SUMMARY AND CONCLUSIONS

Transcription

1 SUMMARY AND CONCLUSIONS

2 ( SUMMARY AND CONCLUSIONS^ Nephrotic syndrome is defined as a clinical state characterised by proteinuria, hypoproteinaemia, hyperlipidaemia and oedema. It is the most common chronic renal disorder observed in children. The occurrence of nephrotic syndrome in children is common between ages VA and 4 yrs and the ratio of males to females is about 2:1 [White et al, 1970; Habib and Kleinknecht, 1971; www merck.com]. The hallmark of nephrotic syndrome can be attributed to a single defect i.e. proteinuria that leads to various clinical and metabolic abnormalities. The other clinical manifestations that arise secondary to proteinuria include hypoalbuminaemia, hyperlipidaemia, and oedema. Hyperlipidaemia is not only a marker of the disease, but it also contributes to the process of glomerulosclerosis. Moreover, elevated level of LDL-C seems to enhance the atherogenesis of LDL-C [Lee et al, 1997; Steinberg et al, 1989]. Dietary antioxidants protect the in vitro oxidation of LDL-C and limits experimental atherosclerosis [Pedraza-Chewerri et al, 1995; Warwick et al,, 2000]. Therapeutic manoeuvres aimed at correction of lipid abnormalities may slow the progression of nephrotic syndrome to end stage renal disease. In children, minimal change disease is most frequently observed and they respond well to corticosteroid treatment. They usually have a favourable long-term prognosis but nearly half of the patients experience frequent relapses [Koskimies et al, 1982; Trompeter et al, 1985] that impair the quality of life and develop complications of the disease [Srivastava et al, 1992]. Immunosuppressive agents have been used traditionally in such cases [Albeitsgemeinscaft fur Padiatrische Nephrologic, 1982], However, the potential side effects of such treatment limit their use. The long-term immunosuppressive effect and the cumulative risk of such treatment are prohibitive in a condition that ultimately has good prognosis. 270

3 Thus, the use of an alternative therapy, which has an antihyperlipidaemic effect and has antioxidants; would be an attractive option for these patients. So in this context, the belief in a non-pharmacological, nutritional and natural cure has prompted this clinical trial of spirulina (which is a store house of all the nutrients along with its amazing other useful properties) in nephrotic patients. The pathobiochemistry of the disease was studied so that the disease could be understood in a better way, which later could help in the management of the disease in an appropriate way. In this regard, eighty patients suffering from nephrotic syndrome were enrolled. Their socioeconomic profile, medical history along with anthropometric measurements and dietary history were collected at baseline. The metabolic profile was studied by analysing the lipids and lipoprotein fractions, apolipoproteins, total protein and its fractions along with various analytes representing the kidney functions in these patients at baseline. The mean age of all the nephrotic patients was 8.4±3.8yrs. The mean age of the onset of the disease was yrs in these patients. The general information of the patient s reveal that most of them lived in a nuclear family while the remaining lived in joint family. All the parents of the nephrotic patients belonged to the low-income group and this can further be affirmed by the occupation of the parents in which they were engaged. Food habits show that 13.7% were ovo-vegetarians, 33.8% vegetarians and 52.5% nonvegetarians. The clinical information depicts that most of the patients had gradual onset of nephrotic syndrome; while in the remaining (31.2%) patients onset of disease was associated with infections. Maximum number of patients fell in the clinical category of infrequent relapsers (36.3%) while 25% were frequent relapses/steroid dependent and only 6.2% were steroid resistant. 271

4 Normal children (n=28) were enrolled and they had normal serum levels of total protein and its fractions, creatinine, urea and BUN. The corrected creatinine clearance of these children was observed to be within the normal ranges. The comparison of the levels of total protein and its fractions of normal children and nephrotic patients exhibited that the nephrotic patients had significantly decreased levels of total proteins (p<0.001), albumin (p<0.001) and A:G ratio (p<0.001) and proteinuria was seen in nephrotic patients. Thus, it was observed that these patients had hypoproteinaemia and proteinuria that are the clinical manifestations of nephrotic syndrome. It has been reported the hypoproteinaemia leads to hyperlipidaemia during nephrotic syndrome. And hyperlipidaemia is another clinical manifestation of nephrotic syndrome [Wheeler and Bernnard, 1994; Ongkingco et al, 1991]. In this study, lipid levels observed in the normal children were in line with the lipid and lipoprotein levels observed by Khalil et al., 1995; Gandhi, 1982; Nerurkar et al., Between normal boys and girls no significant difference was observed within their lipid and lipoprotein concentrations. Similarly, Khalil et al, 1995 observed no significant difference in the lipid profile in relation to gender. In the current study, the lipid concentrations of the nephrotic patients were compared with the normal children. The results revealed that the lipid levels were elevated in nephrotic patients in comparison to normal children. Moreover, it is known, that in nephrotic patients the pattern of increased lipid abnormalities appear to be associated with a higher risk of cardiovascular diseases [Osula etal, 2002; Silva, 2002; Hopp etal, 1994; Grundy and Vega, 1989] and experimental studies have reported that lipid abnormalities accelerate renal injury [Keane et al, 1991; Grundy and Vega, 1989], in the present study, a two-fold increase in the concentration of TG (p<0.001) was observed in comparison to the normal children. Similarly, various authors 272

5 [Skrzep-Poloczek et al., 2001; Kuhn et al., 1998; Ongajyooth et a!., 1993; Keane et al., 1992; Nayak etal., 1990] have observed increased levels of TG in these patients. In the present study, the nephrotics exhibited almost three-fold increase in the level of TL (p<0.001) and TC (p<0.001), three and a half fold increase in the level of LDL-C (p<0.001) and a two-fold increase in the level of VLDL-C (p<0.001) and HDL-C (p<0.001) as compared to the normal children. Various authors have reported similar observations that hyperlipidaemia is noticed in the patients with nephrotic syndrome [Skrzep-Poloczek, 2001; Fogo, 2000; Majumdar and Wheeler, 2000; Nayak et al., 1999; Wheeler and Bernard, 1994; Warwick and Packard, 1993; Keane et al., 1992; Querfeld et al., 1988;Oetliker et al., 1980]. In the past few years, the evaluation of non- HDL-C has gained attention. In the current study, the level of non-hdl-c in nephrotic patients was three-fold higher in comparison to normal children. Nishizawa et al, 2003 reported non-hdl-c as a predictor of cardiovascular mortality in the patients with end stage renal disease (ESRD). In the present study, the atherogenic indices i.e. TC:HDL-C (p<0.001) and LDL-C.HDL-C (p<0.001) ratios in these patients were significantly increased in comparison to the normal children. It has been reported that hyperlipidaemia is an important potential risk factor in the aetiology of cardiovascular disease [Silva et al., 2002; Radhakrishnan et al., 1993; Warwick and Packard, 1993; Bernard, 1988; Mallick and Short, 1981] and also aggravate glomerular damage with a pathological mechanism analogous to atherosclerosis (D Amico et al 1993; Hutchison, 1993; Grone et al, 1993). With regard to apolipoproteins, in the current study, a three-fold increase in the apo B concentration of the nephrotic patients was observed as compared to the normal children. Similarly, Majumdar and Wheeler, 2000; Kuhn et al, 1998 Nayak et al., 1990 observed elevated levels of apo B in these patients. The level of apo A1 (p<0.001) was found to be increased as 273

6 well in comparison to the normal children. The ratio of apo A1:B (p<0.001) was observed to be significantly decreased in comparison to the normal children. Significant increment in these levels in the nephrotic boys and girls was observed as compared to the normal boys and girls. In the present study, dietary intake of the patients revealed that their diet could not provided adequate intake nutrients. One possible reason can be that these patients belonged to the low-income group, thereby plausibly affecting the nutrient intakes. With regard to the levels of total protein and its fractions studied agewise, it was observed that decreased levels of total protein, albumin and A:G ratio were noticed in older children as compared to the younger aged counterparts. When the patients were segregated into boys and girls, they showed the same trend as was witnessed by these patients in toto. The concentration of creatinine, urea and BUN was higher in older patients as compared to their younger counterparts. Increase in the ratio of serum albumin:creatinine was observed older patients as compared to their younger counterparts. Proteinuria and the level of urinary creatinine were found to be higher in older patients as compared to <6yr (p<0.01) and 6-12yr old patients. Age-wise comparison of lipid profile of nephrotic patients showed that the magnitude of increase in the TG level was observed to be higher in 6-12yr and >12yr old (p<0.01) patients as compared to their younger aged counterparts. Similar trend was observed in the VLDL-C levels in all these patients. Increase in the concentrations of TL, TC, LDL-C and non-hdl-c was seen to be higher in older nephrotics as compared to younger nephrotics. The magnitude of increase in these levels was found to be more in nephrotic boys as compared to the girls. The level of HDL-C decreased, as they grew older implying that HDL-C was being lost in the urine. Mani et a/., 1988 observed that nephrotic patients had low levels of HDL-C, as it was lost in urine. Elevated level of TC.HDL-C and LDL-C:HDL-C was seen in 6-12yr old patients followed by >12yr (p<0.01 and p<0.01, respectively) old patients as 274

7 compared to <6yr old patients. In nephrotic boys and girls, same increasing trend was noticed. With regard to apolipoproteins, the level of apo A1 was lower in 6-12yr old patients, followed by patients aged >12yr and higher in <6yr old patients. Significant increase in the concentration of apo B was registered in older (>12yr, p<0.05 and 6-12yr) patients as compared to their younger aged counterpart. In the present study, at three levels of proteinuria (<1g, <2 and' >2g/24hr), the levels of lipid aberrations were studied in nephrotic patients. The concentrations TG and VLDL-C was noticed to be increased in patients with proteinuria of >2g/24hr as compared to patients excreting <2 (p<0.01) and <1g/24hr of proteins in urine. The same trend was seen in the nephrotic boys and girls and it was observed that boys had higher value of TG as compared to the girls. Increment in the concentrations of TL, TC, LDL-C, HDL-C and non-hdl-c was noticed as the level of proteinuria increased from <1 to >2g/24hr in the nephrotic patients. Increment in the atherogenic indices was also observed as the level of proteinuria increased. With regard to apolipoproteins, an increase in the concentration of apo A1 was seen in all these patients. When separately boys and girls were looked into, it was seen the rise in apo A1 was more in girls as compared to boys. Patients with high proteinuria registered increased concentration of apo B (>2g/24hr, p<0.05 and <2g/24hr) as compared to the patients with low level of proteinuria. Moreover, ratio of the lipoprotein fractions i.e. TC:HDL-C and LDL-C: HDL-C and apo A1 :B ratio were all higher in patients with increased level of proteinuria as compared to their counterparts. The present study is in line with the study carried out by Ongajyoth et al, 1993 who observed that the atherogenic indices (i.e. TC: HDL-C and LDL-C: HDL-C) and apo A1:B ratios were all significantly higher in nephrotic patients than normal control (p<0.001) and correlated with urinary protein level. Thus, their study revealed that the nephrotic patients who have persistent heavy proteinuria have dyslipidaemia, which is highly atherogenic and probably increases the incidence of coronary heart disease. Furthermore, proteinuria has been implicated in the genesis of 275

8 lipid abnormalities in nephrotic patients was affirmed by Warwick and Packard, It is known that during proteinuria along with other proteins, albumin is also lost in urine, thereby leading to hypoproteinaemia in these patients. Moreover, it has been reported by GISEN group [1997] that during nephrotic syndrome, the synthesis of albumin may be increased by more than two fold. Thus, the liver synthesises more of proteins in these patients [Zanetti et al, 2001; Gaspari et al, 1995; Ballmer et al, 1992]. In addition, the rate of synthesis of various other plasma lipoproteins, which are not lost in urine, are also increased during nephrotic syndrome and it is also reported that hypoalbuminaemia leads to the genesis of hyperlipidaemia in nephrotic patients [Maugeais et al, 1997; Warwick and Packard, 1993]. To ascertain whether there was any relation between degree of hypoalbuminaemia and abnormal lipid levels, Joven et al, 1990, carried out a study. They arbitrarily divided nephrotic patients into two groups - group 1 (Serum albumin >2.1 g/di) and group 2 (<2g/dl). The results revealed that the mean serum TG and LDL-C concentrations were significantly higher in group 2 than in group 1 as was the level of VLDL-C. Among the apolipoproteins, the level of apo B was significantly higher in group 2 than in group 1. The current study is in line with the above stated findings. Significant increase in the level of TG and VLDL-C, was observed in patients with <2g/dl (p<0.001 and p<0.001, respectively) and 2-3g/dl (p<0.01 and p<0.01, respectively) of serum albumin than in patients with >3g/dl of serum albumin. An increase in the concentrations of TL, TC (p<0.001), LDL-C (p<0.001), HDL-C (p<0.001) and non-hdl-c (p<0.001) in the patients with <2g/dl of albumin was found to be more as compared to their counterparts. Nephrotic boys and girls showed same trend but the level of increase was more in boys than in girls as the level of serum albumin decreased. The atherogenic indices increased as serum albumin level decreased in these patients. With regard to apolipoproteins, increased concentrations, of apo A1 and B was noticed in the patients with decreased level of albumin as compared to their counterparts. The ratio of apo A1:B was 276

9 lowest in the patients with low level (^2g/di) (p<0.001) of albumin as compared to the patients with 2-3g/dl (p<0.001) and >3g/dl of albumin. It has been observed that as the kidney functions deteriorate, the concentrations of proteins decrease and lipid levels increase. In the current study, nephrotic patients in toto were grouped according to the urea levels (urea level <30mg/dl Vs urea level >30mg/dl) and their serum levels of total protein and its fractions and kidney function were assessed along with the lipid aberrations. The results revealed that uremic patients exhibited significantly lower levels of total protein (p<0.05), albumin (p<0.05) and globulin (p<0.05) as compared to non-uremic patients. Increased concentration of creatinine (p<0.001), BUN, BUN:creatinine ratio and albumimcreatinine ratio (p<0.001) was observed in patients with raised level of urea in comparison to patients with low level of urea. In uremic patients the CCrCI was noticed to be slightly lower than in the patients with <30mg/dl of urea. In the current study, it was observed that ureamic patients exhibited elevated levels of lipid profile and atherogenic indices as compared to the patients with <30mg/dl of urea. Lewith and Gabriel, 1975 observed that in the presence of negative nitrogen balance, hyperlipidaemia noticed reflects the result of persistent breakaway of free fatty acid in these patients. With regard to apolipoproteins, the over all picture shows that a slight decrease in the apo A1 level and increase in the level of apo B level was observed in patients with high level of urea as compared to the patients with low level of urea. Concomitantly the ratio of apo A1:B was observed to be low in patients with high level of urea as compared to those patients with low (<30mg/dl) urea level. In the present study, the impact of duration of disease revealed that at all different levels of duration of disease, low level of total protein and its fractions was observed in these patients. A slight rise in total protein and 277

10 albumin concentration was seen in patients with 1-4yr of duration of disease in comparison to the patients with <1yr and >4yrs of duration of disease. The concentration of urea and BUN in the patients with longer duration of diseases was more as compared to the patients with less duration of disease (<1yr, p<0.05 and 1-4yr, p<0.01). Increased level of proteinuria was noticed in the patients with longer duration of disease as compared to the patients with shorter duration of disease. With regard to the lipid levels, increased levels of TG, TC, LDL-C and non-hdl-c were witnessed in the patients with longer duration of the disease, followed by the patients with shorter (^1yr and 1-4yr) duration of disease. The level of HDL-C was observed to be decreasing as the duration of disease increased in these patients. The level of apo A1 decreased as the duration of disease increased whereas, the level of apo B was found to be increased at <1yr and >4yr of duration of disease in these patients. The ratio of apo A1:B decreased in patients with longer duration of disease followed by <1yr and 1-4yr of duration of disease. In addition, it has been observed that the decreased apo A level and increased apo B level at different levels of duration of disease could be important risk factors for the development of coronary atherosclerosis [Hopkins and Williams, 1986]. It was also observed that the atherogenic indices (i.e. TC:HDL-C and LDL-C :HDL-C ratio s) were found to be significantly increased in patients with longer duration of disease (>4yr) as compared to the patients with shorter (<1yr [p<0.05 and p<0.05, respectively] and 1-4yr) duration of disease that may later develop coronary atherosclerosis in these patients. After studying the pathobiochemistry of nephrotic syndrome, we move on further, to study the effect of spirulina supplementation on the protein status, lipid and lipoprotein fractions and apoiipoprotein concentrations of nephrotic patients. 278

11 The enrolled patients were divided into two groups - Control and experimental group. Control Group: Patients in this group were only on medication and they were studied for a period of four months. Experimental Group: This group comprised of patients with medication plus spirulina (1g/day). The number of patients who completed the two months of study included eighteen in control group and thirty-five in the experimental group whereas seventeen in the control group and thirty in the experimental group completed the study after four months. The concentration of serum total protein and its fractions, creatinine, urea, lipids and lipoprotein fractions and apolipoproteins were analysed at baseline and at two monthly intervals. An improvement in the levels of total protein (p<0.001) and albumin (p<0.001) was witnessed at two months in the experimental group patients. In addition, spirulina helped in maintaining the raised total protein and albumin levels in these patients after four months. No change in the globulin concentration was observed after the spirulina supplementation for two and four months. Significant improvement in the A:G ratio (p<0.01) was observed after two months of supplementation but long term (four months) supplementation of spirulina led to slightly more significant improvement in the A:G ratio (p<0.001) in the nephrotic patients. A small rise in the creatinine level (p<0.05) was noticed after two months of supplementation but no change in its level was seen after four months in these patients. A study conducted by Chongviriyaphan et al, 1999 supplemented tuna fish oil (4g for 8 weeks) and observed a non-significant difference in the serum level of creatinine. Decrease in the values of urea, BUN and BUN:creatinine ratio was witnessed after two and four months of supplementation (but four months showed a slightly more decrease as compared to two months of supplementation). A small decrease in proteinuria was also seen in these patients after long term supplementation, which reflects that as compared to baseline less amount of proteins were lost in urine. 279

12 When the nephrotic patients were segregated based on gender, both the genders showed improvements in the levels of total protein and albumin after two months of supplementation. Similar increase in the level of total protein and albumin was seen after four months of supplementation in nephrotic boys (p<0.001 and p<0.001, respectively) and nephrotic girls. But the magnitude of improvements in these values and also in the analytes representing kidney function was noticed to be more in boys as compared to the girls after two and four months spirulina therapy. Moreover, it was seen that the level of proteinuria after long-term supplementation of spirulina decreased in both nephrotic boys and girls. Hyperlipidaemia is a clinical manifestation of nephrotic syndrome and is commonly seen in these patients. Characteristically TG and TC levels are elevated, as are VLDL-C and LDL-C [Querfeld, 1999; Aguilar et al, 1995; Wheeler and Bernard, 1994; D Amico, 1991; Olbricht, 1991; Warwick et al, 1991; Warwick etal, 1990; Vega & Grundy, 1988; Oetliker etal, 1980] and the concentration of HDL-C has been reported to be variable [Querfeld, 1999; de Sain van der Velden, 1998; D Amico, 1991], The results of this study revealed that at baseline all the patients exhibited elevated levels of lipids. After the supplementation of spirulina for four months, significant decrease in the lipid levels was observed. A significant fall in the level of TG (8G.6mg%, p<0.05) was noticed after two months and after four months (126.3mg%, p<0.01) of supplementation. Nephrotic boys showed more decrease in TG level as compared to nephrotic girls. The concentration of TC (p<0.001) in these patients resulted in a significant decrease after two and four (p<0.001) months of supplementation in these patients. The atherogenic lipoprotein, LDL-C and VLDL-C displayed significant reduction after two months (p<0.001 and ' p<0.01, respectively) and four months (p<0.001 and p<0.001, respectively). Same decreasing trend in the level of non-hdl-c was noticed in these patients after two and four months of spirulina supplementations. Various other clinical studies [Mani et al, 2000; Mani et al, 1997; Nayak, 1988; Howard, 1988; Nayaka, 1986] have proved that spirulina effectively reduces 280

13 the elevated cholesterol levels and similar observations have been observed in the current study. With regard to the reduction observed in the TG and VLDL-C levels in nephrotic patients after the spirulina supplementation it could be attributed to decreased VLDL triglyceride production and increased VLDL clearance in the periphery that might have been brought about by the high protein and fibre content of spirulina. During nephrotic syndrome, increased hepatic VLDL-C production leads to increased level of circulating VLDL-C and LDL-C in these patients. But spirulina decreases these levels effectively. As reduction in the VLDL-C levels after spirulina supplementation was noticed there is a simultaneous drop in LDL-C levels in these patients. This may be due to the fact that most of the LDL-C is formed from VLDL-C. In the present study, the over all picture shows a very small decrease in the concentration of HDL-C after two months where as after long-term supplementation, no alteration in the level of HDL-C was observed in these patients. Therefore, it can be postulated that long-term spirulina supplementation was helping slowly in improving the HDL-C concentration in these patients. After spirulina effectively lowered the lipid and lipoprotein fractions, it consequently resulted in significant lowering of the atherogenic indices i.e. TC:HDL-C and LDL-C:HDL-C in these patients. Such changes in the lipid levels have also been associated with a lower incidence of coronary heart disease [Parikh et a!, 2001]. With regard to apolipoproteins, the concentration of apo B in the current study was found to be increased at baseline, a finding reported by various authors. But it is noteworthy, that spirulina supplementation significantly decreased the apo B levels after two (p<0.001) and four months (p<0.001). A reduction in the level of apo A1 (p<0.05) was observed after two and four months of supplementation in these patients. Concomitantly, spirulina therapy exhibited a significant rise in the apo A1:B ratio after two (p<0.001) and four months (p<0.001) in these patients. 281

14 Furthermore, to affirm the beneficial effect of spirulina therapy, the nephrotic patients of the control and experimental -group were matched for age, gender and severity of the disease (for which proteinuria was taken). The results revealed significant improvements in the levels of total protein and albumin in the control and experimental group after two and four months in these patients. But the magnitude of improvement witnessed in the experimental group patients was more as compared to the patients in the control group. With regard to the lipid levels, drop in the lipid profile was seen in control and experimental group patients. But it was noticed that more reduction in the lipid levels was observed in the patients of experimental group as compared to the control group after two and four months. It was seen that the magnitude of decrease witnessed in the experimental group patients was found to be more after two month of spirulina supplementation. In addition, the supplementation of spirulina in the experimental group also showed substantial reduction in the number of relapses. Based on this the clinician has brought down the level of drug to 54% of the initial requirements whereas the control group patients required 87% of the initial level for the treatment of nephrotic syndrome. These observations clearly demonstrate and substantiate that spirulina therapy has shown a remarkable improvement in the prognosis of the patients. Hence, it can be said that spirulina aided in improving the quality of life of the experimental group patients in a better way as compared to the patients in the control group. Therefore, from the results of the present study it can be summarised that 4- Nephrotic patients showed diminished levels of.total protein and its fractions when compared with normal children. Elevated levels of lipid and lipoprotein fractions, apolipoproteins and atherogenic indices were observed as compared to normal children. 282

15 4- As the level of proteinuria increased from <1 to >3g/24hr, the concentration of lipids and lipoprotein fractions, atherogenic indices increased while the ratio of apo A1:B decreased. ^ With the decrease in the level of albumin from >3g/dl to <2 g/dl, the concentration of lipids and lipoprotein fractions, atherogenic indices and apolipoproteins were observed to be increasing in nephrotic patients. Patient with >30mg/dl of urea level exhibited significantly lower level of total protein and it fractions as compared to patients with <30mg/di of urea level. Increased levels of analytes representing the kidney function were noticed in patients with high urea level as compared to patients with low urea ievel. The levels of lipids, lipoprotein fractions and apolipoproteins were elevated in the nephrotic patients with >30mg/di of urea as compared to the nephrotic patients with <30 mg/dl of urea level. 4- The levels of total protein and its fractions and analytes representing kidney function of nephrotic patients based on the duration of disease showed that the level of total protein and its fractions was observed to be more in patients with duration of disease of 1-4yrs and >4yrs than in patients with duration of disease of <1yr. Increase in the lipid and lipoprotein fractions, apolipoprotein and atherogenic indices were observed in patients with longer (>4yrs) duration of disease as compared to the patients with shorter (<1yr and 1-4yrs) duration of disease. ^ Spirulina therapy has helped in improving the protein levels of these patients. 283

16 Significant decrease in the level of TG, TC and its fractions was observed after the supplementation of spirulina for two and four months. ^ The atherogenic indices also exhibited significant reduction after two and four months of supplementation. With regard to the apolipoproteins, the level of apo B reduced significantly and a small decrease in the level of apo A1 was observed in these patients after supplementation. The ratio of apo A1:B showed significant increase after the supplementation of two and four months in these patients. 284