Volume 9 Number 2 August 1995
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MethodsSample Preparation: Grass pea samples of local variety were obtained from a market in Ambo, Shoa. All the samples were cleaned manually to remove foreign matters and aliquots were ground in a cyclone mill for estimation of antinutritional factors. The remaining whole grain samples were used for decortication, boiling and/or cooking processes as described below. Preparation of heat treated grass pea meals Dry heat treated grass pea meals: 10g of finely ground grass pea flour was heated in a beaker at a thickness not exceeding 1 cm in an oven maintained at 200°C for 30 minutes. Autoclaved grass pea meals: the autoclaved grass pea meals were prepared by autoclaving 10g of finely ground grass pea flour in a beaker at a thickness not exceeding 1 cm at 12ooc (15 Ib/in2) for 10 minutes as described by Tan et al (18). The autoclave was preheated to minimize the time required to reach the desired temperature (approximately 7 minutes). Boiled grass pea meals: about 10g of whole grass pea seeds were boiled in 100 ml of distilled water for 20 minutes. The cooking water was drained off, and the seeds were dried in an oven at 80OC overnight and were ground to yield the boiled grass pea meal. Cooked grass pea meals: whole grass pea seeds (10g) were soaked in 30mI of distilled water at room temperature for 24 hours. After the soaking water was drained off, the seed were cooked in sufficient amount of boiling water 4 hours. The Cooked seeds mar were boiled to 'eating soft' were then dried in an oven at 80°C overnight before grinding fur fine flour. Preparation of grass pea tempeh: grass pea tempeh was prepared using the procedure of Ko and Hesseltine (19). The pretreated grass peas were inoculated with a powder-form (10% of the substrate) of inoculum of Rhizopus oligosporus at 10s colony forming units/gram (cfu/g) prepared substrate. The inoculum of R. oligosporus was obtained from the Nutrition Research and Development Centre at Bogor , Indonesia. Determination of .p-N-oxalyl-L-a,.p- diaminopropionic acid: the ODAP content of heat treated grass pea meals were determined using Rao's method (20). A hundred milligram of grass pea meals was extracted with 10ml of 60% ethanol with mechanical sh3;king, for 6 hours at room temperature. After centrifugation, 100p.l of the extracts were taken in duplicate in test tubes. To one set of tubes, 0.2 mI of 3N KOH was added and the tubes were kept in a boiling water bath for 30 minutes. After cooling the tubes to room temperature, the samples were brought to 1 ml with water and 2 ml OPT (o-Phthalaldehyde dissolved in 95% ethanol and mercaptoethanol) reagent was added. The absorbency of the yellow solution was measured after 30 minutes at 420 nm against a reagent blank using Beckman DU-60 spectrophotometer (Beckman, USA). Determination of the tannin content: the method of Bums (21) as modified by Max son and Rooney (22) was used for tannin determination. One gram of grass pea meal was extracted with 10 ml of 1% concentration HCl in methanol for 24 hours at room : temperature. After centrifugation at l0000g e (Sorval, USA) for 5 min, lmI of,supematant was mixed with 5 ml of Vanillin-HCl reagent, a and the absorbency was read at 500 nm after :d 20 minutes. Values of tannins were expressed :r in mg of D-catechin/gm of sample. Determination of the phytic acid content: 19 Phytic acid content was determined using the method of Haug and Lantzsch (23). An aliquot (200mg) of grass pea meal was extracted with 10 mi of 0.2N HCI containing 10% Na2SO4 using a mechanical shaker for 3hours at room temperature. The extract (0.5 mi) was pipet ted into a test tube fitted with a ground glass topper. One mi of ferric solution (ammonium iron (III) sulphate dissolved in 2N HCI) was added. The tubes were heated in a boiling water bath for 30 minutes, and after cooling in ice water for 15 minutes, they were allowed to adjust to room temperature. After centrifugation for 10 minutes at 5000 g, 1 mi of the supernatant was transferred to another test tube and 1.5 mi of bipyridine solution was added. The absorbency was read at 519 Dm (Beckman, USA) against distilled water . Determination of trypsin inhibitor activity (FIA): Trypsin inhibitor activity of heat treated grass pea meals was determined using the method of Kakade et al (24). N-benzoyl-DL- arginine-P-nitroanilide (BAPNA) was used as the trypsin substrate. One trypsin unit (TU) was arbitrarily defined as an increase of 0.01 absorbency units at 410 nm per 10 mi of the reaction mixture. Trypsin inhibitory activity was expressed in terms of Trypsin Units Inhibited (TUI). Total TIA was expressed as TIU/g (dry weight). Analysis of variance was used to all the data. Duncan's multiple range test was used to locate differences between means. ResultsPhytates, tannins TIA and ODAP contents of heat treated grass peas are presented in Table 1. The amount of phytic acid in raw whole seeds was 476.3 p.g/g. Phytic acid was greatly diminished by cooking (59.4%) and substantially removed by autoclaving (21.2%) but decreased only slightly upon boiling (11%). Dry heat treatment, however, effectively eliminated all phytic acid in the grass pea seeds (Table 1). A reduction of about 98.4% in the phytic acid content of grass peas, however, was shown to occur when grass peas were fermented into tempeh (Table 2).
hours in boiling water to 'eating soft' reduced the trypsin inhibitor activity by 81% whereas boiling the whole seed for 20 minutes inactivated 89% of the TIA. Prefermentation treatments of grass pea for tempeh inactivated Table 1: Effect of different heat treatments on antinutritional factors in grass pea*
* Data are mean values of three measurements Means of the same column followed by different letters differ significantly (P<0.05) Table 2: Effect of tempeh preparation process on antinutritional factors in grass pea*
* Data are mean values of three measurements Means of the same column followed by different letters differ significantly (P<0.05) from 88.4 to 93.4 % of the TIA. Fermentation of grass pea into tempeh further reduced the TIA by 94.9% (Table I). Dehulled grass pea seeds contained 23231 IU/g of TIA which was decreased upon fermentation to 88.4%. Tannin content of whole grass pea appeared to be high (6.7 mg/g) (Table 2). Pretreatment of dehulled grass pea prior to tempeh fermentation (soaking and boiling) significantly decreased the contents of tannin (51.2-84.7% ). However, fermentation of pretreated dehulled grass pea into tempeh significantly increased the content of tannins in grass peas (Table 2). The content of tannin in final tempeh product increased 6.6-fold compared with dehulled seeds. The neurotoxin, ODAP, content of raw grass pea was 4763 p.g/g (Table 1). Cooking to 'eating soft' and heat treatment process effectively reduced the ODAP content by about 78 and 77% , respectively. The least effective processing of grass pea for fermentation into tempeh significantly (p grass pea further reduced the ODAP content by 93% (Table 2). loss of TIA of cowpeas was, however, reported by Ologhobo and Fetuga (14) after autoclaving and soaking. Such heat treatments have also been reported to be effective in inactivating the trypsin inhibitors in several food legumes (12,15).
The results also indicate that the ODAP content of the grass peas was greatly diminished by cooking and dry heat treatment but reduced only slightly upon autoclaving and boiling. However, a recent study using chick bioassay with these same processing methods did not indicate the toxic substances in Lathylls sativus to be completely removed or destroyed (32). In a more recent study, the loss of ODAP, however, was 52-82% when grass pea seeds were soaked for three days in different soaking media (33).
value of the seeds of this legume. 2. Davies NT, Nightingale R. The effect of phytate on intestinal absorbtion and secretion of zinc and whole body retention of zinc, copper, iron and manganese in rats. Br J Nutr 1975;34:243-58. 3. Singh M and Krikorian AP. Inhibition of trypsin activity in vitro by phytate. J Agric Food Chem 1982;30:799-802.
4. Liener IE. Significance for humans of biologically active factors in soybeans and other food legumes. J Assoc Oil Chem Soc 1979;56:121-9. 5. de Lumen BO, Salamat LA. TIA in winged beans (Psophocarpus tetragnolobus) and the possible role of tannin. J Agric Food Chem 1980;28:533-6.
6. Rao SNL, Malathi K, Sharman PS. Lathyrism. World Rev Nutr Diet 1962; 10:214- 38. 7. Tekle-Haimanot R, Y, Wuhib E, Kasina A. Kidane Y, Alemu T, Spencer PS. The epidemiology of lathyrism in North and Central Ethiopia. Eth Med J 1993;31:15-24.
8. Dwivedi MP. Epidemiological aspects of lathyrism in India -a changing scenario. In: Spencer P.S. (ed.) Grass Pea: Threat and Promise. New York, Third World Medical Research Foundation. 1989; 1-26. 9. Haque A, Mannan MA. The problem of lathyrism in Bangladesh. In: Spencer PS. (ed.) Grass Pea: Treat and Promise. New York, Third World Medical Research Foundation. 1989;27-35.
10. Teutenico RA, Knorr D. Impact of biotechnology on nutritional quality of food plants. Food Techno11985; 39:127-34. 11. Pardez-Lopez, Harry GI. Change in selected chemical and antinutritional components during tempeh preparation using fresh and hardened common beans. J Food Sti 1989;54:968-70.
12. Ologhobo AD, Fetuga BL. Trypsin inhibitors activity in some lima bean varieties as affected by different processing methods. Nutr Rep IntI 1983;27:41-9. 13. Ologhobo AD, Fetuga BL. Distributionof phosphorus and phytate in some Nigerian varieties of legumes and some effect of f processing. J Food Sci 1984;49:199-201.
14. Ologhobo AD, Fetuga BL. Effect of processing on trypsin inhibitors, haemagglutinins, tannic acid and phytic acid contents of seeds of 10 cowpea varieties. Trop f Agric 1984;64:261-4. 15. Khokar S, Chauhan BM. Antinutritional factors in moth bean, varietal differences and If effects of domestic processing and cooking. J Food Sci 1986;51:591-4.
16. Urga K, Fite A, Gebretsadik M. Influence lof processing methods on cooking time and nutritional quality of grass pea. In: Berhanu Abegaz Molla, Redda Teklehaimanot, Palmer, V.S., Spencer, P.S. eds. Proceedings of the Second International Lathyrus/Lathyrism Conference in Ethiopia. New York: Third World Medical Research Foundation, 1993:119-34. 17. Roy DN and Bhat N. Variation in neurotoxin, trypsin inhibitors and susceptibility to insect attack in varieties of Lathyrus sativus seeds. EnvironPhysiolBiochem 1975;5:172-5.
18. Tan NW, Wang KC, de Lumen BO. Relationship of tannin levels and trypsin inhibitors activity with in vitro protein digestabilities of raw and heat treated winged beans. J Agric Food Chem 1984;32:819-23. 19. Ko SD, Hesseltine CW. Tempeh and related foods. Econ MicrobioI1979;9: 115-40. Rao SLN. A sensitive and specific colorimetric method for the detennination of a-6- diaminopropionic acid and the Lathyrus sativus neurotoxin. Anal Biochem 1978;86:386-95.
21. Burns ME. Method of estimation of tannins in grain sorghum. Agron J 1971;63:511-2. 22. Maxson ED, Rooney LW. Evaluation of methods for tannin analysis in sorghum grain. Cereal Chem 1972; 44:719-29.
23. Haug W, Lantzsch HJ .A sensitive method for the rapid determination of phytate in cereals and cereal products. J Sci Food Agric 1983;34:1423-6. 24. Kakade ML, Rackis JJ, McGhee JE, Puski G. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem 1974; 51:376-82.
25. Chang R, Schwimmer S, Bum K. Phytate removal from whole dry beans by enzymatic hydrolysis and diffusion. J Food Sci 1977 ; 42:1098-111. 26. Sutard: Buckle KA. Phytic acid change in soy;beans fermented by traditional inoculum an4 six strains of Rhizopus oligosporus. J App BacterioI1985;58:539-43.
27. Wang HL, Swain EW, Hesseltine CW. Phytase of moulds used in oriental food fermentation. J Food Sci 1980; 45:1266-9. 28. Reddy NR, Peirson M D, Sathe SK, Salunkhe DK. Dry bean tannins: a review of nutritional implications. J Assoc Oil Chem Soc 1985;62:541-7.
29. Mugula JK. The nutritional quality of sorghum -common bean tempeh. Plant Foods for Human Nutr 1992; 42:247-56. 30. Roozen JP, Groot T. Analysis of low levels of trypsin inhibitors in foods. Lebensm-Wissen Und Technol 1985;20:3305-8.
31. Wang HL, Vesp JV, Hesseltine CW. Release of bound trypsin inhibitors in soy beans fermented by R. olighosporus .J Nutr 1072;102:1495-9. 32. Moslehuddin ABM, Hang YD, Stoewood GS. Evaluation of the toxicity of processed Lathyrus sativus seed in chicks. Nutr Rep Inti 1987;36:851-8.
33. Urga K, Gebretsadik M. Effect of soaking time and soaking solution on the nutritional quality of grass pea seeds. Ethiop J Health Dev 1993;7:79-83. In many parts of Africa, villagers prepare fermented beverages from maize, sorghum, millet, barley or from various mixtures of these cereals. There is some information on the fermentation of a variety of African beverages such as Pito, Burkutu and Obiolor from Nigeria (1-3), Kaffir or Bantu beer from southern Africa (4), Merissa from Sudan (5), .Busaa from Kenya (6) and Tella from Ethiopia (7). who cannot afford a reasonable meal. In most open markets in southern Ethiopia, horde and shamita are available for purchase. ____________________ Table 1: counts of the various microbial groups in shamita and borde pH
log (cfu/ml)
AMC1 Entero-bacteriaceae Yeasts
LAB2 Shamita
Mean
4.2
9.00
6.38
7.01
8.92
SD
0.02
0.63
0.60
0.78
0.67
%CV
2.1
7.0
9.4
11.1
7.5
Borde
Mean
4.1
9.13
6.37
8.27
8.95
SD
0.15
0.75
0.72
0.49
0.49
%CV
3.7
8.4
11.3
5.9
5.4
fermentation as noted by constant gas production. The product turns too sour four hours after being ready for consumption. A laborer usually consumes about three to four liters of shamita and a liter costs about USD 0.15.
MethodsSource and Collection of Samples; This study was carried out in Awassa, a town located 275 km south of Addis Ababa. It has a population of about 63,000. A total of 30 samples each of borde and shamita were collected at random from open-market vendors on different sampling days. Microbiological analysis was conducted within two hours of collection. Microbiological analysis; Twenty-five ml of borde or shamita were sampled aseptically at 12 or 24 hr intervals. They were separately diluted in 225 mI of sterile water and processed for the following microbiological tests. Aerobic mesophilic bacteria were Table 2: Distribution (%) of dominant microorganisms in Shamita and borde
*AMC, Aerobic mesophilic count (log cfu/ml) I Bacillus II Lactobacillus III Micrococcus IV Staphylococcus V Acinetobacter VI Streptococcus analyzed after further dilution of samples in sterile water. Volumes of 0.1 ml of appropriate dilutions were spread-plated in duplicate on pre-dried surfaces of Plate Count Agar (PC; Merck). Colonies were counted after incubation at 30 to 32 °C for 48 hrs. For the enumeration of Enterobacteriaceae volumes of 0.1 mI of appropriate dilutions were spread plated in duplicate on pre-dried surfaces of Violet Red Bile Glucose (VRBG) Agar (Oxoid) plates. The plates were incubated at 30 to 32 OC for 24 h. purple red colonies were counted as members of Enterobacteriaceae. For counting lactic acid bacteria, volumes of 0.1 mI of appropriate dilutions were spread plated in duplicate on pre-dried surfaces of de man, Rogosa sharpe (MRS) agar (Oxoid) plates. Colonies were counted after incubation in an anaerobic jar (Oxoid) at 32 °C for 48 h. For the enumeration of yeasts and mo.lds, volumes of 0.1 ml of appropriate dilutions were spread plated in duplicate on pre-dried surfaces of chloramphenicol-bromophenol-blueagar ( CBB) consisting of yeast extract, 5.0; glucose, 20.0; chloramphenicol, 0. 1; bromophenol blue, 0.01; agar, 15; pH, 6.0 to 6.4 (g/1 in distilled water). Yeast colonies were counted after incubating the plates at 25-27 °C for 5 days. Flora assessment was done as follows. After colony counting, 10-15 colonies were selected at random from countable PC agar plates. The sub-cultures were further purified by repeated plating and differentiated into various bacterial groups by the following characteristics. Phase- contrast microscopy was used to examine cell shape and grouping, presence or absence of endospores and motility. Gram reaction was determined using the KOH test of Gregersen (9). Cytochrome oxidase was tested by the method of Kavacs (10). Catalase test was made with 3% (v/v) H2O2 solution. Glucose metabolism was investigated by the O/F test of Hugh & Leifson (11). Biochemical Analyses: pH was measured by aseptically placing the electrode of a digital pH meter in the samples. Moisture content was determined by drying a sample to constant weight in a ventilated thermostatic oven at 70 °C. Borde and shamita samples were freeze dried and stored at 4 °C until they were further analysed for protein, fat and ash. To determine total protein content, two 9 sample was digested with 15 mI concentrated sulphuric acid for 45 min at 410 °C. A solution of sodium hydroxide -sodium thiosulphate was added to the digest and distilled into a solution of boric acid and titrated with 0.2N hydrochloric acid according to AOAC 981.10 (12). Protein percentage was calculated by multiplying %N by 6.25. For fat determination a two gm sample was solubilized in alcohol (2 ml) and hydrolyzed with 10 ml concentrated hydrochloric acid at 70-80 °C for 40 min. The hydrolyzed fat was extracted with petroleum ether. The ether was evaporated from the extract and the fat was dried to constant weight at 100 °C for 90 min according to AOAC 922.06 (12). Ash was determined by igniting a five gm sample in furnace at 550 °C to constant weight according to AOAC 923.1(12). Protein availability was estimated by the in vitro disappearance of dr)' matter after treating a sample with papain as described by Kazanas and Fields ( 13). Table 3: Protein, fat and ash content of horde andshamita
as determined by Agren and Gibson (17). N.D. notdetermined ResultsBorde and shamita had low pH values(<4.2) with insignificant variation within samples (Coefficient of variation, CY, <4%). Counts of aerobic mesophilic bacteria and lactic acid bacteria were also high (around 109 cfu/ml) for both products. Counts of Enterobacteriaceae was over 106 cfu/ml in both products but yeast counts were slightly higher in horde samples. In all counts variations within samples were markedly low (CY, 5- 11%) (Table 1). A total of 790 isolates were obtained from countable PC plates in this study. The aerobic mesophilic flora was dominated by a variety of bacterial genera (Table 2). Bacillus spp. heavily dominated the microflora in shamita. The major genera that dominated horde were Bacillus, Micrococcus and Lactobacillus spp. The mean moisture contents of horde and shamita were 86.43% and 81.03%, respectively. Shamita had slightly higher content of protein and ash than horde (Table 3). No marked difference was observed in their fat content. Over a third of the total protein in both fermented beverages was soluble. DiscussionHorde and shamita could be considered as acidic beverages. The low pH observed 'in horde and shamita is in agreement with other observations tnade during fermentation of traditional beverages in Sudan (5) and Nigeria (3). The low pH value obtained in a short fermentation time showed that sufficient fermentable sugar was available and the lactic acid bacteria involved were strong acid producers. Despite the high yeast counts, both beverages are known to have low alcohol content due to the short fermentation time. The products are desired to have low alcohol content and, to this effect, are usually consumed within two to three hours. A similar non-alcoholic fermented beverage was reported from Nigeria (3). Longer holding could render the products too alcoholic for meal replacements. The high amount of gas in the fermented products was indicative of the activity of yeasts and other gas producing microorganisms. Lactic acid bacteria and yeasts are important in the fermentation of various African fermented beverages or foods made from cereals (1 ,3,6, 14,15). The count of Enterobacteriaceae in both products was in the order of 106 cfu/ml and these could contribute to acid production in the early stages but could be inactivated at the final pH level. Some members of Enterobacteriaceae were reported to be involved in the fermentation of African maize beverages or fO fermentation of Obiolor, a Nigerian acidic non-alcoholic fermented beverage (3). The high microbial load in horde and shamita could make the products good sources of microbial protein and this might contribute to their role as meal replacements. Some members of Enterobacteriaceae are reported to synthesize some vitamins while the lactic acid bacteria and yeasts could be responsible for production of lactic acid and flavour components, respectively (14). , The protein contents of roasted Ethiopian barley and maize, which are used in the production of shamita and horde, are 9.0% and 8.7 % , respectively ( 17) .The fermented products appeared to have higher protein content. A marked increase was also noted in fat and ash content in the fermented products. The increment in the various nutrients could be due to the proliferation and action of microorganisms during fermentation. Similar increase in crude protein and fat was also observed during the traditional fermentation of maize and sorghum in Nigeria (18). Heat treatment of the grains could also encourage microbial protein production as noted by Abasiekong (18). AcknowledgementsThe technical assistance of Haile Alemayehu and Tsigereda Bekele is acknowledged. Tetemke Mehari did the biochemical analysis at the University of Nebraska, Lincoln during his annual sojourn financed by the Rockfeller Foundation Biotechnology Career Fellowship. References1. Ekundayo JA. 'lne production of pito, a Nigerian fermented beverage. J Food Technol 1969;4:217-25. 2. Faparusi SI. Sugar changes during the preparation of Burkutu beer. J Sci Food Agric 1970;21:79-81. 3. Achi OK. Microbiology of 'obiolor':a Nigerian fermented non-alcoholic beverage. J App Bacteriol 1990;69:321-25. 4. Novellie L. Kaffircorn malting and brewing studies. 3. Determination of amylases in kaffircorn malts. J Sci Food Agric 1959;10:441-49. 5. Dirar HA. A microbiological study of Sudanese merissa brewing. J Food Sci 1978;43:1683-6. 6. Nout MJR. Microbiological aspects of the traditional manufacture of Busaa, a Kenyan opaque maize beer. Chem Mikrobiol Technol Lebensm. 1980;6: 137-42. 7. Sable S and Abegaz BG. The microbiology of tella fermentation. Sinet: Ethiop J Sci 1991;14:81-92. 8. Steinkraus KH. Handbook of indigenous fermented foods. New York, Marcel Dekker Inc. 1983. 9. Gregersen G. Rapid Method for distinction of gram negative from gram Positive bacteria. Eur J Appl MicrobioI1978;5:123-7. 10. Kovacs N. Identification of Pseudomonas pyocyanae by the oxidase reaction. Nature 1956;178:703. 11. Hugh R. and Leifson E. The taxonomic significance of fermentative versus oxidative gram negative bacteria. J BacterioI1953;66:24-6. 12. A.O.A.C. Official Methods of Analysis. 15th ed. Herich, K ed. Association of Official Analytical Chemists, Arlington, Virginia, 1990. 13. Kazanas N. and Fields NL. Nutritional improvement of sorghum by fermentation. J Fd Sci 1981 ;48:819-21. 14. Akinrele IA. Fermentation studies on maize during the preparation of a traditional African starch-cake food. J Sci Food Agric 1970;21:619-25. 15. Sanni A, Loenner C, Marklinder I, Johansson ML and Molin G. Starter cultures for the production of ogi, a fermented infant food from maize and sorghum. Chem Mikrobiol Technol Lebensm 1994;16:29-33. 16. Anon. Microbiol Ecology of Food commodities. New York: Academic Press. 1980. \ 17. Agren G and R Gibson. Food Composition Table for Use in Ethiopia 1. CNU report No.16, 1968. 18. Abasiekong SF. Effect of fermentation on crude protein and fat contents of crushed grains of maize and sorghum. J Appl Bacteriol 1991;70:391-3.
IntroductionIodine is an essential trace element required for human growth and development. It is mainly obtained by way of food and water consumed. Determination of its level in ready- to-eat food provides a reliable estimate of the amount of iodine ingested ( I) .Iodine intake can also be evaluated by indirect indices such as estimation of the thyroid uptake of the radioiodine, measuring thyroid hormones using radioimmunoassay and determination of the urinaJy iodine (2,3,4). Of all these methods urinary iodine measunnent has the advantage of applicability in field conditions. Estimation of daily urinary iodine concentration in a sufficient number of casual samples as urinary- iodine to creatinine ratio or microgram iodine per day (p.gl/day) had been a good approach and remains the most convenient index of iodine intake. Investigators in a large number of studies of iodine deficiency disorders (IDD) have used urinary iodine to evaluate dietary intake of iodine in communities (5,6). Studies in Thailand (7) have found that urinary iodine distribution patterns correlate well with the regional prevalence of goiter. In order to ensure safe plasma inorganic iodine level of 0.1 p.gl/dL, an average intake of 70 p.gl is required. This would allow for 50 p.g of urinary iodine excretion which is considered as the minimum quantity of adequate thyroid hormone synthesis (8) . Several reports (7,9,10) indicate that moderate IDD is generally associated with urinary iodine ranging from 25 to 50 p.g per day while, severe IDD occurs when daily excretion falls below 25 p.g iodine per day. Therefore, urinary iodine assessment, helps in the evaluation of available nutritional iodine in a given community .Iodine content in water is also an indication of the level of iodine consumed with locally produced food. However, drinking water provides ten percent of the average daily body requirement (11). Observation of its level in water could give supportive evidence to the consumption level of iodine and may reflect the urinary iodine excretion in a given locality. This study was part of a baseline survey of IDD, carried out before implementing iodine intervention programme to identify the existing situation of goitre. It was substantiated with determination of VIE which could be used as a reference in evaluating the impact after distribution of iodated salt. ___________________ 1From Ethiopian Nutrition Institute P.O.Box 5664 Addis Ababa MethodsStudy Sites; The country-wide goitre prevalence survey of 1981 (12) was used to select five goitre endemic regions (prevalence greater than 20% ) and four nonendemic regions (prevalence less than 5% ). The endemic regions and their corresponding sampling sites were Shoa (Majetie), GamuGofa (Sawla), Shoa (Gohatsion), Bale (Adaba) and Gojam (Bure) while sites taken as nonendemic regions were Harerghie (Alemmaya), Shoa (Mojo), Sidamo (Yabello), and Arsi (Huruta). The study was conducted from 1988 to 1991. Sampling procedure; In all the survey sites, data on household members were obtained from Kebele (lowest administrative units) and then properly labeled call cards were distributed to households selected systematically for goitre examination and obtaining information like age, sex, and drinking water source. Sub-samples were selected systematically matching to age and sex, from sample populations with predetermined sample size of 50 to 100 for collection of urine samples. A total number of 5399 subjects from areas taken as goitere endemic and 60 10 from sites taken as non- endemic were included in goitre examination. Thyroid size estimation was accomplished according to the criterion established by WHO /PAHO (13). Urine samples were collected in duplicate from subjects in all study sites. Creatinine was determined in the urine samples prior to determinating the urinary iodine (14). Creatinine value was used as a basis for computing urinary iodine as iodine / creatinine ratio in casual urine samples and expressed in microgram iodine per day (p.gl/day) (15). Analysis of urinary iodine was carried out by the method of Sandell and Kelthoff, as described by Barker (15). Water samples were collected from drinking water sources in polyethylene bottles washed with distilled water and then rinsed with deionized water except from Sidamo (Yabello). Iodine content in water samples was determined according to the method of Rogina and Dubravcic (16). Classification of urinary iodine excretion ranges were based on the method of Follis (6) which indicates iodine consumption level and IDD endemicity of a group or community. Statistical analysis: The data were tested for skewedness and transformed to square root. The level of correlation was performed on a SPSS/PC soft-ware package. ResultsTable I shows total goitre rate, urinary iodine excretion and the level of iodine in water. As indicated, Alemmaya (Harerghie) and Yabello (Sidamo) have UIE rates of greater than 100 p.gl/day whereas Mojo (Shoa) and Huruta (Arsi) have UIE between 50 and 00 p.gl/day. Other study sites have UIE values of less than 50 p.gl/day. TGR higher Table 1: Mean urinary lodine in relation to total goitre rate and water iodine by study sites
SE = Standard error Number in parentheses are number of samples taken (r = 0.839 for UIE and water iodine), (4 = -0.5148 for UIE and TGR) than 20% was noted in 80% of the study subjects with VIE less than 50 JLgI/day. Consequently, a mean VIE rate of 58.50 +14.75 JLgI/day was observed against total mean goitre rate of 21.80 +.5.40 was noted for both endemic and nonendemic sites. A positive and highly significant (p<0.05) correlation (r=0.8399) was also observed between UIE and iodine concentration of water sources. Mean urinary iodine excretion values in Bure (Oojam) and Oohatsion (Shoa) were below 25 JLgI/day whereas concentrations of iodine in water in Bure (Oojam) and Oohatsion (Shoa) were 0.4 JLg/1 and 15 JLg/1, respectively. Table 1, shows UIE range, total goitre rate (%TOR) and absolute number of people having goitre out of the biochemical groups, by sex. In females, the highest value, 50.W% TOR, was recorded within the range of 0.01-24.99 JLgI/day. This value was about 33% for male subjects with similar range of UIE rate. For all ranges of UIE, % TOR was greater in females than in males. For all UIE ranges, %TOR was higher in sites taken as goitre endemic than those sites taken as nonendemic (Table 3). However, % TOR in sites taken as nonendemic was found to be greater than 20.0%. DiscussionThis is most probably the first study on VIE to be done in relation to endemicity of goitre in Ethiopia. It was reported by different investigators that no region in Ethiopa under investigation was free from iodine deficiency despite an indication of wide variation in the magnitude of endemicity (12,17). Our study which covers a total of nine sites ascertained the existence of goitre supported by biochemical fmdings. Both endemic and nonendemic sites of the present study selected from the national survey report of 1981 ( 16) were found to have high prevalence of giotre than previously reported (16). The nonendemic sites were also found to be endemic. This indicates that most parts of Ethiopia, including sites regarded as nonendemic (16) were exposed to iodine deficiency problems through time. Of the total of nine study sites, mean VIE in Bure (Gojam), Shoa (Gohatsion) and Sawla (Gamogofa) appears to be quite low (Table 1). The low rate of VIE combined with high TGR is a good indication that these areas are highly affected by iodine deficiency problems. Similarly, Majetie (Shoa) and Adaba (Bale) have also low VIE and high TGR that they Table 2: Urinary iodine excretion by range and Total Goitre Rate by Sex
N = Biochemical groups too are categorized under the area of high IDD endemicity .UIE thus have a negative correlation (r=-0.5148) with total goitre rate in tIle study sites. The TGR in Mojo (Shoa) is relatively high although with a high UIE value. The water iodine value for Mojo was also found to be among the highest and this gives rise to other speculations which again requires further investigation. Mojo lies within the Rift Valley area where high fluoride intake is observed (18). The mechanism of interference of high intake of fluoride has a possible influence upon the amino acid precursors of thyroxin, tyrosine and its metabolites rather than upon iodine (19) .The study resutls also indicate that UIE did not linearly increase with the decrease in TGR, which might be due to the presence of some goitrogenic factors and / or water contaminats (3,20) which are known to contribute to the lack of absolute correlation between iodine deficiency and goitre size. Microorganisms like Escherichia coli and other dietary goitrogenic factors like thiocyanate impose their adverse effects on bioformation of thyroid hormones in the thyroid gland by inhibiting iodine uptake and organification and increasing iodine excretion through the urine. Our study resutls also indicated that with increasing ranges of UIE, TGR decreased in both male and female study subjects. %TGR dramatically decreased in male than in female for UIE ranges of 25.99-49.99 to 50.00-99.99 JLgI/day which could perhaps be explained by ,exceptional exposure of females for high :physiological demand of iodine that could laccelerate its shortage in the body (21,22). There is a concomitant decrease of TGR for both sexes until UIE rate reached a level of 99.9 p.mg/day urinary iodine (Table 2). Beyond this range, the TGR shifted forward with increasing tendency. There is also unexpected increament of TGR with increasing UIE (Above 49.99 (p.mg/day) in regions considered as nonendemic. This might be due to seasonal migration of people for food search and trade from goitre endemic areas, to less endemic regions which may have improved UIE while without significant change in goitre size. Once goitre has developed as a result of poor iodine intake the thyroid enlargement may not disappear when the individual is taken to iodine sufficient environment (23). Our study results also indicate that TGR in females is higher than in males for the same range of UIE. This can be attributed to the higher physiological requirement of iodine and sex- based low thyroxine binding. Pre-albumin (PA) biosynthesis in females than in males would aggravate TOR when dietary intake is insufficient or marginal (22,24). The relationship of UIE to total goitre rate shown both in endemic and in sites taken as nonendemic is also a good indicator of the severe iodine dificiency situation in study sites. There is also an indication of IDD agravating factors besides iodine deficiency. It was reported (6) that the effect of goitrogenic factors can be overcome by iodine intervention. Hence, the findings presented here urge for implementing, control and eradication programme of IDD in the affected areas. Table 3 urinary Iodine excretion by range and Total Goitre Rate by Endemicity
N = biochemical groups AcknowledgementThe financial support of UNICEF for goiter survey and IDD control programme in Ethiopia is gratefully acknowledged. Reference1. Moxon RED and Dixon PJ .Semi-automatic method for determination of total iodine in food. Analyst 1980; 105:344. 2. Jalin T and Escobar FD. Evaluation of Iodine Creatinine ratios of casual Samples as indices of daily urinary iodine output during field studies. J:Clin.Endocrinol.metab. 1965;25:540. 3. Jose R. Varea Teran Nutritional and public health considerations relating to endemic goitre and cretinism. In:F.Delange and R. Ahluwalia,(eds). Cassava toxicity and thyroid research and public health issues proceedings of a workshop held in Ottawa, 31 May- 2 June 1982; p. 55. 4. Dourdoux P, Thilly C, Denlange F, and Ermans AM. A new look at old concepts in laboratory evaluation of endemic goitre. In: John T. Dunn,Eduardo A.pretell,Carlos Hernan Daza,Fernando E. Viteri.(eds). Towards eradication of endemic goitre,cretinism and iodine deficiency PAHO. WHO Scientific publication 1986;502: 115. 5. Jerome M. Hershman,Glenn A. Melnick,and Rosemary Kastner .Economic Consequences of endemic goitre In: John T. Dunn, Eduardo A. Pretell, Carlos Hernan Daza,Femando E. Viteri,(eds). Towards eradication of endemic goitre, cretinism and iodine deficiency. PAHO. WHO Scientific Publication 1986;502:36. 6. Hennart Dourdoux P, Vis HF, Yunga Y, Seghers P and Delange. Epidemiology of goitre and m.ilnutrition and dietary supply of iodine, thiocyanate and protein in Daszaire, Kivu and Yubang In:Delange FB, Itekzand A.M. Ermans,(eds). Nutritional factors involved in goitrogenic action of Cassava. International development research center publ. Ottawa Canada,IDRC -184e 1982 p 25. 7. Richard H, Follis JR. Patterns of Urinary Iodine Excretion in goitrous and non-goitrous areas. The American I. Clin.Nut. 1964;14:253. 8. Wayne PJ, Kentros DA and Alexander WD. Clinical Aspect of Iodine Metabolism,Dlack Well, Oxford,UK. 1964; p 303. 9. De Visscher MC, Deckers HG, Von Den Schrieck, M.De Smet, Ermans AM, Galperin H,and Dastenie PA. Endemic goitre in Uele Region (Republic of Congo) J.clin. Endocrinol Metab. 1961;21:175. 10. Gill MC, Taylor Pe and Suppoery A. new focus of endemic goitre in Mwezi, Tanzania East Africa Medical Journal. 1970;47:66. 11. Koutras DA. Iodine: Distribution, availability, and effect of deficiency on thyroid. In:John T .Dunn,Eduardo A. Pretell, Carlos Hernan Daza, Fernando E. Viteri,(eds). Towards eradication of endemic goitre,cretinism and iodine deficiency P AHO Scientific publication 1986;502: 15. 12. Wolde-Gebriel Z, Demeke T, Clive E. West and Frits Van der Haar. Goitre in Ethiopia. British Journal of Nutrition (in press). 13. Hetzel BS. The prevention and control of iodine deficiency disorders. Nutrition policy discussion paper 1988;3:89. 14. Roscoe MH. Creatinine in serum or urine. J.ciin.path. 1958;11:173. , 15. Barker SB et al. The clinical determination of protein bound iodine J.Clin Invest 1951; 50:30 Lynch MU. et al. Estimation of protein bound Iodine. Medical laboratory technology W.B, Sounds Co. philadelphia 1969; p 542. 16. Rogina B and Dubravcic: Microdetermination of iodine by arresting the catalytic reduction of ceric ions Analyst 1953;78:594. 17. Inter-departmental Committee on Nutrition for National Defence (ICNND). Ethiopian Nutrition Survey Washington DC: US Government printing Office 1959. 18. Teklehaimanot R, Fekadu A, Bushra B. Endemic Fluorosis in the Rift Valley Ethiopia. Trop.Geogr.Med. 1987;39:209. 19. Day TK and Powell-Jackson PR: Fluoride Water Hardness and Endemic Goitre. the Lancet 1972;1:1135. 20. Bourdoux p, Mafuta M, Hanson A and Ermans AM. Cassva toxicity the role of linamarin in: A.M. Ermans,N.W Mobulamoko, F. Delange,endemic goiter and cretinism. IDRC- 136e 1980; p 15. 21. Delange F, Thilly CH and Ermans AM. Endemic Goitre in kivu area Africa: FOCUS on cassava In: A.M. Ermans, N.M. Mbulamako, F.Delange, and Ahuluwalia, (eds). Role of cassava in the etiology of endemic goitre and cretinism. IDRC-136e 1980; I' 29. 22. Ingenbleek Y and Visscher DM. Hormonal and nutritional status: Critical conditions for endemic goitre epidemiology? Metabolism 1979;28:9. 23. Thilly CH, Delange F, Ermans AM: Five years follow-up in the treatment of endemic goitre with iodized oil. Acta Endocrinol (Suppl) (kbh) 1973;74:11. 24. Braveman LE, Foster AE, Ingbar SH: Sex related differences in the binding in serum of thyroid hormones. J.Cli. Endocrinol Metab. 1967;27:227.
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