CONTINUOUS AMBULATORY PERITONEAL DIALYSIS IS RESPONSIBLE FOR AN INCREASE IN PLASMA NOREPINEPHRINE

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1 Peritoneal Dialysis International, Vol. 20, pp Printed in Canada. All rights reserved /00 $ Copyright 2000 International Society for Peritoneal Dialysis CONTINUOUS AMBULATORY PERITONEAL DIALYSIS IS RESPONSIBLE FOR AN INCREASE IN PLASMA NOREPINEPHRINE John G. Vlachojannis, Sotiris Tsakas, Sotiria Alexandri, Chrysanthi Petropoulou, and Dimitris S. Goumenos Department of Internal Medicine Nephrology, University Hospital, Patras, Greece Objective: To investigate the reason for increasing norepinephrine (NE) levels reported in continuous ambulatory peritoneal dialysis (CAPD) patients. Methods: Norepinephrine was measured in the plasma and peritoneal dialysate of CAPD patients (n = 22) and in the plasma and the urine of healthy subjects (n = 20). It was also measured in the plasma of patients with chronic renal failure (CRF) (n = 15) and patients on hemodialysis (HD) (n = 15). Results: It was found that NE was increased in CAPD patients compared with healthy individuals (687 ± 221 pg/ml vs 199 ± 25 pg/ml, p < 0.01). The daily removal of NE from the peritoneum of CAPD patients was lower compared with the amount of NE excreted in the urine of healthy subjects. Plasma NE increased after infusion of peritoneal dialysate. In 15 new patients on CAPD, it was found that NE plasma levels increased from 329 ± 67 pg/ml before initiation of dialysis, to 584 ± 173 pg/ml after 12 months of treatment (p < 0.01). Finally, plasma NE in CAPD patients (687 ± 221 pg/ml) was significantly higher compared with the already increased levels in patients on HD or with CRF (406 ± 143 pg/ml and 378 ± 142 pg/ml, respectively). Conclusions: It is concluded that CAPD in patients with end-stage renal disease is responsible for a progressive increase of plasma norepinephrine. Correspondence to: J. Vlachojannis, Internal Medicine Nephrology, University Hospital of Patras, Patras, Greece. Received 13 April 1999; accepted 18 November KEY WORDS: Norepinephrine; sympathetic system. End-stage renal failure appears to be accompanied by sympathetic dysfunction (1,2). Reliable tests have been used for the estimation of relevant disorders (3). The Valsalva test, the orthostasis challenge test, and the handgrip test have been used for this purpose and have demonstrated significant autonomic nervous system dysfunction in patients on continuous ambulatory peritoneal dialysis (CAPD) (4). Researchers have also approached the issue of the autonomic system disorder by studying the major biological products of the sympathetic system, that is, catecholamines (5 7). Catecholamines are synthesized from tyrosine via the following biosynthetic pathway: tyrosine dihydroxyphenylalanine dopamine norepinephrine (NE) epinephrine (8). The adrenal medulla contains all the necessary biosynthetic enzymes; epinephrine is the final product. Other sites, such as the sympathetic postganglionic neurons, lack the final enzyme, so NE is their major product (9). As NE is released in the synaptic cleft, a small amount (less than 20%) escapes into the circulation (10). Plasma NE half-life is very short (1 2 min) due of its rapid clearance through cellular uptake and metabolism (9). Alteration of NE clearance from blood can change the impact of NE release on plasma concentration (11). Contradictory results have been reported concerning the plasma levels of catecholamines in renal patients on dialysis. Elevated NE plasma levels have been found in patients on hemodialysis (HD) and in patients on CAPD (5). Some authors have reported elevated plasma NE only before HD; levels decreased after the dialysis session (6). These results are probably due to the different methods used for the measurement of plasma catecholamines. Epinephrine and dopamine plasma concentrations have been found to be in the normal range in HD and CAPD patients (5,6). In the present study we examined the plasma NE concentration in patients treated by CAPD, and we also investigated the influence of CAPD on plasma NE levels. SUBJECTS AND METHODS SUBJECTS Plasma and peritoneal dialysate catecholamine concentrations were determined in 22 patients on CAPD (14 males, 8 females; age years, average age 42 years). Patients were treated with four exchanges of 2 L dialysate daily (Baxter, Utrecht, Holland; or Fresenius, Hamburg, Germany) and were 322

2 PDI MAY 2000 VOL. 20, NO. 3 NOREPINEPHRINE IN CAPD on CAPD from 1 5 years (average 2 years). None of the patients suffered from amyloidosis, alcoholism, or psychiatric illness, and none had undergone nephrectomy or parathyroidectomy. In 15 new patients on CAPD, catecholamines were determined just before the introduction of the method and again 12 months later. Catecholamines were also measured in the plasma and urine of 20 normal subjects (12 males, 8 females; age years, average age 40 years). In addition, plasma catecholamines were determined in 15 patients (9 males, 6 females; age years, average age 45 years) with chronic renal failure (CRF) (creatinine clearance 21 ± 6 ml/ min). Catecholamines were also measured in 15 patients (10 males, 5 females; age years, average age 46 years) on HD for 1 4 years (average 2.5 years). SAMPLE PREPARATION Subjects were placed in the supine position for 20 minutes after cannulation. Blood (6 ml) was collected in heparinized tubes, 2 hours after the dialysate infusion and centrifuged for 10 minutes at 1500g, at 4 C. Plasma samples (2.5 ml) were removed and stored at 20 C until assayed for epinephrine and NE. All patients kept 24-hr urine and dialysate collections in their refrigerator (4 C) at home and brought them to the hospital the next day. Aliquots of 2.5 ml from 24-hr urine collection of healthy subjects as well as from 24-hr peritoneal dialysate collection of CAPD patients were stored at 20 C. Sodium metabisulfite 5 mg was added as an antioxidant to every sample. METHODS Catecholamines were measured by cation-exchange high performance liquid chromatography (HPLC) with electrochemical detection (BioRad, Hercules, CA, U.S.A.). Samples were absorbed onto mg of alumina and finally extracted with 150 μl 2 mol/l phosphoric acid prior to HPLC (12,13). Plasma, urine, and peritoneal dialysate creatinine was determined with picric acid assay. PERITONEAL EQUILIBRATION TEST The peritoneal equilibration test (PET) is used to assess the permeability of the peritoneal membrane. Dialysate samples were collected at 0, 2, and 4 hours (equilibration time) after infusion. Blood was drawn just prior to the dialysate infusion (0 time)and at 0.2, 2, and 4 hours after dialysate infusion. All patients were cannulated and remained in the supine position for 20 minutes before each blood sample was drawn. This was done to exclude any NE plasma increase due to sympathetic response. Creatinine and urea were determined in the plasma (P) and dialysate (D). The D/P ratio (P at 2 hr) for each substance was then plotted over a 4-hr period to estimate peritoneal transport characteristics (14). CALCULATIONS The renal or peritoneal removal of NE was calculated according to this equation: NE daily removal (total amount of NE excreted in the urine or removed by the peritoneum in 24 hr) = NE urine or peritoneal dialysate concentration total urine or peritoneal dialysate volume in 24 hr. STATISTICAL ANALYSIS The results were expressed as mean ±SD. Differences between the patient groups and the healthy subject group were determined by comparison of their mean values using Student s t-test for unpaired data. Differences before and during CAPD were determined using Student s t-test for paired data. A p value of less than 0.05 was considered significant. RESULTS NE PLASMA LEVELS IN NORMAL SUBJECTS AND CAPD PATIENTS Catecholamines were measured in the plasma of 22 patients with end-stage renal disease treated with CAPD and also in the plasma of 20 healthy individuals. Norepinephrine plasma levels were significantly increased in the CAPD patients group compared with the control group (687 ± 221 vs 199 ± 25 pg/ml, p < 0.05) [Figure 1(a)]. On the other hand, epinephrine levels were not significantly different between the two groups (34 ± 10 pg/ml vs 31 ± 9 pg/ml). Dopamine plasma levels were in the normal range (< 25 pg/ml) in both groups. NE REMOVAL IN NORMAL SUBJECTS AND CAPD PATIENTS The daily removal of NE from the peritoneum was determined in order to explain the elevated concentration of NE in the plasma of patients on CAPD. Norepinephrine was measured in the daily total peritoneal dialysate of 22 patients on CAPD and compared with the daily urinary excretion of NE by 20 healthy individuals. It appeared that the removal of NE (5.3 ± 1.9 μg/24 hr) from the peritoneum, was significantly lower than its removal from the kidneys of healthy persons (33 ± 9 μg/24 hr) [Figure 1(b)]. The daily NE 323

3 VLACHOJANNIS et al. MAY 2000 VOL. 20, NO. 3 PDI Figure 1 Plasma norepinephrine concentration is higher in CAPD patients than in normal controls (A). Norepinephrine daily removal is lower in CAPD patients (Group PD, n = 22) than in normal subjects (Group N, n = 20) (B). removal through the peritoneum in CAPD patients was about sixfold lower compared with its removal by the kidneys in healthy subjects. On the other hand, plasma NE was only threefold higher. To investigate these contradictory results, the peritoneal NE removal rate was compared with the respective rates of creatinine and urea. NE REMOVAL RATE FROM THE PERITONEUM The PET was performed in 15 patients on CAPD, as described in Subjects and Methods. Results showed that creatinine and urea follow similar rates of removal, leading to a decrease in the slope of the line in the second period of the graph (2 hr 4 hr), compared with the slope in the first period (0 hr 2 hr) (Figure 2). On the other hand, NE removal rate presented an increasing slope in the second period of the graph, compared with the slope in the first period (Figure 2). The peritoneal transport graphs for urea and creatinine were characteristic of substances with relatively stable plasma concentration. The increasing course of NE in the second period could be on line with increasing NE plasma levels found while dialysate was present in the peritoneal cavity. To investigate this possibility, blood samples were collected from 15 patients just prior to the infusion of dialysate into the peritoneal cavity (Po), as well as at 0.2-, 2-, and 4-hr dwell times (P). The graph of P/Po versus time showed that plasma NE increased significantly during the dialysis session, compared with its initial plasma concentration before infusion of dialysate into the peritoneal cavity (Figure 3). Urea and creatinine levels remained practically unchanged. CHANGES IN NE PLASMA LEVELS WITH TIME ON CAPD Since plasma NE increases during the exchange session in CAPD patients, the question arises as to whether the application of the method itself is responsible for this significant increase. Norepinephrine plasma levels were measured in 15 end-stage renal patients before the initiation of CAPD and 1 year later. Plasma NE before initiation of the method was significantly higher compared with normal values (p < 0.01), and it increased significantly after 12 months of CAPD: from 329 ± 67 pg/ml to 580 ± 173 pg/ml (p < 0.01) (Figure 4). NE PLASMA LEVELS IN PATIENTS WITH CRF, PATIENTS ON HD, AND PATIENTS ON CAPD To further investigate whether the increased NE plasma levels in CAPD patients were due to the method itself, NE was measured in the plasma of 15 patients with CRF and in 15 patients on HD. These values were compared with those of CAPD patients and healthy controls. It was found that plasma NE levels did not differ between CRF and HD patients (378 ± 142 pg/ml vs 406 ± 143 pg/ml, respectively). 324

4 PDI MAY 2000 VOL. 20, NO. 3 NOREPINEPHRINE IN CAPD Figure 2 Peritoneal equilibration test (PET) at 0-hour, 2-hr, and 4-hr dwell times for urea, creatinine, and norepinephrine in patients on CAPD (n = 15) is plotted. D = peritoneal dialysate concentration at 0-hr, 2-hr, and 4-hr dwell times; P = plasma concentration at 2 hr. Figure 4 Norepinephrine plasma levels change in patients with end-stage renal failure, before and after 12 months on CAPD (n = 15). On the contrary, these values were significantly higher compared with the healthy subjects (199 ± 25 pg/ml, p < 0.01), and were significantly lower compared with the CAPD patients (687 ± 221 pg/ml, p < 0.01) (Figure 5). DISCUSSION Figure 3 Plasma changes of urea, creatinine, and norepinephrine at 0 hr, 0.2 hr, 2 hr, and 4 hr during peritoneal equilibration test (PET) in patients on CAPD (n = 15) are compared. P = plasma concentration at 0-hr, 0.2-hr, 2-hr, and 4-hr dwell times; Po = plasma concentration at 0 hr, before the dialysate infusion. Sympathetic dysfunction has been established by several reports on patients with end-stage renal failure (1,2,4). Blood pressure and heart rate are related to other factors rather than to sympathetic system dysfunction (4). The clinical usefulness of plasma catecholamine levels is limited to the evaluation of autonomic insufficiency or suspected pheochromocytoma. These measurements have been carried out mainly by radioimmunoassay, and the results appear to be contradictory with respect to plasma NE concentration in patients on either HD or CAPD. Differences in NE plasma levels varied from pg/ml (6,15), and up to pg/ml (5). In the early 1990s, radioimmunoassay was replaced by a new, more accurate technique, HPLC. Using this technique, we studied the plasma concentration of NE in end-stage renal disease patients treated with CAPD. In the present study, it was found that plasma NE was significantly increased up to fourfold of normal values in CAPD patients [Figure 1(a)]. Although blood pressure was well controlled and within the normal range in all patients, plasma NE 325

5 VLACHOJANNIS et al. MAY 2000 VOL. 20, NO. 3 PDI TABLE 1 Norepinephrine (NE) Plasma Levels in Correlation with Peritoneal Transport Capability, Gender, Age, and Antihypertensive Medication in CAPD Patients Norepinephrine (pg/ml) Group N Group HD Group CRF Group PD Figure 5 Plasma norepinephrine concentration in normal subjects (Group N, n = 20), in patients with chronic renal failure (Group CRF, n = 15), in patients on hemodialysis (Group HD, n = 15), and in patients on CAPD (Group PD, n = 22) are compared. NS = not statistically different. values varied from pg/ml. We found no correlation between NE plasma concentration and peritoneal transport capability, or drug use, or gender, or age (Table 1). As for the volume status, all patients were euvolemic. The daily removal of NE from the peritoneal membrane was six times lower than the respective removal in the urine of healthy subjects [Figure 1(b)]. The question then arose as to the influence of the low peritoneal removal of NE on its plasma levels. It is known that NE plasma concentration is mainly regulated by cellular uptake and metabolism, and is cleared from the circulation within 1 2 minutes after its release (9). Thus, the speculation that the increased NE plasma levels are due to its low removal rate from the peritoneum may be excluded as a cause for the increased plasma levels. To investigate the cause of the increased plasma NE, the rate of NE removal from the peritoneum was studied in parallel with creatinine and urea removal during a PET. Norepinephrine removal showed an increasing slope in the second period of the PET (2 hr 4 hr) compared with the first period (0 hr 2 hr), while creatinine and urea showed a decreasing slope during the same periods (Figure 2). This increasing rate fits more likely to an increasing NE plasma concentration, in contrast to the stable plasma levels of creatinine and urea. To confirm this suggestion, plasma NE was determined during a single dialysis session. It appeared that NE increased in the plasma of CAPD patients immediately after infusion of dialysate into the peritoneal cavity, and remained unchanged throughout the dialysis session (Figure 3). It seems that the increasing levels of NE in the plasma Peritoneal transport capability Low average (0.60±0.06) (n=9) 676±195 High average (0.69±0.07) (n=13) 714±213 Gender Male (n=14) 687±229 Female (n=8) 627±187 Age years (n=9) 658± years (n=13) 697±215 Antihypertensive medication Yes (n=12) 695±192 No (n=10) 671±204 Statistical analysis revealed that norepinephrine plasma levels do not differ significantly in each category between the two groups of CAPD patients. are caused by the presence of dialysate in the peritoneal cavity. For some reason, during a CAPD session, an increased escape of NE into the circulation occurs, which cannot be balanced by the cellular uptake mechanisms. Dialysate glucose and blood insulin did not seem to interfere with the increase in plasma NE. Serum insulin levels (19 ± 13 mu/l) and the insulin/ glucose ratio (18 ± 9 U/g glucose) were within the normal range during the dialysis session. It is also important to note that plasma NE in CAPD patients was significantly higher compared with the already increased levels in patients on HD and in patients with CRF (Figure 5). A hypothesis may be that the infusion of dialysate into the peritoneal cavity leads to an increased escape of NE into the plasma (Figure 3). The plasma NE remains increased possibly due to the inadequacy of the cellular mechanisms to reuptake the extra amount of released NE and decreases in the absence of dialysate from the peritoneal cavity (0 time). This continuous imbalance presumably leads to the permanent increase in plasma NE levels that we observed in end-stage renal disease patients after 1 year on CAPD. It is concluded that the sympathetic system is most likely overstimulated rather than malfunctioning with respect to regulation of the plasma NE concentration in CAPD patients. The increased plasma NE is presumably due to its higher release or escape from the synaptic cleft following the infusion of dialysate into the peritoneal cavity. Dialysate infusion can be regarded as a stressful event leading to a rise in plasma NE in CAPD patients. 326

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