Nephron 45: 196 201 (1987) & 1987 S. Karger AG. Hasel 0028-2766/87/0453 0I96S2.75/0 Protein Losses in Patients Receiving Continuous Ambulatory Peritoneal Dialysis G.A. Young, A.M. Brownjohn, F.M. Parsons Renal Research Unit, General Infirmary, Leeds, UK KeyWords. Continuous ambulatory peritoneal dialysis ■Blood proteins • Albumin • Prealbumin • Transferrin ■ Globulins • Glycoproteins • Haptoglobin • Peritoneal dialysis Abstract. Total protein and 12 specific proteins were measured in dialysates from 8 patients on continuous ambulatory peritoneal dialysis during training. Mean daily loss of total protein was 10.5 g and this included 5.2 g albumin, 805 mg of the immunoglobulins G, A and M, 323 mg transferrin and 530 mg of the remaining 7 proteins measured. The plasma to dialysate ratio of protein concentrations correlated with the natural logarithm of molecular weight, suggesting that proteins in dialysate are an ultrafiltrate of plasma. A greater loss of proteins overnight was due to longer dwell time as the mean rate of loss was similar for all exchanges. Losses were similar with 1.36% and 3.86% dextrose fluids, suggesting that the initial effects of hypertonicity are diminished or reversed by dilution and absorption of dextrose. Daily outflow volumes for 4 patients correlated inversely with the quantities of several proteins removed, probably due to effects of osmolality. It is concluded that protein losses are related to plasma concentration, molecular weight and osmolality of the dialysis solution and to the physiology of the patient. Continuous ambulatory peritoneal dialysis (CAPD) is a valuable alternative to haemodialysis for the treatment of end-stage renal disease. The loss of proteins into the dialysis fluid was first measured during intermittent peri toneal dialysis (IPD) [1] and has since been studied clin ically and experimentally. Comparisons between IPD and CAPD have shown similar losses of 30-100 g/week [2], Recently there have been several investigations into the causes and consequences of protein losses during peritoneal dialysis and these have been reviewed [3] but very few detailed studies of specific proteins have been made. In this study we have evaluated the losses of total protein and 12 specific proteins into dialysis fluid during CAPD and have identified some of the factors contribut ing to these losses. Patients and Methods Patients Eight patients, 4 females and 4 males, aged 28-58 years (table 1) were studied during their initial 10- to 13- day training period whilst in hospital. All patients had an OreopoulosorTenckhoff peritoneal catheter and had received intermittent peritoneal dialysis for be tween 5 days and 22 months. They were dialysed continuously with 4 exchanges daily using 2 I plastic bags of Travenol® Dianeal 137 except for 1 small patient (No. I) who was prescribed 1 I bags. Dextrose (anhydrous) concentration was usually 1.36 g/100 ml (356 exchanges) although a 3.86 g/100 ml dehydrating dialysis solution was used intermittently in 4 patients to maintain constant daily weight (23 exchanges). Mean dietary protein intake was 1.4 g/kg of body weight and calories 27 Kcal/kg in addition to calories derived from dextrose in the dialysis fluid. Vitamin supplements and alumi nium hydroxide were prescribed for most patients. Methods Body fat and arm muscle circumference (AMC) were calculated from anthropometric measurements [4] and body surface area was calculated from height and weight [5). Osmolality of dialysate was measured using an Osmometer (Fisons). Total protein in dialysate and plasma was measured using a Beckmann® Trace I analyser and specific proteins measured using a Beckmann® Immunochemical analyser or by radial immunodiffusion. The recovery of proteins from 1.36 and 3.86% dextrose dialysis fluid containing added plasma was between 95 and 104% except for immunoglobulin M (85%), p2 microglobulin and caeruloplasmin (75%). All proteins were measu rable in dialysis fluid without any concentration. Heparinised blood samples were collected and the plasma separated and stored at -70 °C. During the study all dialysates were collected at the completion of each exchange, mixed and measured. Aliquots from each ex- Downloaded by: Vanderbilt University Library 129.59.95.115 - 10/26/2017 11:20:14 PM Introduction Protein Losses during CAPD 197 Table I. Sex, age. previous l PD and anthropometric data for 8 patients at the start of CAPD Patient Sex Age years Previous treatment on IPD, months Height cm Weight kg Body surface area, m2 Fat kg AMC cm 1 2 3 4 5 6 7 8 F F F F M M M M 28 49 57 58 38 51 52 55 < 1 <1 22 3 18 <1 12 <1 142 164 155 160 168 161 185 163 30.4 63.5 57.0 65.4 78.7 52.0 80.0 60.0 1.1 1.7 1.6 1.7 1.9 1.5 2.0 1.6 5.1 18.5 20.2 22.3 19.5 8.6 26.2 16.5 14.9 24.3 21.6 24.6 29.6 23.4 28.0 26.3 162 ±12 60.9 ±15.7 1.64 ±0.27 17.1 ±7.0 24.1 ±4.5 48.5 ±10.4 M ean±SD Table II. Losses of total protein and 6 specific proteins in CAPD dialysate during training; plasma protein concentrations at the start and end of the study are also shown 1 2 3 4 5 6 7 8 M ean±SD Study days 10 10 13 13 13 13 10 13 Start of CAPD End of study Outflow volume l/day 4.4 ±0.5 9.1 ±0.1 8.5 ±0.5 8.2 ±0.6 9.0 ±0.3 9.4 ±0.7 9.0±0.6 8.3 ±0.2 8.2 ±1.6 Dialysate protein losses total protein g/day albumin g/day transferrin mg/day IgG mg/day IgA mg/day IgM mg/day 8.0 ± 2.1 9.8 ±1.0 10.7 ±3.6 10.9 ± 1.8 11.4 ± 1.6 11.6 ±2.3 10.7 ± 1.5 11.0 ± 1.4 10.5 ± 1.2 Plasma protein 4.8 ±1.5 5.4 ±0.5 4.7 ± 1.8 5.2 ±1.2 5.3 ±1.4 4.5 ± 1.1 5.5 ± 1.7 5.9 ±0.6 5.2 ±0.5 concentrations 299 ±80 323 ±4.6 325 ±116 389 ±78 361 ±93 267 ±56 249 ±67 373 ±58 323 ±5.0 718 ±299 684 ±91 572 ± 312 675 ±177 489 ±17 672±238 596± 162 579 ±68 623 ±77 106 ±41 130 ± 13 99 ±49 132 ±42 207 ± 51 91 ±28 82 ±28 147 ± 15 124 ± 40 42 ±21 51 ±21 51 ±23 40 ± 7 103 ±45 50 ±23 48 ± 14 76± 16 58 ± 21 g/dl g/dl mg/dl mg/dl mg/dl mg/dl 3.9 ±0.4 3.7 ±0.4 264 ±40 269 ±47 914 ±212 864 ±205 253 ±54 221 ±54 192 ±45 184 ±27 6.9 ±0.8 7.0 ±0.7 change and the pooled samples containing all the days’ exchanges in proportion to their volumes, were stored in containers without preservative at 20 °C. The Student t test was used for comparison of both paired and unpaired values. Results Palien I Data: Table I shows the sex, age, previous dialysis treatment, height, weight, calculated body surface area, fat and AMC for the 8 patients. There were no significant differ ences between the sexes for age, height, weight, body suface area, fat or AMC. No peritonitis or other illness occurred during the study. All the patients had previously been on IPD; 4 for periods of 3-22 months and 4 for less than 1 month. Patient 4 alone had had a single attack of peritonitis 2 months before the start of CAPD. Protein Losses in Dialysates: Mean daily losses of total protein and 12 specific proteins into the dialysate during the training period for each of the 8 patients are shown in tables II and III. The mean daily loss and mean initial and final plasma con centrations for each protein in the 8 patients are also shown. The mean loss of total protein was 10.5 g and included 5.2 g albumin (49%), 805 mg immunoglobulins Downloaded by: Vanderbilt University Library 129.59.95.115 - 10/26/2017 11:20:14 PM Patient Young/Brownjohn/Parsons 198 Table III. Losses of 7 specific proteins in CAPDdialysale during training: plasma protein concentrations at the start and end of the study are also shown Patient 1 2 3 4 5 6 7 8 M ean±SD Start of CAPD End of study Study days 10 10 13 13 13 13 10 13 Dialysate protein losses, mg/day prealbumin AAG haptoglobin AMG caerulo plasmin (i2 micro globulin C3 32 ±12 40 ± 13 46 ± 10 46 ± 11 55± 16 64 ± 19 41 ± 7 44 ± 9 46 ± 10 Plasma protein 97 + 26 95 ±11 167 ± 66 119 ±20 176 ±40 204 ± 91 158 ±29 222 ±25 155 ±47 80 ±30 44 ± 7 121 ±78 52 ± 19 35 ± 10 160 ±39 62 ±18 156 ±22 89 ±50 87 ±39 67 ±12 127 ± 102 47 ± 17 67 ±21 73 ±22 51 ±18 95 ±20 77 ±26 19 ± 4 26 ±5 34± 16 23 ±6 29 ± 11 34 ± 12 24 ±10 41 ±6 29 ±7 17 ± 2.8 12 ± 1.9 31 ±3.7 29 ±3.8 24 ±2.9 21 ±3.5 25 ±2.7 24 ±2.9 23 ± 6 76 ±24 97 ± 18 120 ±65 85 ±20 169 ± 65 150 ±35 120 ± 31 124 ± 14 115 ±32 concentrations, mg/dl 39 ±4.0 39 + 4.0 109 ± 31 107 ±36 249 ±74 256± 74 54 ± 13 47 ±10 2.6 ±0.7 2.7 ±0.6 155 ±32 166 ±31 2 10 ± 116 207 ±108 this low-molecular-weight protein were 27.3 mg/day and 18.5 mg/day (p< 0.025). Plasma Concentrations.The mean loss for each protein for all the patients correlated with their mean concentra tions in plasma at the start of training (r = 0.99: p < 0.001), suggesting that the proportions are similar in dialysate and plasma. The wide ranges of concentrations contri buted to this high correlation coefficent and when the highest concentrations for albumin and lgG are excluded then we found r = 0.59; p<0.05. Molecular Weight. The concentrations of the proteins in plasma are ranked in figure I and the corresponding Factors Contributing to Protein Losses amounts in dialysate and the molecular weights are Sex. Differences in mean protein loss between the shown. Several proteins in dialysate are in smaller sexes were not significant except for a t acid glycoprotein amounts than expected from plasma concentrations and (p<0.02) and complement C3 (p<0.02). The use of 1 1 this can be attributed to higher molecular weights, e.g., a2 exchanges and the smaller body size of the female patient macroglobulin, IgM and caeruloplasmin. In contrast, 1may have contributed to the smaller mean loss for these larger amounts in dialysate are associated with lower two proteins. molecular weights, e.g., albumin, transferrin, a, acid gly Age and Anthropometry. The losses for each of the 12 coprotein and p2 microglobulin. This relationship is specific proteins were unrelated to age ot to anthropo shown in figure 2. The plasma :dialysate ratio of protein metric measurements of the patients although total pro concentrations correlated with the natural logarithm of tein did correlate with AMC (r = 0.78; p<0.05). molecular weight (r = 0.92; p<0.001). This suggests that Previous Intermittent Peritoneal Dialysis. There was no proteins in dialysate are an ultrafiltrate of plasma. significant difference between protein losses in the 4 Losses during Each Exchange. Losses of 3 proteins, patients who had previously been on I PD for the longest albumin, transferrin and haptoglobin were measured for periods of 3-22 months and those for less than I month the 8 patients during 270 daytime exchanges and 83 except for p2 microglobulin. The comparative losses of overnight exchanges using 1.36% dextrose. The mean Downloaded by: Vanderbilt University Library 129.59.95.115 - 10/26/2017 11:20:14 PM G, A and M (8%), 323 mg transferrin (3%) and 530 mg of the remaining 7 proteins (5%). The daily loss for each protein was equivalent to 31 142 ml plasma, i.e., a clear ance of 0.02-0.10 ml/min [loss per day or per minute divided by plasma concentration (g/ml)] with the excep tion ofp2 microglobulin which was equivalent to 938 ml plasma per day or 0.65 ml/min. There was no significant change in the daily loss or the concentration in plasma for each protein during the training period. Over the 13 days the coefficient of variation for the mean daily total pro tein loss was 18%. Protein Losses during CAPD 199 Molecular weight, In Fig. 1. Initial plasma concentrations for each of 12 proteins are ranked and the corresponding amounts in dialysate and their molec ular weights are shown. Alb = Albumin; IgG = Immunoglobulin G; Trf = transferrin; IgA = immunoglobulin A; AMG = a : macroglob ulin; Hapt = haptoglobin; IgM = immunoglobulin M; C3 = complement C3; AAG = ct| acid glycoprotein: Caer = caeruloplasmin: PreA = prealbumin; [LMG = microglobulin. rates of protein loss during daytime (±SD ) exchanges were: albumin 218± 60 mg/h, transferrin 13.7± 4.0 and haptoglobin 4.2 ±3.0 mg/h and those at night time were not significantly different (unpaired t test). The total losses at night time were greater because the exchanges were longer, i.e., 9.5 compared with 4.8. The first 2 day time exchanges at the start of training were not included in the calculation of daily losses because of residual protein and dialysate in the peritoneum from previous IPD. The Effects of Osmolality o f Dialysis Fluid on Protein Loss During a total of 377 exchanges in the 8 patients, 3.86% dextrose was required for only 24 exchanges in 4 patients. The mean rates of loss for albumin, transferrin and haptoglobin were not significantly different using either 1.36 or 3.86% dextrose during the overnight or daytime exchanges. The effects of the movement of water into the peritoneum during CAPD on the amounts of protein removed were evident in 4 patients maintained on 1.36% dextrose fluid. The daily losses of several proteins were inversely correlated with the volumes of dialysate removed. In patient 1an inverse correlation was observed between volumes of fluid removed and total protein (r = 0.75; p<0.05), albumin (r = 0.72; p<0.05) as shown in figure 3 and also with caeruloplasmin (r = 0.70; p < 0.05). Similar correlations were observed in patient 6 for transferrin (r = 0.79; p<0.01), caeruloplasmin (r = 0.56; p<0.05) and haptoglobin (r = 0.73; p <0.01), and for albumin and haptoglobin respectively in patients 2 and 5. The volumes removed also correlated with the osmolality of the dialysate in patient 1. This suggests, that the accumulation of excess fluid in the peritoneum de creased the osmolality and may have contributed to a reduced loss of proteins. Outflow volume was unrelated to protein loss in the other 4 patients. Downloaded by: Vanderbilt University Library 129.59.95.115 - 10/26/2017 11:20:14 PM Fig. 2. Plasma: dialysate ratio correlated with the natural loga rithm of molecular weight. Abbrevations as in figure 1. 200 Young/ Brownjohn/Parsons (820,000) are retarded and low-molecular-weight pro teins such as ¡)2microglobulin (11,820) are increased. This is characteristic of the ultrafiltration process that occurs across the other body fluid barriers such as plasma to cerebrospinal fluid [6], synovial fluid [7], amniotic fluid [8] and also ascitic fluid [9]. Some effects of molecular weight on protein loss during CAPD have been suggested in a study of 6 proteins [10] and from a review of previous data [3]. The quantity of |)2microglobulin in dialysate is very high and the possibility of synthesis by macrophages within the peritoneum, as previously suggested for col lagen [11] and glutathione peroxidase [12], cannot be ex cluded. Effects o f Physiology on Protein Loss The amount of protein removed during CAPD is re Fig. 3. Inverse correlations between losses of total protein, lated to certain anatomical and physiological features of albumin and osmolality against outflow fluid volume in patient I. the patient [3]. In a previous study the total protein in dialysate correlated with body surface area and this was equated with anatomical surface area [15], We did not Discussion observe this relationship but protein loss correlated with arm muscle circumference. Smaller protein losses in Protein Losses relative to Plasma Concentration and women may be associated with the smaller peritoneal Molecular Weight surface area although such differences between men and The daily losses of total protein, albumin, transferrin, women were confined to ct| acid glycoprotein and com immunoglobulins G, A and M, and complement C3 were plement C3 and other causative factors cannot be ex of a similar order to that found by Blumenkrantz et al. [2] cluded. The comparability of losses between patients although comparable measurements for prealbumin, a, suggest that these were unaffected by any clinical differ acid glycoprotein, haptoglobulin, a 2 macroglobulin, ca ences or by previous treatment with I PD. (12 Microglobueruloplasmin and IT microglobulin are not available. The lin was the only protein lost in greater amounts in those concentration in dialysate was related not only to plasma patients previously maintained on 1PD for longer peri concentration but also to molecular weight. Our results ods, suggesting that any increase in porosity of the perito suggest that loss of higher-molecular-weight proteins neum to proteins was limited to those of low molecular such as immunoglobulin (950,000) and a2macroglobulin weight. None of the patients had peritonitis or other Downloaded by: Vanderbilt University Library 129.59.95.115 - 10/26/2017 11:20:14 PM The Effects o f Dialysis Fluid on Protein Loss The effects of osmolality were evident only in patients maintained solely on a 1.36% dextrose solution where the amounts of proteins removed were inversely related to the outflow volumes of the dialysate. This suggests that the transfer of excess fluid into the peritoneum may decrease osmotic pressure and reduce protein loss. Des pite this relationship with osmolality the use of hyperos molar dextrose solution, both in this and several previous studies, did not cause a greater loss of protein [2, 13]. Clearly any effects of osmolality are small and if in creased protein losses occur during hyperosmolar dialy sis over the first 3 h [14] they could subsequently decrease with the fall in osmotic pressure caused by the absorption of dextrose and expansion of the fluid volume. Protein LossesduringCAPD 8 9 Consequences o f Protein Loss The consequences of protein losses during CAPD have not been fully elucidated. During this study there was no decrease in the plasma concentrations of proteins measured and in previous short-term studies nutritional status and albumin haemostasis were maintained in wellnourished patients [17]. During long-term studies a de crease in plasma proteins, body muscle and total body nitrogen may occur [18,19], particularly when the dietary protein intake is less than 1.2 g/kg body weight [19], Any depletion of plasma proteins will inhibit many vital func tions, particularly transport mechanisms and response to infection [19]. Replenishment of specific proteins will also depend on their rates of biosynthesis. Consequently, if the daily loss equals or exceeds the amount synthesised, depletion will occur. This is most likely for low-molecu lar weight compounds where the clearance is high, e.g., pi microglobulin. Further studies of the loss of small pro teins are required. Acknowledgements We wish to thank Mrs Shirley Hobson, Mr Albert Taylor and Miss Mandy Thompson for their invaluable help. We are grateful to the nursing staff of the General Infirmary at Leeds and the Leeds Area Health Authority who kindly supported this work. 10 11 12 13 14 15 16 17 18 References 1 Berlyne, G.M.: Jones, J.H.; Hewitt, V.: Nilwarangkur, S.: Pro tein loss in peritoneal dialysis. Lancet, i: 738-741 (1964). 2 Blumenkrantz, M.J.: Gahl, G.M.; Kopple, J.D.; Kamdar. A.V.; Jones, M.R.; Kessel, M.; Coburn, J.W.: Protein losses during peritoneal dialysis. Kidney int. 19:593-602 (1981). 3 Dulaney, J.T.; Hatch, F.E.: Peritoneal dialysis and loss of pro teins: a review. Kidney int. 26:253-262 (1984). 4 Durnin, J.V.G.A.: Womersley, J.: Body fat assessed from total body density and its estimation from shinfold thickness: mea surements on 481 men and women aged from 16 to 72 years. Br. J. Nutr. 32:77-97 (1974). 5 Sendroy, J.; Cecchini, L.P. Determination of human body sur face area from height and weight. J. appl. Physiol. 7:1-12 (1954). 6 Felgenhauer. K.: Protein size and cerebrospinal fluid composi tion. Klin. Wschr. 52:1158-1164(1974). 7 Levick, J.R.: Permeability of rheumatoid and normal human 19 synovium to specific plasma proteins. Arthritis Rheum. 24: 1550-1560(1981). Burnett, D.; Wood, S.M.: Studd, J.W.W.; Lewis, B.V.: Bradwell. A.R.: A study of the relationship between maternal serum and amniotic fluid proteins in early and late pregnancy. Protides biol Fluids 24:161-164 (1976). Henderson, J.M.; Stein, S.F.: Kulner, M.: Wiles. M.-B.: Ansley, J.D.; Rudman. D.: Analysis of twenty three plasma proteins in ascites. The depletion of fibrinogen and plasminogen. Ann. Surg. 192:738-742 (1980). Giangrande, A.: Limido. A.tCanto. P.; Allaria. P.: SDS-Polyac rylamide electrophoresis of protein loss during continuous am bulatory peritoneal dialysis. Clin.Lab. 10:117 12 (1980). Kowalewski, J.: Tomaszenki, J.: Hanzlik, J.; Zawislak. H.:Zbikowska. A.: The elimination of free, peptide-bound and proteinbound hydroxyproline into dialysate during peritoneal dialysis in patients with renal failure. Clin. Chim. Acta. 34: 123-126 (1971). Young, G.A.; Brownjohn, A.M.; Turney, J.H.: Glutathione per oxidase and tocopherol in patients on continuous ambulatory peritoneal dialysis (CAPD). Kidney int. 26:612 (1984). Katirtzoglou, A.: Oreopoulos, D.G.: Husdan, H.; Leung, M.: Ogilvie. R.: Dombros, N.: Reappraisal of protein losses in patients undergoing continuous ambulatory peritoneal dialysis. Nephron 26:230-233 (1980). Rubin, J.; Nolph. K.D.; Arlania, D.; Prowant. B.; Fruto, L.; Brown, P.: Moore, H.: Protein losses in continuous ambulatory peritoneal dialysis. Nephron 2<Y: 218— 221 (1981). Twardowski, Z.: Ksiazek, A.; Majdan.M.: Janicka, L.: Bochenska-Nowacka, E.; Sokolowska, G.; Gutka, A.; Zbikowska, A.: Kinetics of continuous ambulatory peritoneal dialysis (CAPD) with four exchanges per day. Clin. Nephrol. 75:119-130 (1981). Rubin. J.; Nolph, K.D.: Arfania, D.: Brown. P.: Follow-up of hourly peritoneal clearances with and without nitroprusside augmentation in patients undergoing CAPD. Kidney int. 16: 619-633 (1979). Kaysen. G.A.; Schonfeld, P.Y.: Albumin homeostasis in pa tients undergoing ambulatory peritoneal dialysis. Kidney int. 25:107-114(1984). Williams, P.: Kay, R.; Harrison, J.; McNeil, K.; Pettit, J.; Kelman, B.; Mendez, M.; Klein, M.; Ogilvie, R.: Khanna, R.: Carmichael, D.: Oreopoulos, D.: Nutritional and anthropomet ric assessment of patients on CAPD over one year: contrasting changes in total body nitrogen and potassium. Perit. Dial. Bull. 7:82-87 (1981). Young, G.A.: Young, J.B.; Young, S.M.: Hobson, S.M.: Hil dreth, B.; Brownjohn. A.M.: Parsons, F.: Nutrition, and delayed hypersensitivity during CAPD in relation to peritonitis. Neph ron 43:177-186(1986). Accepted: May 28,1986 Dr. G.A. Young, Scientific Director, Renal Research Unit, General Infirmary, Leeds LSI 3EX (UK) Downloaded by: Vanderbilt University Library 129.59.95.115 - 10/26/2017 11:20:14 PM infections during the study, which could have increased the clearance of proteins [2,16). The similarity of the rate of protein clearance during overnight and daytime ex changes suggest that any effects of diurnal rhythms or physical activity were small. 201
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