A AND B-CARQTENE IN NON PREGNANT NON LACTATING WOMEN Plasma retinol levels vary considerably among apparently well-nourished individuals living in industrialised countries Cotfee leaves(dried) 2360) Cowpea immature seeds 150 leaves raw 7770 Hares 1ettuce(raw) (1 eaves) 1430 Okra (raw) 185 Peanut (groundnut) <1 eaves) Peppers (hot)(raw) 330 red raw 7140 dr i ed 14250 ? sweet raw < ir ? immature 180 dried 2840 Pumpkin(raw) 3565— 3600 Sweet potatoes 2290- 7050 Cocoyam 1800 Tomatoes (raw) ripe who1e 360—700 Group 6 Frui ts Atrican locust bean 4 Aprlcot a Avocado iS Bush mango 310 Cashew(raw > 760 Date(raw) 145 Grape 50 Guava 290 Mango 3200 Orange 250 Palm oi1 ( 6150 42420-168800 - Pawpawr(ar£ayw) 950 - Watermelon 250 — Group 7. Meat- Poultry and Insects Liver -beef 180 810 Eggs -raw 300 350 Fish Tiger-raw 550 2465 Cheese 1 70 - Milk whole 345 400 Cow milk 630 625 Group 8 Oils Butter 545 630 Others Chili 1060 Vinegar 3050 SOURCE:FAO. United Nation (1968). Food composition table ■for use in Africa complied by Woot—Tsu.en Wu Leung. U.S. Department of Health, Education and Welfare Public Health Service. 51 H A P T H R M A T E R I A L S AND M E T H O D S SUBJECTS The study was carried out in three phasea T 1) A cross-sectional (CS) study of pregnant women which involved the study of 171 pregnant women recruited during the three trimesters of pregnancy and 35 non­ pregnant non lactating (NF'NL) proli ferative phase of their cycle- 2) Vitamin A absorption test carried out on 10 NPNL women and 30 pregnant women also from the different trimesters of pregnancy. 3> A longitudinal study of the effect of oral vitamin A supplementation carried out on 28 pregnant women from the 14th week of pregnancy to the 6th week post partum. SECTIONAL CRITERIA All subjects were seen by a physician at the begining of , the study. Medical history and clinical examination were carried out on each subject. Furthermore biochemical indices such as Packed cell volume (PCV), Liver function test (LFT), urinalysis were also carried out on each patient at admission. This was necessary in order to exclude those patients with abnormally low PCV and abnormal LFTs. Subjects were admitted into the study based on the -following criteria: 1) Absence of any respiratory, cardiovascular, hepatic diseases, and other systematic diseases associ ated wi th precjnancy were al so exc 1 uded , 2) Informed consent of the subjects. □ n a d m i s s i o n e a c h s u b j e c t s h a d t h e of the study explained in detail to her. The detail1 of her participation were carefully enumerated and the need for cooperati on and comp1i ance i n the ex©cuti on of the study was explained- Ethical commi.tft.ee approval was obtained b e f o r e t h e c o m m e n c e m e n thi' s t u d y - At admission each subject was required to complete a questionnaire on her personal, educational, vocational, and nutr i t i onal^tatus ̂Appendi x 11). Q y At thiis A irnitial stage the physical measurements of weight, height, and blood pressure were taken by the observer. CROSS-SECTIONAL STUDY he subjects in this phase were recruited from both the Uninversity College and Adeoyo hospitals, Ibadan. They were seen on every antenatal clinic day for a period of nine months. The subjects were classified into five soci o economic groups usi ng i ncome, education and o c c u p a t ion based o n a m o d i f i e d ri e t h o d o f Tay1 or and Akande (1975). Fast i ng bi ood samp1es were coll ected f rorn each sub j ect i n thi s study group i n the morning between 8 a.m. and 9 a.m. -from the ante cubital vein into heparinized and E.D.T.A. bottles- These samples were kept in black polyethylene bags in a refridgderator for 2hrs on each occassion. This was to prevent light penetration and destruction of the Vitamin A content of the samples. The blood samples were separated and plasma samples were then stored frozen at : 0u un t i1 analyzed RELATIVE DOSE RESPONSE (RPR) This phase of t:l h eVlst lid y sp an i\ e d a period of 5 weeks. 40 subjects were ad mitted and were selected from the University College Hospital, Ibadan- The subjects coftipri sed non pregnant nan-1actating women and 30 pregnant women. The non pregnant non—1actating women were normally cycling women studied in the proliferative phase of their cycle- The pregnant subjects were cruited from the ante natal clinic of the department of Obstetrics and Gynaecology, Ibadan- The control subjects were healthy staff members of the U.C-H, Ibadan. Three subj ect s at a t i me wer e admi 11ed into the Metabolic Research Unit early in the morning on the day of the study after an overnight test for a period of 5hr s. They were put in the recumbent position on arrival at the Metabolic Research Unit- An indwelling canula was inserted in the ante cubital vein, which was kept patent with normal saline infusion. Blood was drawn at -15 and 0 minutes after which 450ug Vitamin A in oil was fed to each subjects. Blood samples were taken after 5 hrs of the dosing. The relative dose response was calculated from the following formulas A5 - A0/A5 X 100 where AO is the fasting plasma Vitamin A, h5 - a second specimen taken after 5 hr LONGITUDINAL S_TUDY < L .S.>- A total of fifty two pregnant subjects in the first half of the second trimester were recruited into this phase of the study. This period was chosen because of the supposed teratogenic effects of large doses of vitamin A in early pregnancy during ceil differentiation. The study 1ast^ & or a per i od of 18 mon t h s. The subjects were divided into two groups and were seen fr :he 14th week of pregnancy and every 7 weeks thereafter until 6 weeks after delivery. The two groups were matched for Age, parity, and weight. They were required to have plasma Vitamin A level < 30 ug/dl and normal liver function tests. 7,000 IU (2000 ug> of Vitamin A in oil or lactose prepared in gelatin capsule was given to the supplemented and the placebo groups respectively- The study was single blinded and the subjects were not given any dietary advice. They were however instructed not to take any other multivitamin preparations apart from folic acid and fersolate. Blood samples were obtained from each subjects at every appointed visit, immediately after delivery and 6 weeks after delivery- Cord blood samples wer& also obtained at delivery. The subjects were informed to report any symptoms experienced during the study^: were computed. Weighed food samples (breakfast, lunch. supper) were collected from ecj^^j^sub j ec t in their various homes during the study period. Samples of each food item for each meal were collected as eaten and stored frozen at - 20oC until analyzed- 24 hour dietary recall was also carried out on each visivt Vto ensure that the dietary pattern of the subjects was not altered significantly during the weighed dietary procedures. 56 3.1 ANALYSIS OF SAMPLES 1■ SERUM B-carotene and Vi tami n A The procedure is a modification of the methods of Kimble (1939) and Kaser and Stekol (194(3) except that Trif1oroacetic Acid (TFA) is substituted for SBC13 (Neeld and Pearson, 1963) 5 * The principle of this method involves the reaction of ,,-electrons in the conjugated double bonds of Vitamin A with trifluoroacetic acid tq^jhrm a chemical compound with a blue colour. $ Procedure for B-carotene arid Vi tami n A 1ml of serum was transferred in duplicate into 16 x 25 mm glass stoppered test tubes. With mixing 2ml of absolute ethanol/ascorbic acid in water lOOg/1 was added followed by 3. Ihl of petroleum ether (boiling range 30 to 40°C). Ascorbic acid was used to prevent the oxidation ofF V*ifteamin A during the extraction process (Driskell et a1 * 1985). TThhee mmiixx1ture was stoppered and shaken vigorously for two mintlutes to insure complete extraction of carotene and Vitamin A. The tubes were centrifuged slowly for three minutes. 2ml of the petroleum ether (upper) 1ayer was pipetted into a Coleman 75 x 100 mm cuvette. The cuvette was stoppered immediately and the 57 carotene was read immediately at 450mu against a petroleum ether blank in the Coleman Jr. spectrophotometer. The cuvette was removed and the petroleum ether was evaporated to dryness in 40°C water bath. The residue was taken up immediately in 0.iml of chloroform. The Coleman Spectrophotometer 620nm and set to zero optical density consisting of 0.1ml of chloroform and 1.0 TFA reagent The sample cuvette was placed in the spectrophotometer, and 1.0ml of TFA reagent added and the reading was taken at exactly 30 seconds after addition of the reagent. B-carotene St an d ar di z at i on The B-carotene working standards obtained were run seven times. The inter assay coefficients of variation bromocresol purple (BCP) to form a stable blue purple color complex with an absorption maximum at 600nm. The intensity of the colour is proportional to the serum a1burni n concent rati on i n t he samp1e. Procedure 1 ml of Albumin reagent (BCP) was added to tubes labelled blank, standard and test. To standard, test, an d h 1 an k 0. 01 rn 1 Alb urn in standard, ser um, and saline = A3 x ug retinol standard/cuvet X 100 X 0-872 A620 retinyl acetate standard where 3 ~ volume of the petroleum ether extract of 1.0ml serum. 2- aliquot o-f the petroleum ether extract: uased -f for the assay. 100-convert i on of ug reti nol/ml to cre/t:i no■l/1OOml. 0.872- ratio of molecular mass £)f retinol to molecular mass of re■tinyl acetate. Thus this factor corrects tor the use o-f retinyl acetat^ Instead of retinol as the standard. Albumin The method employed is the modification of that of Doumas and Biggs (1975) optimised for Sigma Catalog No 625 2. The principle of the method involves the reaction of human serum albumin specifically with ▼ ■ bromocresol purple (BCP) to form a stable blue purple color complex with an absorption maximum at 600nm. The intensity of the? colour is proportional to the serum albumin concentration in the sample. Procedure 1 ml of Albumin reagent (BCP) was added to tubes labelled blank, standard and test- To standard, test, and blank 0.01 ml Albumin standard, serum, and saline 64 w e r e a d d e d r e s p e c: t i v e 1 y . T h e s o i u t i o n s w e r e m i x e d together and the absorbance of standard and test read a q a;!. n s 1 t h e b 1 a n k a t 6 0 0 m m » CALC U LAI. IONS A1 b u rn i n r:; o n c e n t r a t. i o n (g / d 1 > o f s a m pie — A Test x canc en t r at i on of stangar d A E>td Reting 1 bi ndi nq protei n , and transth^l y:r.E±,iri The method employed in the analysis of serum retinol binding protein, and transthyretin was a modification of t h e radi al immunodiffusion procedure described by Mancinni (1965)- The Co:ommm is checked by means of control serum for Nor—partigen and the batch-dependent precipitate ring diameter was within t he conf i denc e r ange < D = +0.3mm). Standards for each parameter were prepared aitt y2!5'5%, 507., 75%, a n d 10 0 % c o n c e n t r a t i o n s » T h e D v a 1 u e s w e r e r e a d and p 1 o t t ed ( f i g s « 3 - 4 and 3« 5) « T h e ^ v a l lies f o r t he samp 1 es wer e r ead of f the ca 1 i br at i on jcur ves f or t he t w a parameters, ANALYSIS OF FOOD SAMPLES•jF Each food sample was dri ed Di n an oven at 60 C for 72 h o u r s u n t i 1 a c o n s t a n t w#i|jht was obtained- Each samp1e was then analysed for lipid, nitrogen, calorie, B~~ c a r cj t e n e a n d V i t a m i n A ! IQIAl l i p i d The method emp1oyed was the ether ex tract i on method» The principle? of the method is based on the fact that n o n - p m r components of samples are easily extracted into organic solvents- Procedure A s cd x h let a p p a r a t u s e x t r a c t o r w i t hi a r e f 1 u x c cd n d e n s e r and a round bottom flask was set up- 5g of each dried food sample was weighed into an oven dried fat-free extraction thimble of a known weight (Wl). The thimble was placed in the extractor and petroleum ether (BP- 40 FIG. 3 -4 ■ RETINOL BINDING PROTEIN. Standard Curve. FIG. 3 - 5 : transthyretin standard CURVE _____ 69 CONCENTRATION m g / 100 m is checked by means of control serum for Nor-partigen and the batch-dependent precipitate ring diameter was within the confidence range CD — +0.3mm). Standards for each parameter were prepared at 257-, 50/-, 75%, and 1007. cone eritr at i ons. The D values were read and p1otted (fig s. 3.4 and 3.5). 7he va1uesVor t he samp1es wer e read off the ca11 br at ion curves for t h e two par ameters. 3.3 ANALYSIS OF FOOD SAMPLES Each food sample was dried in an hours until a constant weight was obtained. Each sample was then analysed for lipid, nitrogen, calorie, B~ carotene and Vita TOTAL LIPID The method employed was the ether extraction method. The principle of the method is based on the fact that non-polar components of samples are easily extracted into or . c solvents. soxhi et apparatus extractor with a reflux condenser and a roun d bottom flask was set up. 5g of each dried food sampl e was weighed into an oven dried fat-free ex tract ion thimble of a known weight u'p to volume. 20ml aliquots was distilled using Markham distillation procedure. 0.1ml HC1 was employed in the receiving flask to trap the- liberated ammonia. The distillate was mixed with 20 parts methyl red and then titrated with 0.1 NaOH. Calculati on The Nitrogen content of the sample was calculated in percentage based on the dilution factor: lml 0. 1M HC1 «= 1ml 0. 1M NaOH lml 0.1 NHrr = 1.4 mg M Total protein = Volume of acid neutralized by titration x 1.4 factor 71 7. protein (dry matter) mg protein X 100 mg sample Recovery Standard ammonium solution was dried in an oven at 105° overnight. A 27. solution of ammonium sulphate was prepared and 2 ml of it was treated like the sample. The percentage nitrogen recovery was cal culated. The recovery in this case was 96.8%. ENERGY v\> The calorie content of the food samples was analyzed using the Bomb Calorimetry method. Procedure The dried food samples were thoroughly mixed before weighing to ensure a homogenous and representative sample. 0.5g of the dried food sample was weighed on a balance into a dry labeled crucibles of known weights. 0.5 of benzoic acid was used as the standard and put into another crucible while an empty crucible was used for blank reading. the crucibles weretplaced on the pillar of the base of the bomb calorimetry one after another and a length of 5 centimetre serving thread that was supplied with the apparatus was inserted between the coils of the platinum 72 wire and the other end was dipped into the centre of the sample in the crucible. Same cotton length was used for all the samples standard and blank. The bomb cal orimetre was then lowered and locked by rotating it for the thread to engage firmly- The thermo couple was plugged into the hole and the bomb body and the valve on the bomb calorimetre was closed while the inlet on the front panel of the control box was opened by 1/4 turn. The pressure was allowed to rise to 25 atmosphere by opening the oxygen cylinder for about 20 seconds and the valve was then closed and the cylinder locked. It is assumed that the pressure i s enioouugh to ensure adequate combustion of any biological materials. By means of the Calvo Zoro Knobs of the control box the light spot index of the galvanometre was brought to zero and the firing button of the bomb was then released. Maximum deflection of the galvanometre was recorded after each bombing. The used gas was then released from the bomb by opening the pressure release valve at the right side of the base of the bomb. The body of the bomb was released and placed in cold water to cool so that heat is not transferred to the next sample bombed. After cooling, the body of the bomb was then wiped with a clean piece of cloth and the next sample treated as the first 73 Calculati on E value of benzoic acid standard = 0.596 KCal/0.58 E value of food = Kcal/0.58 Correcting for standard X * 0.596 B^Carotene and Vi tamin A 50mg of each powdered food sample was homogenized with 2ml of water. Saponification of the lipids Gw«*a s carried out using 4ml ethanol ic potash nm for its carotene content. Aliquots (4ml) were dried in the water bath at 37°C;I they were dissolved in 0.1 chloroform and trifluoroacetig acid was added for the development of the Vitamin A colour complex which was read at 620nm. ug B-carotene (per 100ml) « ug B~ carotene per mg of mg of tissue used tissue g Vitamin A (per 100ml) « ug Vitamin A per mg of tissue mg of tissue used 74 STATISTICAL ANALYSIS All data collected were fed into an IBM compatible personal computer and analysed using the Oxstat and packages. Mean +/- Standard deviations, Unpaired t-test, paired t-test, Analysis of variance, Pearson’s correlation and Regression methods of data analysis were employed as appropriate. < r o r & 75 C H A P T E R F O U R R E S U L I S This study was carried out in three phases and the results will be presented as such under the following headings: 1) Cross Sectional study (C S S> 2) Relative Dose Response Test (R D R 3> Longitudianl Study c 2 9 . 4 6 + / - 1 . 1 7 + / - 7 . 5 1 . 4 8 T 1 2 2 2 6 . 9 5 + / - 1 . 4 0 + / - 11 • /J~Ll •j“L*} 7̂ T T 2 8 8 2 7 . 0 2 + / - 1 . 6 5 + / — oT6 4 1 . 3 T 3 6 1 2 7 . 7 5 + / - 1 . 9 2 + / - 6.4 1.59 T 1 1st Trimester T2 2nd " T3 3rd " ----------- A - ------------------------------------------ From the analysis of the questionaire, the subjects and the controls were further classifiedi nto the different socioeconomic classes based on profession, educational attainment and income using the method by, Table 3 shows the different classes in each group. The husbands of the subjects and the controls were traders (both petty and large scale), carpenters, farmers, teachers, medical 77 TABLE 2 Age di stribution of the subjects and the controls in the cross-sectional study. Subjects controls Age range T1 T2 T3 c 13-24 9 34 2-3 14 25-31 10 37 n n 9 32-33 1 11 13 3 39-45 1 6 3 sQ 346-51 1 - i T1 - 1st trimester of pregnancy T2 - 2nd " " ' T3 - 3rd " " ' _C _-_ C_on_t_ro_l_s ___ f > ......... ................ practitioners, architects, administrators, and civil servants of various caders. The women on the other hand were engaged in various profit making activities such as those of the men but majority were teachers, civil servants and petty traders. DIET , < r The analysis of the dietary patterns and habits of the subjects and the controls revealed that 547. of the study population consumed 3 meals per day and all of these belonged to the social classes one to three. 30% ate twice and the remaining 137. once daily. The reasons 78 given for this attitude varied and it ranged from lack of fund to such symptoms as nausea. Table 4 shows the various reasons given for missed meals. A 1ist of B- carotene and vitamin A rich foods and the consumption pattern is given in Table 5. TABLE 3 Soci o-economi c c1assi f i cati on of sub jects and controls in the cross sectional study CLASS SUBJECTS CONTROLS T1 T3 C 1 5 11 6 2 a j rVV 20 14 6 3 3 V 19 9 10 4 & 22 11 6 5 17 16 7 < 0 Total 22 as 61 35 Class 1- Acadaemic professionals, senior administrators, proprietors. Class J — Non academic professionals. Nurses, Secretaries and Teachers. Class 3- Non manual skilled workers, Clerks, Typists, Police officers. Class 4- Manual skilled workers, Drivers, Carpenters, Goldsmith. Class 5- Semi-skilled (unskilled workers) labourers, small-scale farmers. 79 TABLE 4 The reasons qiven for mi ssed meals by women. Reasons Number Lack of fund 72 Lack of time 105 Nausea 44 Loss of appetite 41 Other illnesses Weight reduction 20 A list of B-carotene_ and Vit amin A rich foods and their consumption pattern. T2 T3 C 7. N C^2 88 61 35 Food ite y $ f Palm oi1 20 76 56 32 86 Carrots 12 28 17 16 37 Mangoes 14 52 33 22 58.5 Liver 3 10 6 9 14 Milk 11 39 25 11 41.7 Eggs 14 32 23 8 37.4 80 The consumption of B-carotene rich foods was highest for red palm oil followed by mangoes and lastly carrots. The intake of vitamin A containing foods on the other hand was highest for milk followed by eggs with liver coming 1 ast. 86% of the total population used red palm oil more than four times a week, whilst only 147. consumed any form of liver once a week. 37% took carrot£ d 58.57. had mangoes twice a week, Analysis of the vitamin A deficiency signs and symptoms showed that 6(3.5%) subje and 2(5.7)controls had night blindness. 01d corne al’ scars were observed in 23 <117.) of the total population. BLOOD AND PLASMA a The mean packed cel 1 volume (PCV)9 B-carotene (BC)* Vi tamin A> and albumin (ALB) values +/— SD for the subiects and the controls are shown in Table 6. The pev values for the controls ranged between 327. and 40% while those of the subjects was between 28% and 38%. Fig. 4.1 shows the pattern in the subjects and the controls. 2% of the subjects in the 3rd trimester of pregnancy had PCV levels less than 30%. Analysis of r. ' variance as shown in Table 7 revealed that the subjects in the different trimesters had lower plasma values than 81 & Controls in the Cross-Sectional Study* 82 the controls 0.5). Analysis of variance of the F'CV values showed no difference within the subjects and between the subjects and the controls. B-carotene levels were between 45 ug'/i and 120 ug7. for both the subjects and the controls. Table 6 shows the mean +/- S.D while Fig. 4.2 shows the pattern of plasma 83 Mean Plasma B Carotene values of subjects. and controls in the Cross-Sectional study 130­ 120- r io- i o o - 90­ 80­ 3? 70 ­ 6 0 - T1 T2 T3 controls and pregnant subjects B-carotene levels in the subjects and the controls. Only 2% of the total population had B-carotene levels less than 50ug/dl. The mean plasma vitamin A levels ranged from 19.lug to 41.9ug/dl for the controls and 9.1 to 52.3 for the subjects. The controls had vitamin A levels those of the subjects 0.5; P < 0.05). Further analysis o > data showed that only 1 (0.5%) of the total population had plasma vitamin A less than lOug/dl and she belonged to the 3rd trimester of (11.7%) had values in the range of 10 The latter group belonged to the 2nd and 3rd of pregnancy. 14 (23.3%) of the subjects in ?ster had plasma vitamin A levels between 10 and 19.6ug/dl. 60.2% of the pregnant subjects had plasma vitamin A levels between 20 and 29ug/dl majority of which belonged to the 3rd trimester of pregnancy. The cut off levels used are based on the WHO criteria for identifying both clinical and subclinical vitamin A 85 Mean Plasma Vitamin A Value of Subjects and controls in "the Cross-Sectional s tudy 86 Vitamin A ug/cH. deficiency in any population. Plasma albumin levels ranged between 2.9 and 4.2g/dl in the controls while in the pregnant subjects it was 2.2 to 4.0g/dl. The mean +/--S.D are shown in Table 6. Plasma albumin levels were significantly higher in the controls than the subjects 0.5). 47"/. of the pregnant subjects had plasma albumin levels lower than 3.5g/dl Fig.4.4. shows the levels in the controls and the subjects. & 87 TABLE 7 Anal vsi s o-f variance between control s and subjects and wi thin subjects i n each trimester. PARAMETERS CALCULATED TABULAR LEVEL OF TESTED F VALUE F VALUE SIGNIFICANCE Wei ght C vs S 6. 82 2.68 O. 01 Bet S 6.38 3.07 0.01 F'CV C vs S 21.53 2.648 0. 01 Bet S 3.71 X .c07 0.05 BC C vs S 0.97 2 .68 N.S Bet S 0.78 3.07 N.S VIT A C vs S •VA3.53 2.68 0.01 Bet S 2.56 3.07 0.01 ALB C vs S 6.57 2.68 0. 01 Bet S 3. 98 3.07 0.05 N.S — Not significant at P < 0.05 C - Non pregnant non lactating mothers S - Pregnant subjects in the different trimesters F’CV - Packed cell volume B-C - B-carotene VITA“ Vitamin A vs ~ versus. 88 89 RELATIVE DOSE RESPONSE TEST Vitamin A status of women was determined using fasting plasma vitamin A levels and relative dose response . The RDR was calculated as RDR = A5 - AO/ A5 X 100. Table 8 shows the mean +/- S.D o-f the ages, weight, height, basal plasma vitamin A, RDR values of the subjects and the controls. v \ / The RDR values of the subjects revealed that 4 (13.37) of the 30 subjects studied had RDR greater than 207. and all of these had basal plasma values less than 20ug/dl. Three out of the four subjects belonged to the 3rd trimestery while the remaining one belonged to the 1st trimester of pregnancy. 12 subjects had basal plasma vitamin ft levels between 21 and 29 ug/dl. Out of these 2(16.77) had RDR >207. The two subjects were in the 3rd trimester of pregnancy. The remaining 14 subjects who had plasma vitamin A >30ug/dl also had RDR values < 20ug/dl. In the control group none of tahe subjects had RDR > 207. 90 TABLE 8 Mean age weight hei ght basal piasma vitamin A and RDR val Lies of pregnant sub jects and the NF'NL control s. N AGE WT HT BASAL RDR C 10 30.2+/- 57.5+/- 155.6+/- 28.6+/- 15.2+/ 2.9 4.0 ‘ 5:1 T1 lO ’9.7+/- 59.2+/- 156.7+/- 27 .0+/- <\24­. 8+/ 4.: 4.4 4.0 4.8. y 3 7 T2 10 28.9+/- 63.7+/- 156.9+/- 26 w V 16.1+/ 3. 4 4.0 4.7 T3 10 29.1+/- 67.6+/- 157.4+/- 23..:3+/- 19.4+/ 4.0 2 . 8 .0 3.! 4.0 WT - Weight HT - Height RDR - Relative dose response T1 - 1st trimester of pregnancy T2 - 2nd T3 - 3rd ~ Non pregnant non lactating controls 91 LONGITUDINAL STUDY: VITAMIN A*. SINGLE BLIND PLACEBO- CONTROLLED STUDY OF ITS SUPPLEMENTATION EFFECT ON HEALTHY PREGNANT NIGERIAN WOMEN 29 pregnant subjects and controls with normal liver function tests were carried to the end of the study bringing the drop out to 23 (44.27.). 15 subjects and 14 controls were taken through the study but a report will be presented on 28 (14 subjects and 14 controls). The controls were the placebo group wh he subjects were the treated group fed 7,000ita vitamin A in oil daily from the 14th week of pregnancy to 6weeks post partum. They were properly matched for age, age of pregnancy, parity, weight, h _it. (table 9 shows the means +-/-S.D). Blood samples were obtained from each subject and 14th week of pregnancy before supp1ementaiton until 6weeks post partum and also from their babies at paturition. They were bled six times durinrCj'the course of the study. The 1st and the 2nd bleeds represented the 1st and the 2nd halves of the 2nd trimester; the 3rd and the 4th represented the 1st and 2nd halves of the 3rd tirmester; and the 5th and 6th bleeds represented immediate post partum and 6 weeks post partum periods. The plasma sarnies were analysed for B-carotene, Vitamin A, albumin, retinol binding protein (RBF‘) and transthyretin (TTR) . The food intake of 14 of the subjects and the controls was assessed 92 TABLE 9 Mean ( + /-S.D) age, parity, wei qht; of the subjects and the controls (14 subjects in each group AGE PARITY WEIGHT (yrs) (k g > using the method outlined in the ̂ chapter on materials and method- COMPLIANCE AND SIDE EFFECTS We assessed compliance o-f the subjects and the controls by pill count. Overall 987, and 947. of the subjects and the controls respectively took their pill regularly but only those that had 1007. compliance are included in this report. Side effects were almost non existent except for one subject who complianed of persistent headache and was l mmecdi atel y removed from the study. PACKED CELL VOLUME The PCV values of the subjects were maintained with oral vitamin A (7,000 I.U) in oil throughout the study 93 period. Those of the placebo controls decreased . Table 11 shows th^ iftean +/-S.D of plasma albumin levels in the supplemented and placebo groups . The pattern of change in plasma albumin for the two groups are shown in Fig. 4.8 96 97 FiG. Mean Plasma B - tene (BC) 4 -7 values of subj and controls in the Longitudinal study 3 64 DO 63*“ Bleeds L.i — S 98 TABLE 10 Mean +/-S.D. PCV. V IT. and B-C. 1evels i n the supplemented sub 1ects and piacebo controls (14 sub 1ects i n each group Mo of : 1 2 3 4 bleeds PCV (•/.) S 34.1+/- 33.3+/- 34.7+/- 34.0+/ 36. 3+/- 2.3 2.3 1.4 1.7 C 33.1+/- 32.9+/- 32.6+/- 52. 1+7- %300.. 9 +/— 33.6+/- 3.8 3.1 3. 03 2.97 2.3 1.86 VITA(ug/dl> S 25.6+/- 27.9+/- 52.3+/ 1.2+/- 30.8+/- 32.8+/- 6.1 5.7 4.4 .9 4.6 4.4 C 27.2+/- 24.7+/- 24 ,8+/ 22. 8+/- 22.5+/- 25.0+/- 6.9 5.1 5.6 6 . 2 5.5 5.8 B-C(ug/dl) S 60.2+/- 62.1+/- 63.0+/- 65.4+/- 63.0+/- 68.1+/- 16.9 17.7 18.9 13.7 13.8 13.1 C 66.0+/— 64.7+/— 65.6+/— 63.4+/- 64.3+/- 64.3+/- 14.8 < 12"i4 11.3 10.3 10.1 10.1 PCV -Packed cell volume £h-C -B^fecbtene VITA -Vitamin A S -Pregnant subjects supplemented with oral vitamin A C -Praegnant women supplemented with oral placebo 4 ? RETINOL BINDING PROTEIN supplemented and the piacebo groups (14 subjects i n each group No. of: 1 4 5 6 b1eeds ALB S 19.1-^- 18.6 + /- 18.8+/- 18.6+/- 18.2+/- 19.7+/- 3.02 3.4 3.0 3.2 2.4 2.4 19.2+/- 18.1+/- 17.7+/- 17.7+/- 16.05+/- 19.43+/- 3.3 3.2 3. 1 3. 0 ,68 ALB -Albumin RBP -Retinol Binding Parotein TTR -Transthyretin S -Vitamin A supplemented pregnant women C -Placebo supplemented pregnant women 101 FIG. Mean PLASMA RETINOL BINDING k- 9 PROTEIN (RBP) values of —s■—ub cjoenctrtosls ian nthde Longitudinal s tud y--------- 102 PLASMA TRANSTHYRETIN Table 11 shows the means +/-S.D of the TTR levels in both the subjects and the controls. There were no differences between the TTR levels of the subjects when compared to those of the controls except at the 5th bleed when the subjects had significantly higher values than the controls (mg) (g> 3 21.02+/-• 59.36+/- 2.93+/- 12.71+/- 3.36+/- 2.02 11.67 0.16 3. 77 0 .22 18.12+/- 59.36+/- 2.93+/- 11.61+/- 3.34+/- 3.52 9.46 0.38 3. 15 0. 32 VITA -Vitamin A B-C B-carotene ALB Albumin TTR Transthyreti n RBP Retinol Binding Protein S Neonates of the vitamin A group C Neonates of the placebo group 106 u1 FOOD ANALYSIS The food consumption pattern of the pregnant women in the longitudinal study revealed the the during the week­ day 3 (21.5) of all the subjects surveyed consumed 2 meals daily on a regular basis this was reduced to 2 over the weekend. The others ate 3 times daily. Appendix 4.1 shows the typical menu of the population. JThel food samples were analysed using chemical analysis and Food composition Table (FAQ, United Natl:i . Di et history and 24 hour dietary recall were carried out to validate the data on food habits of the subjects. The procedure is outlined in the chapter on materials and methods. The mean+/~ S.D of the various nutrients are shown on Table 14. The results showed that the calories and protein intake of the women ranged between 1452 and 2655 calories and 33 to 131g per day respectively. The fat intake of the subjects varied from 30 to 74 g/day and maj ori ty & was provided by palm oil and groundnut B-carotene intake of the women ranged between 298 and 1399 ug/day. The foods that contributed high B-carotene values were mostly palm oil containing foods such as vegetable soup, bean portage, yam portage, okra soup, moinmoin and akara. The cereals and tubers contained no 107 B-carotene at all. The intake of vitamin A was from 0 to 385ug per day with a median of 168ug. The foods that contributed to the vitmain A intake of the women included milk and eggs. None of the women consumed liver throughout the survey period. The different levels of intake for the different nutrients are shown in Table 14. TABLE 14 Mean +/ — S. D Calories protein, fa<., i-ni m-cne and vi tami n A i ntake of pregnant subjects i n the 1ongi tudinal ts in each group CALS I B-C VITA (cals) (ug) (ug) S 2115.64+/- 6. 47.29+/- 697.36+/- 46.79+/- 309.02 7.89 123.69 177.4 C 2029.59+/- 4: 53.43+/- 438.14+/- 116.71+/ 23" — * ^ 1 . 2 9 5.74 130.51 124.80 Cal -Calori i PRO -Protein FAT -Fat B-C -B-carotene 108 D I S C U S S I O N C H A P T E R V E CROSS-SECTIONAL STUDY SUBJECTS . The ages of the subjects and controls were observed to be in the range of 18 to 48 years with a mean value of 28.6yr. Majority of the women were in the range 25-35 years indicating that the peak of repprr and is comparable to that obtained by Venkatachalam et al, 1962). Plasma Vitamin A levels observed in this study are within the normal range <20 - 50 ug/dl) for the controls as observed by Tiez (1986) but 13(6.3"/!) of the subjects had values below the normal levels. Venketachalam et al. (1962) and Baker et al. (1977) observed mean plasma Vitamin A levels similar to those observed in this study but Gal and Parkinson (1974) observed hi gher levels in their subjects. The di fference may be attri buted to the difference in the socio-economic background of the subject population. Whereas Venkatachalam et al. (1962) studied women in 1 13 India, Gal and Parkinson (1974) studied women from Queen Charlottes Hospital, London- The subjects in this study were from the high, intermediate and low socio economic classes- The mixture of the social classes might explain the similarity of the findings in this study and those of Venkatachalam et ai (1962). The plasma Vitamin A levels decreased as pregnancy progressed. This finding agr•e es v«D from the 1st to the 3rd trimester of pregnancy. Gal .and Parkinson however observed a fall in ea^rly pregnancy, followed by an increase and a few wefl̂ ks before term the levels dropped but not significanly. The significant progressive fall in vitamin A from the 1st to the 3rd trimester observed in this study may be due to the fact that half of the population studied belonged to the intermediate and low socio economic classes. The rise observed by Gal and Parkinson, (1974) was attributed to the circulating p r o g e s t e r o n e levels, The findings of a progressive drop in plasma Vitamin A as pregnancy progressed is further validated by the observation of other workers. 114 H i r 5 1 an cl Shoemater ( 1 94 1.) f oun d t h at 40% of 35 pregnancies had plasma Vitamin A levels below normal r a n g e » Bodansky ?t al (1943) also showed a siqni fi cant difference between the mean values of the first 6 months ar*d the 1 ast 3 rnonths of preqnancy - They ajjLr i buted t his t o t h e s t a rage o f V :i. t a m i n A :i. n thet-? Tf oU e t a 1 1 i v e r a n d u t i 1 i s a t i o n b y t h e f o e t a 1 t i s s u. e„ Lund and Kumb1e (1943) alsa suq ̂ seed t ha t ac t iv e1y growl nq t i ssue may al so ut i 1 i se consi cler ab 1 e amount s of V i t a m i n A „ 1 hi e b a s a 1 m e t a b a 1 i c: r a t e i n c: r e a s e s d u r i n q t hi e 1 a 11 e r h a 1 f o f r e* qnancy and there i s gener a 1 a g r e e m e n t t h a t rn o n-1 Qt t h i s i. n c r e a s e is due to the g r cj w t. h o f t h e f o e t u /. VI h i s rn a y a 1 s o b e i r * part accou n t. e d f or by a possib1e act i vation of maternal endocri ne q1 and ([ )\ \ B o i s , 193yb) - K r The other jf^ason given for a reduction in plasma Vitamin A .LevelVs V in the 3rd trimeser include the possible i n rences with the release of Vitamin A from the liver associated with a probable derangement of the liver during pregnancy (Bodansky et al, 1943). It has been observed that when the liver Vitamin A reaches marginal levels,, there is a conservation mechanism invoked to protect the remaining Vitamin A and the 115 release o-f Vitamin A i s greatly reduced (Underwood, 1990)- Bodensky et. al (1943), suggested that the foetus may make two kinds of demands upon the depot of Vitamin A i n the normal adult 1i ver (500,000 IU to 11,000,000) These include the storage of "Vitamin A in the foetal liver and utilisation of Vitamin A by actively growing foetal tissues. Foetal liver during the 3rd trimester contains considerable amount of Vitamin A while a total store of 12,W O IU is found in the livjer& orf the newborn infant (Lewis et al, 1941). They\suggested that the d ep os 1 1 i on of 12,000 IU in f o e t ? ^ i ver may entail the release of several fold that amount from the maternal 1lver. Though the fate of b 1 ood Vitamin A, arriving either from ingestion .77. of the total population with initial plasma Vitamin A levels between 21 and 29 ug/dl had RDR >/=N one of the subjects with positive RDR (i.e. RDR 207.) had plasma vitamin A levels below 15ug/dl. This observation agrees with the findings of Flores et al (1984). They showed that all the subjects with initial serum retinol of retinyl acetate (Amedee Manesme et al, 1984). The principle behind this test has been described. As liver reserves of Vitamin A become progressively depleted due to chronically inadequate dietary supply conservation mechanisms are invoked to increase the efficiency of Vitamin A utilisastioorn among tissues and to maintain the level that i A t t a i ating to the target tissues (Underwood, 1990). When the liver reserve is depleted below a critical threshold, the rate of release of the remaining reserve is diminished, synthesis of the carrier protein RBP continues and results in the accumulation of a pool of preformed RBP. Providing an exogenous wsource of Vitamin A causes the release of hoIo-RBP at a level and in a characteristic time course relative to the amount of accumulated preformed carrier protein (Loerch et al. 1979. In the light of the available information in the literature and the findings in the present study it is. evident that RDR will predict Vitamin A reserve in an individual with normal liver functions. It can, therefore be concluded from the present study that 13X of the pregnant population examined had liver reserve 307. and therefore none of them was anaemic at the start of the study unlike the children studied by Bloem et al. (1990) who had lower than normal haemoglobin levels. The observation in this study suggest a true beneficial effect of Vitamin A on PCV levels because the controls had a progressive decline in their hematocrit levels while those of the subjects were mai ntai ned ■ v\ > The haematocrit levels were B,9n ificantly correlated to the albumin levels throughout the study period. The reason for this association is not clear. It may however be attributed to increased need for albumin in the synthesis of heme for Hb formation and subsequent increase in the red blood cells. The effect of supplementation was not observed on the haemotocrit levels of the neonates. There was no difference in PCV levels of the neonates born to the subjects when compared to those born to the controls. The factors that cause anaemia in neonates are two-fold haemolytic or haemorrhegic (Behrman and Vaughan, 1987). None of the neonates suffered from any of these disorders. Also the synthesis of red blood cells in the 126 foetus is well controlled and buffered against dietary assaults (Hehrman & Vaughan, 1987). The plasma vitamin A levels in the subjects increased sigificantly than those of the controls from the 1st half of the 3rd trimester until 6 weeks post partum. This observation is consistent with the finedings of various other workers. Wald et al. (1985) observed a small1 but si gi ni f i cnt~ed controls. A-fter 18, 24, 36 months o-f supplementation the mean difference between the groups was 1070, 1220, 1690 IU/L respectively equivalent to 321, 366 and 507 ug/1. All these studies taken together make it clear that Vitamin A supplementation increases serum retinol levels. These studies also show that there is a dose - 128 response relationship as the largest doses of Vitamin A observed the largest increases in serum retinol. Willet et al (1984) concluded that supplementation was more effective among subjects with initially low serum retinol concentrations. This observation agrees with the findings in this study. The pregnant women studied were those who had low levels (between 20 and 30 ug/dl) of Vitamin A to start with and these levels have been associated with marginal deficiency. Most of the studies carried out on the effect of supplementation in pregnancy are consistent with observation in the non pregnant non lactating women. Lewis et al (1943) observed a significant increase (29 IU or 8.7 ug/dl) and also a maintenance of plasma Vitamin A levels in pregnant women receiving 10,000 IU of Vitamin A in the 3rd trimester of pregnancy. More recently, Vi 1 lard and Bates (1983) observed a significant (197.) increase in the plasma Vitamin A levels of supplemented group when compared with the unsupplemented group. The observation from this study and other studies show that Vitamin A supplementation increases the plasma Vitamin A levels either in pregnant or non pregnant 129 women. Its beneficial effect is also evident from the fact that it improved the haematocrit values of the pregnant mothers. The supplemental Vitamin A in this study (7,000 IU is about 3.5 times higher than the recommended dietary allowance (2000 IU or 600 ug/day) . Lewis ejQal (1983) observed that supplemental Vitamin A higher than 10,000 IU is only of little additionaal benef it and this level is adequate to maiantain the plasma w level. is throughout pregnancy. The finding in this study conf i rnms the fact that an additional Vitamin A over and above the normal intake will maintain normal plasma levels throughout pregnancy without the fear of toxicity. Evidence has shown that intake between 25-50,000 IU/day for periods of several months can produce multiple adverse effects (Hathcock et al. 1990). However daily dose as low as 10-20,000 IU over iod of 2 years has been found to produce intracranial hypertension in an 18 year old mal e Vollbracht and Gilroy, 1976). sS In pregnancy, various levels of supplemental Vitamin A have been associated with birth defects. Vitamin A between 18 - 500,000 IU taken acutely or chronically have been associated with several toxic symptoms 130 (Mounoud et al, 1975, Von Lennepet et al , 1985; Bernhardt and Dorsey, 1974). The U.S. RDA for pregnant women is 4000 mg/kg respectively. In the rats the LDc^ values for retinyl palmitate, all-trans RA, 13-Cis RA and etretinate given by oral intubation are 7910 (14.4 >; 10“ IU), 2000, >4000, and >4000 mg/kg respectively (Kamm et 131 al . 1984). The LDc--, for monkeys was estimated to be 168mg <0.56 x 1r0t 6 IU retinol/kg The highest dosage for each specie was 250 times higher on a body weight basis than the human RDA for Vitamin A which is approximately 0.06 mg <110 IU) retinyl palmitate per kg/day. However this dose should not be construed to indicate that an intake of 250 times the RDA is safe for humans because there is difficulty in extrapolation between species. In the light of all these findings, it is safe to advocate supplementation of Vitamin A in pregnancy to the tune of 10,000 IU per day for pregnant women especially in the 3rd trimester. This suggestion is firmly supported by the autopsy findings in the Swedish and Ethiopian infants. The Ethiopian infants were observed to have liver reserves sufficient for 5 — 6 days compared with two months^ available to the Swedish group (Gebre-Medhin and Valquist, 1984). This suggested both the capability of the early infants to build stores as well as the influence of the mothers Vitamin A status on the infant. 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GIVE CONSENT TO PARTICIPATE IN THE ABOVE NAMED F'ROJEb C^1 I HEREBY DECLARE THAT I WAS INFORMED OF THE PROTOCOL OF THE STUDY PRIORt TO MY CONSENT _________________________________________ SIGNATURE OF PARTICIPANT WITNESS DATE--------- V,---------- d a t e- A P P E N D I X 1 1 QUESTIONAIRE FOR VITAMIN A STUDIES IN PREGNANT AND NON PREGNANT NON LACTATING NIGERIAN WOMEN PLEASE SPECIFY OR TICK THE APPROPRIATE ANSWERS t TlO THE QUESTIONS BELOW: 1. CODE NO------------ 2. HOME ADDRESS------- 7 3. AGE/BATE OF BIRTH.- - — 4. EDUCATION 0 1 HUSBAND WIFE 5.OCCUPATION: HUSBND & WIFE 6. INCOME:: <1100 0 Q \ 1V-33000 3-6000 6-9000 >9000 HUSBAND WIFE 7. ARE YOU PREGNANT ? YES NO a. IF YES TO Q7 PLEASE STATE AGE OF PREGNANCY/ EXPECTED DATE OF DELIVERY: 9. PARITY-------------------------------- 10. 24 HR DIETARY RECALL B/FAST LUNCH SUPPER SNACK 11.DIETARY HABBIT HOW OFTEN DO YOU EAT DAILY ?--------------------- IF YOU MISS ANY MEAL WHICH ONE DOY OFTEN MISS? WHAT IS/ARE YOUR REASON/S FOR MISSING THE MEALS WHAT FOODS DO YOU EAT DAILY--------------------------- WHAT FOODS DO YOU EAT OCCASSIONALLY----------------- WHAT FOODS DONT YOU EAT AT ALL---------------------- GIVE REASONS PLEASE---------------------------------- 12. ASSESSMENT OF VITAMIN A INTAKE HOW OFTEN DO YOU EAT THE FOLLOWING FOOD ITEMS WEEKLY? FOOD ITEMS jQv FREQUENCY/WK LIVER------------------------------------ ^ ________________________________________________________________________ CARROTS-----— ----- ------------------- MANGOES-- -r---------------------------- SWEET POTATOES— 13. KNOWLEDGE ABOUT VITAMIN A DO YOU KNOW WHAT VITAMIN A IS? YES NO IF YES TO Q13 DO YOU KNOW WHAT DISEASE ITS LACK CAN CAUSE? YES NO 14. ARE YOU CURRENTLY ON ANY MULTIVITAMIN TABLETS YES NO 160 15.IF YES WHY? 16. DO YOU SEE PROPERLY IN THE EVENINGS IN A DIMMLY LIT ENVIRONMENT YES NO 17. IF HOW LONG DOES IT TAKE YOU TO ADJUST YOUR SITE WHEN YOU ENTER A DIMMLY LIT ENVIRONMENT? 1MIN iMIN >5M IN 10MIN 18.HEALTH ASSESSMENT WEIGHT(KG)--------- HEIGHT (CM)--------- o r EYE EXAMINATION SCAR ULCER DRYNESS BITOT SPOT CONDUCTIVA j p CORNEA $ SCOT------------ $ SGPT------ URINALYSIS- S ' - PLASMA VITAMIN A PLASMA B--CAROTNENE---- ac;MAA VAZLVIBUMIN— ---------- PLASMA TRANSTYRETTIN------------ PLASMA RETINOL BIND £ NG PROTEIN-~ 161 B--CAROTENE AND VITAMIN A CONTENTS OF VARIOUS FOOD ITEMS COMMONLY CONSUMED (u q /IOOq ) FOOD ITEMS B--CAROTENE VITAMIN A Ri ce Bean portage 460 Bread Arnal a Eba (gari) P1 ant ai n (-fried) Pap (eko) Yam Egg (raw) ■fri ed boi1ed A k a r a Moinmoi n Bee-f Fi sh Ewedu Okra Vegetable Mi 1 k 580 520 Soft drink Margari ne 80 162 P O S T G R A D U A T E I N S T I T U T E FOR M E D I C A L R £ S E a - _ ".-■vw.>7 AWD T R A I N I N G “v- m COLLEGE OF MEDICINE, UNIVERSITY OF IBADAN, IBADAN, NIGERIA REF. Cables & Telegrams: I M B fiD t ftrf ~ AJ Telephone: I t Em 2 S 3 lr - Direct Line 415453 25 - u j u s t 1989 H is s ly a b o d e A d e y e fa D ep artm en t o f Human N u t r i t i o n C o l l e g e o f M e d ic in e U n i v e r s i t y o f Ib ad an D e a r M iss A d e y e fa R e : VITAMIN A STUDIES IN PREGNANT T h e t e x t o f t h e s t u d y p r o t o c o l o f t h e a b o v e p r o j e c t h a s b e e n r e v ie w e d b y t h e C o m m ittee . The m e th o d o lo g y i s i n c o n fo r m it y w it h t h e a p p r o p r ia t e r u l e s and g u i d e l i n e s l a i d down f o r c o n d u c t in g r e s e a r c h i n t h e U n i v e r s i t y . O I t h e r e f o r e a p p ro v e on b e h a l f o f t h e J o in t U n i v e r s i t y o f Ib a d a n / U n iv e rs j C o l l e g e H o s p it a l E t h i c a \ C o mini 11 e e t h a t you may p r o c e e d w it h t h e s t u d v . B e s t r e g a r d s You G y e w a l cX Tornori D i r e c t o r A C hairm an E t M e ul Commi11 ee