Arch Dis Child: first published as 10.1136/adc.48.12.927 on 1 December 1973. Downloaded from

Archives of Disease in Childhood, 1973, 48, 927.

Serum protein and transferrin determinations to distinguish kwashiorkor from iron deficiency anaemia AARON E. J. MASAWE and JULIUS RWABWOGO-ATENYI From the Department of Medicine and Paediatrics, Makerere University, Kampala, Uganda Masawe, A. E. J., and Rwabwogo-Atenyi, J. (1973). Archives of Disease in Childhood, 48, 927. Serum protein and transferrin determinations to distinguish kwashiorkor from iron deficiency anaemia. Serum proteins and serum transferrin levels were studied in 38 African children of whom 10 were normal controls, 18 had severe iron deficiency anaemia, and 10 had kwashiorkor. The results showed that the patients with iron deficiency anaemia had relatively higher total serum proteins and serum globulins than a comparable normal control group. Conversely, patients with kwashiorkor had lower total proteins and normal globulins. The albumin fraction was raised above the control in 50% of the patients with iron deficiency anaemia and diminished in the other 50%. In kwashiorkor the levels were uniformly diminished. The transferrin levels were uniformly raised in the iron deficiency anaemia group and uniformly diminished in the kwashiorkor group. The transferrin level was deemed the best screening test to distinguish the two disorders. copyright.

Kwashiorkor and iron deficiency anaemia are it is imperative to have a good criterion for screening common causes of morbidity and mortality in patients with iron deficiency anaemia from those childhoodin the tropics (World Health Organization, with kwashiorkor. 1968, 1971). Kwashiorkor results from early Reported here is a study of serum proteins, serum weaning followed by an overall inadequate protein transferrin, and haematological profiles in normal intake. Iron deficiency anaemia is a consequence of control children, in patients with severe kwashiorkor

inadequate dietary intake of iron, chronic blood loss, (grade IV malnutrition), and in patients with severe http://adc.bmj.com/ or both. Heavy hookworm infestation is the iron deficiency anaemia to attempt to define a good commonest cause of blood loss and iron deficiency screening criterion. anaemia in East Africa (Blackman, 1962; Vanier, Material 1966; Nhonoli, 1969). From 2 to 100 ml blood is lost in faeces per patient per day depending on the For this study, 18 children with severe iron deficiency anaemia, 10 with kwashiorkor, and 10 healthy controls load of the worms (Roche and Layrisse, 1966; were selected. The children included those attending Martinez-Torres et al., 1967). the paediatric services of Mulago Hospital between Often the two conditions coexist, their features January and May 1971. The selection criteria included on September 25, 2021 by guest. Protected overlap, and differentiation of one from the other is (a) iron-deficiency anaemia, clinical evidence of anaemia: usually difficult. Recently McFarlane et al. (1970) Hb concentration of <8 g/100 ml, absent stainable iron reported that patients with severe kwashiorkor have in bone marrow, positive hookworm ova, and heavy diminished serum transferrin and that early iron occult blood in stools; (b) kwashiorkor, clinical features of therapy may be dangerous. Treatment should be severe protein-calorie malnutrition: retarded growth are (low weight for expected height), generalized oedema, deferred until the levels of the transferrin flaky-paint skin lesions, and woolly depigmented hair; improved by intensive dietary management, and this (c) controls, healthy-looking children with Hb con- usually takes a few days (Gabr, El-Hawary, and centrations of 11 g/100 ml and over. El-Dali, 1971). In iron deficiency anaemia, on the These were selected from specialized clinics (surgical other hand, iron replacement is the cornerstone of and skin) and had no apparent evidence of malnutrition treatment and must not be delayed. Accordingly, or infections. The serum protein fractions were in addition comparable to those reported by Edozien and Received 11 June 1973. Udeozo (1960) on 231 h-althy Nigerian children. 927 Arch Dis Child: first published as 10.1136/adc.48.12.927 on 1 December 1973. Downloaded from

928 Masawe and Rwabwogo-Atenyi TABL Haematological and biochemic

Haematology No. of cases M:F Age (yr) Hb (g/100 ml) PCV MCHC

Iron deficiency anaemia 18 5:1 Mean 4*2 4*76 19*0 24 *5 SE - 0 34 1 17 0-46 Range 1-9 1*7-7*6 7-27 21-29 Control 10 3:2 Mean 3*1 12*12 39 *5 31 *25 SE - 0*36 0*93 0 *63 Range 1-7 11-14 34-44 28-35 Kwashiorkor 10 3:2 Mean 2 -8 9 *57 30-8 31 2 SE - 0*53 1 66 0 70 Range 1-8 6-12 22-40 28-34

*Mean percentage saturation 17-4%. SE, standard error; PCV, packed cell volume; MCHC, mean corpuscular haemoglobin concentration.

All three categories were closely matched for age and Haematological profiles. Haemoglobin sex. packed cell volume (PCV) and the mean corpuscular Methods Hb concentration (MCHC) were markedly diminished below the normal control in the iron Blood specimens were collected by venepuncture in heparinized and plain bottles. The plain bottles were deficiency anaemia group and only moderately so in and the serum was stored at -20 °C the kwashiorkor group. 3 children with iron then centrifuged copyright. until used. Haemolysed specimens were discarded. deficiency anaemia had homozygous sickle cell All the specimens were collected before starting therapy. haemoglobin (Hb SS), and 2 of those with kwashiorkor had megaloblastic anaemia. Assays. Total serum proteins were assayed by The peripheral picture was hypochromic micro- modified biuret method (Wootton, 1964) and protein cytic in all patients with iron deficiency anaemia, fractions were determined by electrophoresis (Varley, normocytic normochromic in the control children, 1967). Cellulose acetate membrane 10 cm long and and variable in the children with kwashiorkor. In barbitone buffer pH 8 * 6 were used for electrophoresis. 5 and jig of the specimens was applied and run for 1 hours at the last group, some films were hypochromic stabilized DC current of IOV/cm length. The strips microcytic, others were normocytic and normo- http://adc.bmj.com/ were then stained with Ponceau reagent and quantified by chromic, and others still were macrocytic Beckman microzone densitometer R-110 model. normocytic. The transferrin concentrations were determined by radial immunodiffusion technique of Mancini, Stool examinations. All 18 children with iron Carbonara, and Heremans (1965). deficiency anaemia had heavy hookworm infestation Haematological profiles in all cases were carried out as and strongly positive occult blood test. 5 control described by Dacie and Lewis (1968). Stools were children had mild hookworm infestation. Of these, examined for occult blood by Hematest tablets. 4 had MCHC of less than 310%. All 10 children on September 25, 2021 by guest. Protected with kwashiorkor had moderate to severe parasitic Results infestations. The parasites included hookworms, Ascaris, Trichuris trichiura, and tapeworms. Occult The Table summarizes the haematological and blood test was positive in all. biochemical findings for the three categories of subjects. Serum transferrin. The results are summarized both in the Table and the Fig. The Age. Age distribution was similar in the three concentrations were increased above the normal groups. 12 patients with iron deficiency anaemia, 9 control in the iron deficiency anaemia group, and with kwashiorkor, and 9 control children were aged were markedly diminished in the kwashiorkor 5 years or less. group. The difference between the iron deficiency anaemia and the kwashiorkor groups is significant Sex. Males predominated in all three categories. (P<0001) by Student's 't' test. Arch Dis Child: first published as 10.1136/adc.48.12.927 on 1 December 1973. Downloaded from

Serum protein and transferrin determinations to distinguish kwashiorkor from iron deficiency 929 ta in 38 children

Serum proteins (g/100 ml) ~rum transferrin l (ptgIlOO ml) Total Albumin oel-globulin ax-globulin ,3-globulin y-globulin

328.7* 6 87 2-55 0*47 0*85 0-88 2-12 11*66 0-25 0-15 0 04 0 05 0 07 0 15 257-419 4-48-3 1 3-3-8 0-16-0 75 0-441-36 0-53-1-74 1*2-3-5 273-95 6-33 2-96 0-36 0*67 0 75 1*57 27 0*22 0*18 0 03 0*08 0*07 0*13 248-317 54-7 9 2-1-42 0-2-0-5 0-3-1*26 0 36-1*22 1*08-2-61 170 59 5 0 1 62 0 37 0-72 0 75 1 49 6 18 0*15 0*13 0 03 0*13 0*10 0*19 139-200 3 9-5 5 1 2-2 4 0 23-054 0 36-1-59 0-24-117 0-58-2 4

Serum proteins. Albumin. The results are summarized in the Total. Total proteins for the iron deficiency Table and Fig. Taking 25 g/100 ml arbitrarily as anaemia group were similar to those in the control the lower range of normal, 9 (50%) of the children children, but the total proteins for the kwashiorkor with iron deficiency anaemia, 2 (20%) of the control group were significantly less than the control. 4 children, and all 10 children with kwashiorkor had subnormal albumin levels. patients with iron deficiency anaemia had low copyright. plasma proteins and classical clinical features of Globulin. Results are summarized in the Table kwashiorkor. and Fig. The globulin fractions were slightly

oAlbumin (q/100ml) o Transferrin ( u M/D ml) A ,B- globul in ( g/ lOOml ) 450 -

4-0 0 http://adc.bmj.com/ 4 0 400

3-5 350 I0 0 3*0 1 300 E 225 2 500 i 0 00 2-0 2008 on September 25, 2021 by guest. Protected 3 15- i00k 150 05 - 100 50 05 0 Kwash iorkor Control Chronic iron defici'ency Cross bars indicate mean ± SD

FIG.-Serum albumin, serum transferrin, and serum fl-globulin in patients with kwashiorkor, in control children, and in patients with chronic iron deficiency anaemia. Transferrin concentrations are in ,ug/lOO ml on the right-hand scale, while the other fractions are in g/100 ml on the left hand scale. Arch Dis Child: first published as 10.1136/adc.48.12.927 on 1 December 1973. Downloaded from

930 Masawe and Rwabwogo-Atenyi increased in the iron deficiency group and were deficiency anaemia. Finally, the albumin levels within normal limits in the kwashiorkor group, but were uniformly diminished in kwashirokor but the differences are not significant. bimodal in iron deficiency anaemia-in some patients the levels were raised and in others they were diminished. The albumin findings in the Discussion patients with iron deficiency anaemia were very The above results confirm the introductory similar to those reported by Blackman et al. remarks that iron deficiency anaemia and (1965) on adult patients with similar anaemia in kwashiorkor often overlap. 4 children with iron Kampala. deficiency anaemia had classical features of Transferrin estimation has been recommended kwashiorkor and 3 children with kwashiorkor had for assessing prognosis in patients with kwashiorkor; iron deficiency anaemia. In a study ofiron stores in reduced levels bear a close relation to severity and healthy and malnourished children in India, bad prognosis (McFarlane et al., 1970; Gabr et al., Manchanda, Lal, and Khanna (1969) also found that 1971). A further practical application is suggested 56% of the healthy children and 85% of the by the study, namely screening of patients with malnourished children had iron deficiency. kwashiorkor from patients with iron deficiency Previously, both conditions have been considered anaemia before iron therapy. The procedure is together as nutritional disorders and iron replace- technically simple and straightforward and can be ment therapy has been advocated for both. Iron carried out in many laboratories in the tropics. therapy is also advocated for mass campaigns in various parts of the world (World Health Organization, 1968). Recently, evidence has We are greatly indebted to the Medical Super- intendent and to all paediatricians, Mulago Hospital, for emerged to show that the liberal use of iron may not allowing us to study their patients, and to Dr. H. be rational, and that careful distinction of patients Nsanzumuhire for his comments. This project was with kwashiorkor from those with iron deficiency supported jointly by the WHO Regular Funds and by anaemia may be necessary before therapy is given. Makerere University Research Grant No. 622. In kwashiorkor the serum transferrin levels are copyright. depleted (Edozien and Udeozo, 1960; Antia, McFarlane, and Soothill, 1968; McFarlane et al., REFERENCES 1970; Gabr et al., 1971) and early replacement of Antia, A. U., McFarlane, H., and Soothill, J. F. (1968). Serum siderophilin in kwashiorkor. Archives of Disease in Childhood, iron may inflame underlying infections and 43, 459. accelerate death (McFarlane et al., 1970). The Blackman, V. (1962). Some observations on the severe anaemias of is bacteriostatic and protective Dar es Salaam. East African Medical Journal, 39, 235. transferrin Blackman, V., Marsden, P. D., Banwell, J., and Craggs, M. H. (Weinberg, 1966; McFarlane, Okubadejo, and (1965). Albumin metabolism in hookworm anaemias. Reddy, 1972). In iron deficiency anaemia, on the Transactions of the Royal Society of Tropical Medicine and http://adc.bmj.com/ Hygiene, 59, 472. other hand, the transferrin levels are raised and in Dacie,J.V.,andLewis, S. M. (1968). PracticalHaematology, 4thed., vitro growth of bacteria in blood from such patients p. 36. Churchill, London. Edozien, J. C., and Udeozo, I. 0. K. (1960). Serum copper, iron and is much less than the growth in blood from patients binding capacity in kwashiorkor. Journal of Tropical Pediatrics with kwashiorkor (A. E. J. Masawe and and African Child Health, 6, 60. H. Gabr, M. M., El-Hawary, M. F. S., and El-Dali, M. (1971). Serum Nsanzumuhire, unpublished data). transferrin in kwashiorkor. Journal of Tropical Medicine and Of the screening criteria studied, only the Hygiene, 74, 216. transferrin estimations appear promising. The McFarlane, H., Okubadejo, M., and Reddy, S. (1972). Transferrin and Staphylococcus aureus in kwashiorkor. American Journal of on September 25, 2021 by guest. Protected levels were uniformly raised above the normal Clinical Pathology, 57, 587. control in iron deficiency anaemia and uniformly McFarlane, H., Reddy, S., Adcock, K. J., Adeshina, H., Cooke, A. R., and Akene, J. (1970). Immunity, transferrin, and survival diminished in kwashiorkor. The secondary in kwashiorkor. British MedicalJournal, 4, 268. kwashiorkor changes in some patients with iron Manchanda, S. S., Lal, H., and Khanna, S. (1969). Iron stores in deficiency anaemia did not influence the transferrin health and disease; bone-marrow studies in 1134 children in Punjab, India. Archives of Disease in Childhood, 44, 580. levels, nor did the secondary iron deficiency in some Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965). patients with kwashiorkor alter the transferrin levels Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235. appreciably. Martinez-Torres, C., Ojeda, A., Roche, M., and Layrisse, M. (1967). The other criteria were less specific. The Hookworm infection and intestinal blood loss. Transactions of the Royal Society of Tropical Medicine and Hygiene, 61, 373. haematological profiles were abnormal in both Nhoqoli, A. M. (1969). Iron deficiency anaemia. M.D. Thesis. groups and tended to overlap. Total plasma University of East Africa. proteins were uniformly diminished in patients with Roche, M., and Layrisse, M. (1966). The nature and causes of hookworm anemia. AmericanJournal of Tropical Medicine and kwashiorkor but variable in patients with iron Hygiene, 15, 1032. Arch Dis Child: first published as 10.1136/adc.48.12.927 on 1 December 1973. Downloaded from

Serum protein and transferrin determinations to distinguish kwashiorkorfrom iron deficiency 931 Vanier, T. M. (1966). Causes of severe anaemia in young children Fortifications, Protein Calorie Malnutrition (1971). World admitted to Mulago Teaching Hospital. Tropical and Health Organization. Technical Report Series, No. 477. Geographical Medicine, 18, 287. WHO, Geneva. Varley, H. (1967). The plasma proteins. In Practical Clinical Wootton, I. D. P. (1964). Total proteins. In King's Microanalysis Biochemistry, 4th ed., p. 252. Heinemann, London. in Medical Biochemistry, 4th ed., p. 138. Churchill, London. Weinberg, E. D. (1966). Roles of metallic ions in host-parasite interactions. Bacteriological Reviews, 30, 136. World Health Organization Scientific Group on Nutritional Correspondence to Dr. A. E. J. Masawe, Faculty of Anaemias (1968). World Health Organization. Technical Report Series, No. 405. WHO, Geneva. Medicine, University of Dar es Salaam, P.O. Box 20693, World Health Organization/FAO Expert Committee on Food Dar es Salaam, Tanzania, East Africa. copyright. http://adc.bmj.com/ on September 25, 2021 by guest. Protected