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Detergent induced lysis of erythrocytes in kwashiorkor
CCA 03889
Detergent induced lysis of erythrocytes in kwas~urkor Aravinda
Rao a, Christiana
U. Onuora
b,* and Annie Cherian b
~~part~g~t~ of il Chemical Fatho~o~ and b Paedlatrks, A. R. U. Hospital, Zaria (Nigeria)
(Received 12 July 1985; revision received 4 April 1987; accepted after revision 29 april 1987) Key words: Kwashiorkor; Detergent induced hemolysis; Osmotic fragility; Erythrocyte membrane
Summa~ The effect of the non-ionic detergent Nonidet P40 on lysis of erythrocytes in children suffering from kwashiorkor was studied. The concentration of the detergent causing 50% haemolysis was sig~fi~antly reduced in these patients. Detergent haemolysis was more sensitive than osmotic fragility (which was reduced). The abnormality was only slight in marasmic children.
IIlhOdUCtiOll
Phenomena related to cell membrane dysfunction like renal aminoaciduria [l] and altered intracellular electrolytes [Z] have been observed in protein energy malnut~tion. Study of the structural and functional changes in the cell membrane could be useful in understanding the changes in membrane related functions. Erythrocytes show decreased osmotic fragility [3] and the erythrocyte membrane contains increased amounts of lipids [4] in malnutrition. We investigated to see if the membrane changes would alter the susceptibility of the erythrocytes to lysis in the presence of the non-ionic detergent Nonidet P40 (an octyl phenol ethylene oxide condensate). Methods The study was approved by the Ethics Committee of the A.B.U. Hospital, Zaria. We studied 20 kwashiorkor patients (including marasmic kwas~orkor) and 8
* Present address: Civil Service Commission, P.M.B. 1048, Calabar, Nigeria Correspondence to: A. Rao. Present address: Dept. of Laboratories, Biochemistry Hospital, P.O. Box 4078, 13041 Safat, Kuwait. 0009-8981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Section, Sabah
2
marasmic patients, above the age of 1 yr. 14 well nourished children served as controls. The Wellcome criteria [5] were used to diagnose the patients. Heparinised blood was collected and detergent induced lysis and hypotonic lysis were carried out within 2 h as described below. Plasma albumin concentration was measured by the method of Spencer and Price [6]. Detergent lysis Nonidet P40 was prepared as a 0.4 g/l solution in 154 mmol/l saline. 0.7, 0.8, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2 and 2.0 ml of this solution were pipetted into different centrifuge tubes. The volume in each tube was made up to 4.0 ml with 154 mmol/l saline. 0.05 ml of well mixed blood was added into each tube. The tubes were gently shaken to mix and incubated at 25 o C for 45 min. The tubes were centrifuged for 10 min at 1000 X g at room temperature and the absorbance of the supernatants was measured at 540 nm. The absorbance of the tube containing 0.2 g/l Nonidet P40 was taken to represent 100% lysis and the percentage lysis at each of the concentrations was plotted against the concentration used. The 50% lysis concentration was found by interpolation. Hypotonic lysis Into centrifuge tubes containing 4 ml of 0, 38, 43, 47, 51, 56, 60, 64 and 68 mmol/l sodium chloride solutions 0.05 ml of the blood was added. After gentle mixing the tubes were incubated at 25’ C for 30 min, centrifuged at 1000 x g for 10 min at room temperature and the absorbance of the supemates was measured at 540 nm. Taking the absorbance at zero concentration of sodium chloride as lOO%, The percentage lysis was plotted against sodium chloride concentration and 50% lysis concentration was calculated by interpolation. Student’s t-test for unpaired samples was used to assess the difference between the means of the different groups. Results Table I gives the range and mean values for the 50% lysis concentrations of sodium chloride and of Nonidet P40. It can be seen that there was a marked reduction in the stability of the erythrocytes against the detergent in kwashiorkor. It is also evident that the erythrocytes show increased resistance to hypotonic lysis, confirming the previous reports. The marasmic children, who differ from the kwashiorkor children in not having oedema of hypoalbuminaemia, showed only a slight reduction in the mean 50% lysis concentration of Nonidet P40 (99.0 mg/ml; p < 0.02); resistance to hypotonic lysis was also only slightly increased ( p -C 0.05). Albumin concentration in these children was not reduced as against the marked reduction in the kwashiorkor group. 12 of the 20 patients were anaemic (haematocrit < 30%). The mean 50% lysis concentrations of Nonidet P40 and sodium chloride in these children were not significantly different from those in the remaining 8 patients.
3 TABLE
I
50% lysis concentrations respectively.
of sodium
chloride
and
Sodium chloride, mmol/l
lysis and
P40,
Range
Mean f
56.2 rtO.37
Kwashiorkor (n=20)
52.0-57.2
53.6kO.39 (p <10-q
Marasmus (n=X)
51.4-56.6
SE
group
Mean f 106.4fl.5
90.4-104.0
92.0f0.7 (p
93.0-110.0
54.7 f0.57 (p < 0.05) between
Range 95.0-116.0
=
mean and control
detergent
Plasma albumin,
mg/l
53.1-57.5
of difference
P40 in osmotic
Nonidet
Control (n =14)
a Significance
Nonidet
Mean f
SE
fysis,
g/l SE
39.0 f 0.6
16.0+0.4 =
99.0 zbo.54 ( p < 0.02)
35.OkO.62
mean.
We tested the possibility of using detergent lysis and hypotonic lysis as simple tests for kwashiorkor. From Fig. 1 it can be seen that at Nonidet concentration of 95.0 mg/l there is the least overlap between control and patient values for percentage lysis; 19 of 20 kwashiorkor patients showed greater lysis than the upper limit of the control range (mean _t 2 SD, 43.5% lysis) at this concentration. Similarly from Fig. 2, at 56.0 mmol/l sodium chloride concentration there is minimal overlap of percentage lysis values between controls and kwashiorkor. However, only 11 of
90
1 6 S
i
mg/L Fig. I. Mean and normal.
SD
Nor&t of percentage
PLO lysis at various
detergent
concentrations.
f-e-f
, control;
f-+---j
mmol Sodium chloride/L Fig. 2. Mean and SD of percentage w , kwashiorkor.
lysis at various
concentrations
of sodium
chloride
+o+
, control;
the 20 patients showed less lysis than the lower limit of control (mean k 2 SD, 36% lysis) at this concentration. Precision of the two tests was determined by multiplicate determinations on the same sample. The C.V. for detergent lysis was 5.8% and that for hypotonic lysis was 6.5%. Discussion Our study shows a marked reduction in the stability of the red cell membrane against detergent. It has been reported that the cholesterol and phospholipid concentration per unit area of the red cell membrane is increased in kwashiorkor [4], which means that there is a relative decrease in the density of protein on the membrane. The increased susceptibility to detergent lysis and increased resistance to hypotonic lysis could be related to these alterations in the composition of cell membrane. The abnormality appears to be related to a predominant protein malnutrition, attended by hypoalbuminaemia and oedema which characterise kwashiorkor as against marasmus, since the increased susceptibility to detergent lysis in marasmus is insignificant (p < 0.02) when compared to kwashiorkor (p -c lo-‘). Anaemia appears not to be causative by itself, as anaemic kwashiorkor patients did not behave differently from the non-anaemic ones towards either detergent or hypotonic lysis. Our observations suggest that detergent lysis is potentially a simple test which can be useful in kwashiorkor, particularly where there are no facilities to determine albumin. In this respect it is much more sensitive than hypotonic lysis.
5
References 1 Alleyne GAO. The effect of severe Protein Calorie Malnutrition on the renal function of Jamaican children. Paediatrics 1967;39:40&411. 2 Kaplay SS. Erythrocyte membrane Na+-K+-activated ATPase in protein calorie malnutrition. Am J Clin Nutr 1978;31:579-584. 3 Brown KH, Suskind RM, Lubin B, Kulapongs P, Leitzman C, Olson RE. Changes in red cell membrane in Protein Calorie Malnutrition. Am J Clin Nutr 1978;31:574-578. 4 Coward WA. Erythrocyte membrane in kwashiorkor. Brit J Nutr 1971;25:145-151. 5 FAO/WHO Expert Committee on Nutrition. Eighth Report 1971; WHO Tech Rep Ser No. 477. 6 Varley H, Gowenlock AH, Bell M. (Eds) Practical clinical biochemistry, vol. 1. London: William Heinemann. 1980.
Detergent induced lysis of erythrocytes in kwas~urkor Aravinda
Rao a, Christiana
U. Onuora
b,* and Annie Cherian b
~~part~g~t~ of il Chemical Fatho~o~ and b Paedlatrks, A. R. U. Hospital, Zaria (Nigeria)
(Received 12 July 1985; revision received 4 April 1987; accepted after revision 29 april 1987) Key words: Kwashiorkor; Detergent induced hemolysis; Osmotic fragility; Erythrocyte membrane
Summa~ The effect of the non-ionic detergent Nonidet P40 on lysis of erythrocytes in children suffering from kwashiorkor was studied. The concentration of the detergent causing 50% haemolysis was sig~fi~antly reduced in these patients. Detergent haemolysis was more sensitive than osmotic fragility (which was reduced). The abnormality was only slight in marasmic children.
IIlhOdUCtiOll
Phenomena related to cell membrane dysfunction like renal aminoaciduria [l] and altered intracellular electrolytes [Z] have been observed in protein energy malnut~tion. Study of the structural and functional changes in the cell membrane could be useful in understanding the changes in membrane related functions. Erythrocytes show decreased osmotic fragility [3] and the erythrocyte membrane contains increased amounts of lipids [4] in malnutrition. We investigated to see if the membrane changes would alter the susceptibility of the erythrocytes to lysis in the presence of the non-ionic detergent Nonidet P40 (an octyl phenol ethylene oxide condensate). Methods The study was approved by the Ethics Committee of the A.B.U. Hospital, Zaria. We studied 20 kwashiorkor patients (including marasmic kwas~orkor) and 8
* Present address: Civil Service Commission, P.M.B. 1048, Calabar, Nigeria Correspondence to: A. Rao. Present address: Dept. of Laboratories, Biochemistry Hospital, P.O. Box 4078, 13041 Safat, Kuwait. 0009-8981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Section, Sabah
2
marasmic patients, above the age of 1 yr. 14 well nourished children served as controls. The Wellcome criteria [5] were used to diagnose the patients. Heparinised blood was collected and detergent induced lysis and hypotonic lysis were carried out within 2 h as described below. Plasma albumin concentration was measured by the method of Spencer and Price [6]. Detergent lysis Nonidet P40 was prepared as a 0.4 g/l solution in 154 mmol/l saline. 0.7, 0.8, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2 and 2.0 ml of this solution were pipetted into different centrifuge tubes. The volume in each tube was made up to 4.0 ml with 154 mmol/l saline. 0.05 ml of well mixed blood was added into each tube. The tubes were gently shaken to mix and incubated at 25 o C for 45 min. The tubes were centrifuged for 10 min at 1000 X g at room temperature and the absorbance of the supernatants was measured at 540 nm. The absorbance of the tube containing 0.2 g/l Nonidet P40 was taken to represent 100% lysis and the percentage lysis at each of the concentrations was plotted against the concentration used. The 50% lysis concentration was found by interpolation. Hypotonic lysis Into centrifuge tubes containing 4 ml of 0, 38, 43, 47, 51, 56, 60, 64 and 68 mmol/l sodium chloride solutions 0.05 ml of the blood was added. After gentle mixing the tubes were incubated at 25’ C for 30 min, centrifuged at 1000 x g for 10 min at room temperature and the absorbance of the supemates was measured at 540 nm. Taking the absorbance at zero concentration of sodium chloride as lOO%, The percentage lysis was plotted against sodium chloride concentration and 50% lysis concentration was calculated by interpolation. Student’s t-test for unpaired samples was used to assess the difference between the means of the different groups. Results Table I gives the range and mean values for the 50% lysis concentrations of sodium chloride and of Nonidet P40. It can be seen that there was a marked reduction in the stability of the erythrocytes against the detergent in kwashiorkor. It is also evident that the erythrocytes show increased resistance to hypotonic lysis, confirming the previous reports. The marasmic children, who differ from the kwashiorkor children in not having oedema of hypoalbuminaemia, showed only a slight reduction in the mean 50% lysis concentration of Nonidet P40 (99.0 mg/ml; p < 0.02); resistance to hypotonic lysis was also only slightly increased ( p -C 0.05). Albumin concentration in these children was not reduced as against the marked reduction in the kwashiorkor group. 12 of the 20 patients were anaemic (haematocrit < 30%). The mean 50% lysis concentrations of Nonidet P40 and sodium chloride in these children were not significantly different from those in the remaining 8 patients.
3 TABLE
I
50% lysis concentrations respectively.
of sodium
chloride
and
Sodium chloride, mmol/l
lysis and
P40,
Range
Mean f
56.2 rtO.37
Kwashiorkor (n=20)
52.0-57.2
53.6kO.39 (p <10-q
Marasmus (n=X)
51.4-56.6
SE
group
Mean f 106.4fl.5
90.4-104.0
92.0f0.7 (p
93.0-110.0
54.7 f0.57 (p < 0.05) between
Range 95.0-116.0
=
mean and control
detergent
Plasma albumin,
mg/l
53.1-57.5
of difference
P40 in osmotic
Nonidet
Control (n =14)
a Significance
Nonidet
Mean f
SE
fysis,
g/l SE
39.0 f 0.6
16.0+0.4 =
99.0 zbo.54 ( p < 0.02)
35.OkO.62
mean.
We tested the possibility of using detergent lysis and hypotonic lysis as simple tests for kwashiorkor. From Fig. 1 it can be seen that at Nonidet concentration of 95.0 mg/l there is the least overlap between control and patient values for percentage lysis; 19 of 20 kwashiorkor patients showed greater lysis than the upper limit of the control range (mean _t 2 SD, 43.5% lysis) at this concentration. Similarly from Fig. 2, at 56.0 mmol/l sodium chloride concentration there is minimal overlap of percentage lysis values between controls and kwashiorkor. However, only 11 of
90
1 6 S
i
mg/L Fig. I. Mean and normal.
SD
Nor&t of percentage
PLO lysis at various
detergent
concentrations.
f-e-f
, control;
f-+---j
mmol Sodium chloride/L Fig. 2. Mean and SD of percentage w , kwashiorkor.
lysis at various
concentrations
of sodium
chloride
+o+
, control;
the 20 patients showed less lysis than the lower limit of control (mean k 2 SD, 36% lysis) at this concentration. Precision of the two tests was determined by multiplicate determinations on the same sample. The C.V. for detergent lysis was 5.8% and that for hypotonic lysis was 6.5%. Discussion Our study shows a marked reduction in the stability of the red cell membrane against detergent. It has been reported that the cholesterol and phospholipid concentration per unit area of the red cell membrane is increased in kwashiorkor [4], which means that there is a relative decrease in the density of protein on the membrane. The increased susceptibility to detergent lysis and increased resistance to hypotonic lysis could be related to these alterations in the composition of cell membrane. The abnormality appears to be related to a predominant protein malnutrition, attended by hypoalbuminaemia and oedema which characterise kwashiorkor as against marasmus, since the increased susceptibility to detergent lysis in marasmus is insignificant (p < 0.02) when compared to kwashiorkor (p -c lo-‘). Anaemia appears not to be causative by itself, as anaemic kwashiorkor patients did not behave differently from the non-anaemic ones towards either detergent or hypotonic lysis. Our observations suggest that detergent lysis is potentially a simple test which can be useful in kwashiorkor, particularly where there are no facilities to determine albumin. In this respect it is much more sensitive than hypotonic lysis.
5
References 1 Alleyne GAO. The effect of severe Protein Calorie Malnutrition on the renal function of Jamaican children. Paediatrics 1967;39:40&411. 2 Kaplay SS. Erythrocyte membrane Na+-K+-activated ATPase in protein calorie malnutrition. Am J Clin Nutr 1978;31:579-584. 3 Brown KH, Suskind RM, Lubin B, Kulapongs P, Leitzman C, Olson RE. Changes in red cell membrane in Protein Calorie Malnutrition. Am J Clin Nutr 1978;31:574-578. 4 Coward WA. Erythrocyte membrane in kwashiorkor. Brit J Nutr 1971;25:145-151. 5 FAO/WHO Expert Committee on Nutrition. Eighth Report 1971; WHO Tech Rep Ser No. 477. 6 Varley H, Gowenlock AH, Bell M. (Eds) Practical clinical biochemistry, vol. 1. London: William Heinemann. 1980.