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					                                                      FIBRONECTIN AND FIBRINOGEN IN THE PLASMA AND
                                                             DIAL YSATE OF PATIENTS ON CAPD

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            Simon J. Davies                     The weaker correlation for FG and its lower        The formation of macroscopic strands of fibrin
          Aduke Animashaun                      dialysate loss:plasma concentration ratio,      within dialysate effluent is a problem well
            Albert E. Taylor                    (equivalent to 34.9 ml plasma cleared/24        recognised by both patients and clinicians. In
          Gerald A. Young and                   hrs) when compared with FN (51.4 ml, p <        addition to the temporary inconvenience of
           John H. Turney                       0.01), despite its lower molecular weight,      blocked catheters, fibrin provides a potential
                                                indicated a lower concentration of FG in the    nidus for the attachment of bacteria, and has been
                                                dialysate than expected; this would appear      implicated in recurrent peritonitis resistant to
ABSTRACT                                                                                        antibiotics ( 11) . Fibrin presumably results from
                                                to be due to intraperitoneal conversion of
Plasma and dialysate fluid from 18 patients     fibrin, which may be prevented by addition      intraperitoneal conversion of FG -a protein more
treated with continuous ambulatory              of heparin to the dialysate.                    easily measured due to its solubility.
peritoneal     dialysis    (CAPD)      while                                                        This study was designed to estimate plasma
                                                Peritoneal protein loss and the consequent
peritonitis-free,    were    analysed     for                                                   and dialysate levels of these two proteins,
                                                alterations in albumin homeostasis and
fibronectin (FN), fibrinogen (FG), albumin                                                      comparing the kinetics of their peritoneal loss
                                                nutrition are cause for concern in patients
(ALB) and total protein (TP). Mean plasma                                                       with that of albumin and total protein, in patients
                                                treated with CAPD ( 1-6) . However it is
FG (6.24 g/l) was higher than for normal                                                        treated with CAPD .
                                                likely that some of these proteins have
controls (4.23 g/1; p < 0.001) whereas FN       actions, beneficial or otherwise. within the
(0.31 g/l) was lower than for normal (0.36      peritoneal cavity. Two proteins for which       PATIENT S AND METHODS
g/l; p < 0.02). Twenty-four hour losses of      such roles may be important, fibronectin
proteins into the dialysate were as follows:    (FN) and fibrinogen (FG), are the subject of    Eighteen patients were studied, 14 males and 4
TP = 10.9 g, ALB = 5.2 g, FN = 15.6 mg          this study.                                     females, median age 59, (range 31-71); all were
and FG = 221 mg. Mean dialysate                     Dialysate effluent has a weak opsonic       receiving CAPD for end-stage renal failure of
concentration for FN was 2.09 mg/1 and          activity, and this appears to be proportional   varied etiology, excluding diabetes mellitus.
FG 31.1 mg/l. Dialysate FN and FG               to its protein content (7). In addition to      Mean time on peritoneal dialysis before the study
correlated with dialysate ALB (r = 0.92; p      immunoglobulins and complement, both            was 15 weeks (range 1      -90). No patient had
< 0.001, r = 0.60; p < 0.01 respectively)       present in dialysate effluent (3) , FN          evidence of peritonitis at
suggesting an ultrafiltrate rather than         appears to behave as an opsonin within the       the time of study. or for at least the preceding
synthesis within the peritoneum.                systemic circulation (8) and may perform a       two months.
                                                similar function at the peritoneal                  Each patient was investigated for two
                                                membrane. Whereas most proteins in               consecutive days while an inpatient. Blood was
From the Renal Unit and Renal Research          dialysis effluent represent a plasma             drawn into lithium heparin-coated bottles at
Unit, General Infirmary, Leeds, LSl 3EX.        ultrafiltrate (3, 6) local production of F N     09.00 on both mornings, separated and stored in
                                                may occur within the peritoneum (9) , as in      small aliquots at -80°C. For each exchange

Key Words: Protein loss, Fibronectin,           other extracellular compartments (10).
of dialysate during the 48-hour period, record
was made of the dwell time, volume of dialysate,
dextrose concentration and whether the patient
had injected heparin (500 IU/I). All dialysates
were examined for the presence of macroscopic
fibrin and aliquots were taken and stored at -
80°C. Plasma and dialysates remained frozen for
a maximum period of five months before
     Total protein n plasma and dialysate was
measured by the biuret reaction using a Beckman
Trace I analyser and albumin using a Beckman
Immunochemical analyser. FN and FG were
assayed by radial-immunodiffusion using plates
prepared from commercially available antisera
(Hoechst Phannaceuticals, Middlesex, England).       FG in males and females. Student's t-test was       dialysates containing fibrin, and elevated 8.55 (p
     A previous study recommended the use of         used for the comparison of unpaired data.           < 0.002) in those with heparin, when compared
citrated plasma for the measurement of FN (12).
                                                                                                         with bags without either, 3.91 (Mann-Whitney U
These authors observed an inconsistent decrease
                                                     RESULTS                                             Test). There was no difference between these

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of FN in heparinised samples subjected to se-
                                                                                                         three groups in the FN:ALB ratios, which were
quential freezing and thawing. We repeated this
                                                     Mean plasma concentrations and dialysate losses     2.68,2.72 and 2.62 respectively. This would
study and observed a similar decrease after more
                                                     for proteins studied are summarised in Table I.     suggest that there is conversion of FG to fibrin
than three sequential thawings when visible pre-
                                                     Plasma FG was 6.25 g/1 and was higher than for      within the peritoneum, more marked in those
cipitation occurred. However no significant
                                                     the normal group, 4.23 g/1 (p < 0.001), whereas     dialysates containing macroscopic fibrin, which
change in concentration of FN or FG occurred in      FN was 0.31 g/l, slightly lower than for normals,   can be prevented by addition of heparin. The
unopened samples after six months storage. The
                                                     0.36 g/1 (p < 0.02).                                effect of heparin on dialysate fibrinogen
mean coefficient of variation for duplicate de-
                                                        A total of 134 dialysates were studied. 14 of    concentration in a patient who developed fibrin-
terminations in plasma ( 12 samples) for FN was
                                                     these (5 patients), contained macroscopic strands   related problems during the course of the study
3.5% and FG 4.1%.
                                                     of fibrin, whereas heparin had been added pre-      are shown in Fig I.
    Dialysates     were      concentrated       by
                                                     exchange to 10 dialysates (3 patients). The            The mean dialysate FN loss correlated
ultrafiltration using Centricon 10 micro-
                                                     FG:ALB ratio was significantly reduced 2.03 (p      closely (Fig 2) with that of albumin (r = 0.92, p
concentrator membranes (Amicon, Gloucester,
                                                     < 0.0014) in                                        < 0.001); there was
England). The recovery of FN and FG from
plasma dilutions in water after concentration by
this technique were 105% and 93% respectiveIy,
indicating that binding of either protein to the
microconcentrators was minimal. The recovery
of FN was similar when dilutions were prepared
in dialysis fluid (1.36% or 3.86% dextrose)
although the recovery of fibrinogen was slightly
less (88%). The mean coefficients of variation for
repeated determinations of dialysate (6) were
4.9% for FN and 5.4% for FG.
    Fourteen members of hospital staff , seven
males and seven females served as normal
controls. There were no significant differences
between the mean concentrations for either FN
                                                                                           in this study. The peritoneal membrane appears
                                                                                           to be a poorly selective barrier to proteins, loss
                                                                                           in dialysate or ascitic fluid being proportional to
                                                                                           their plasma concentrations and inverse of
                                                                                           molecular weight (3). Further factors affecting
                                                                                           dialysate protein concentrations would include
                                                                                           synthesis or breakdown of the protein within the
                                                                                           peritoneal cavity.
                                                                                               The close correlation between dialysate FN
                                                                                           and albumin observed strongly suggests that
                                                                                           dialysate FN represents an ultrafiltrate of
                                                                                           plasma. Peritoneal macrophages derived from
                                                                                           CAPD patients are able to synthesise FN (9), but
                                                                                           the present study would suggest that this
                                                                                           synthesis does not contribute significantly to
                                                                                           dialysate FN content. The plasma FN
                                                                                           concentrations of our patients were significantly
                                                                                           reduced when compared with nonnal volunteers,
                                                                                           as has been found in a population of

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                                                                                           maintenance hemodialysis patients (13). The
                                                                                           resulting concentration ofFN within dialysis
                                                                                           effluent is consequently low -on average less
                                                                                           than 1% of that of plasma.
                                                                                               FN acts, at least partially, as an opsonic
                                                                                           protein (7, 14) promoting the removal of
                                                                                           particulate material from the circulation. A
                                                                                           similar important role for this protein may be
                                                                                           part of the defence mechanism of the nonnal
                                                                                           peritoneum. Experimentally produced peritoneal
                                                                                           inflammation results in high FN concentrations
                                                                                           in the peritoneal space in rats (15), and mortality
                                                                                           from Staphylacaccus aureus -induced peritonitis
                                                                                           is greater in FN-depleted animals (16). CAPD
                                                                                           patients with higher dialysate FN content may
                                                                                           have less peritonitis 9), and this may be of
                                                                                           particular importance in infections due to
                                                                                           Staphylacaccus aureus .
                                                                                               The dialysate FG levels observed in this
                                                                                           study were lower than would be expected from
                                                                                           its molecular weight, and exhibited a weaker
                                                                                           correlation with dialysate albumin. The loss
                                                                                           ofFG from dialysate during analysis by
                                                                                           concentration through a synthetic membrane was
                                                                                           insufficient to account for the low concentrations
                                                                                           in this study, but suggests that similar losses may
                                                                                           have occurred in the peritoneum. Similar
                                                                                           discrepancies have been found for FG and
a weaker correlation between dialysate FG     51.4 ml, despite its lower molecular         plasminogen in ascitic fluid from women
and albumin (r = 0.61, p < 0.01). No          weight, (p < 0.01), (figure 3).              undergoing laparoscopy for
correlations were observed between               No correlations were observed with
plasma and dialysate concentrations.          plasma or dialysate protein concentrations
   Calculation of the dialysate loss/day:     and age, length of time on CAPD, or
plasma concentration/mI gives the hy-         previous episodes of peritonitis.
pothetical volume of plasma cleared in a
day. For poorly selective membranes such
as the peritoneum, this figure is primarily
detennined by the molecular weight of the     Peritoneal protein loss in patients
protein (3, 6) .For FG this volume was        undergoing CAPD varies between 5 and
34.9 ml, less than for FN,                    15 g/day (1-6) similar to that seen
sterilisation ( 17) .FG may cross the peritoneal                                                                   II. Scheunemann B, Schwartz P, Mergerian H et at.
membrane less efficiently than other proteins                                                                           Fibrin coating of the peritoneal catheter as a cause
due to its long, thin shape. A more likely              I. Blumenkrantz MJ. Gahl GM, Kopple JO et at.                  of recurrent peritonitis. Proc 2nd Int Symp Perit
                                                           Protein losses during peritoneal dialysis. Kidney           Oial 1981; 296-298.
explanation is conversion to fibrin and/or
                                                           Int 1981; 19: 593-602.                                  12. Bowen M. Muller T. Influence of sample
fibrinolysis. This appears to be the case in the                                                                       preparation on estimates of blood fibronectin
                                                       2. Twardowski Z, Ksiazek A. Majdan M et at.
normal peritoneum where fibrinolysis is                    Kinetics of continuous ambulatory peritoneal                concentration. J Clin Patho11983; 36: 233-235.
indicated by the finding of fibrin degradation             dialysis (CAPO) with four exchanges per day.            13. Eriksen L, Tranabjaerg I. Clemmensen I et at.
products in ascitic fluid (18, 19). This is                Clin Nephrol 1981 ; 15: 119-130.                            Plasma fibronectin concentration in patients with
                                                       3. Oulaney JT, Hatch FE. Peritoneal dialysis and loss           chronic renal failure and treated with
supported by the fibrinolytic coagulopathy
                                                           of proteins: A review. Kidney Int 1984; 26: 253 -           hemodialysis. Scand J Clin Lab Invest 1983; 43:
associat ed with LeVeen shunts (20). Within the                                                                        723-726.
peritoneum of the CAPD patient conversion to           4. Kaysen GA, Schoenfield PY. Albumin homeostasis          14. Leading Article: Fibronectin and infec
fibrin would seem more likely. The increase in             in patients undergoing continuous ambulatory                 tion. Lancet 1983; i: 106-107.
the FG:ALB ratio observed when heparin was                 peritoneal dialysis. Kidney Int 1984;25: 107-114.      15. Richards PS, Saba TM. Fibronectin levels during
                                                       5. Young GA. Young JB, Young SM et at. Nutrition                intraperitoneal inflamma tion. Infect Immun 1983;
added to the dialysate would suggest there is
                                                           and delayed hypersensitivity during continuous              39: 14111418.
some formation of fibrin in all patients , not just                                                               16. Lanser ME, Saba TM. Opsonic fibronectin
                                                           ambulatory peritoneal dialysis. Nephron 1986; 43:
those forming macroscopic strands. The ability             177-186.                                                    deficiency and sepsis: cause or effect? Ann Surg
of bacteria to bind to fibrin and so gain              6. Young GA, Brownjohn AM, Parsons FM. Protein                  1982; 195: 340-345.
protection from phagocytic cells (21) is of                losses    in   patients   receiving     continuous     17. Henderson JM. Stein SF, Kutner M et at. Analysis
                                                           ambulatory peritoneal dialysis. Nephron 1987; 45:           of twenty-three plasma proteins in ascites. Ann
obvious significance to CAPD patients, particu-
                                                                                                                       Surg 1980; 192: 738-742.

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larly those with peritonitis.                                                                                     18. Pat tins on HA. Koninckx PR, Brosens IA et at.
                                                       7. yerburghHA, Keane WF, HoidalJR et at. Peritoneal
    To summarise, this study has documented                macrophages and opsonins: antibacterial defence             Clotting and fibrinolyti c activities in peritoneal
the presence, and relative concentrations of two           in patients undergoing chronic p eritoneal dialysis.        fluid. Br J Obstet Gynaecol1981; 88: 160 -166.
proteins within dialysate effluent for which               J Infect Ois 1983; 147: 1018-1027.                     19. Patrassi GM, Martinelli GM, Sturniolo GC et at.
                                                       8. O.Ardenne AJ. McGee JO.O. Fibronectin in                     Fibrinolytic study in plasma and ascitic fluid of
intraperitoneal actions can be postulated; FN is
                                                          disease. J Pathol 1984; 142: 235 -251.                       cirrhotic patients before and after ascites
present as an ultrafiltrate of plasma, whereas                                                                         concentration; reinfusion technique. Eur J Clin
                                                       9. Goldstein CS, Garrick RE. Polin RA et at.
FG, probably an ultrafiltrate undergoes a degree          Fibronectin and complement secretion by                      Invest 1985; 15: 161-165.
of intraperitoneal conversion to fibrin.                  monocytes and peritoneal macrophages in vitro           20. Ragni MY, Lewis JH, Spero JA. Ascites induced Le
                                                          from patients undergoing continuous ambulatory               yeen shunt coagulopathy. Ann Surg 1983; 198:
                                                          peritoneal dialysis. J Leukocyte Bioi 1986: 39:              91-95.
                                                           457-464.                                               21. Rotstein OO. Pruett TL. Simmons RL. Fibrin in
                                                      10. yartio T, yaheri A, Yon Essen R et at. Fibronectin           peritonitis. V: Fibrin inhibits phagocytic killing of
                                                           in synovial fluid and tissue in rheumatoid                  E coli by human polymorphonuclear leukocytes.
                                                           arthritis. Eur J Clin Invest 1981; II: 207-212.             Ann Surg 1986; 203: 413-419.

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