L i f e S c i e n c e s V o l . 1 8 , pp. P r i n t e d i n t h e U.S.A.

947-952

Pergamon P r e s s

POSSIBLE RELATIONSHIP BETWEEN pH OF PLASMA AND RHEUMATOID ARTHRITIS John Nell McArthur Orthopaedic and Arthritic Hospital, 43 Wellesley Street East, Toronto, M4Y IHI, Canada. (Received in final form March 17, 1976)

Summary The free level of tryptophan in untreated Rheumatoid Arthritic patients is lower than normal controls and normal subjects have a daily fluctuation in the concentration of the amino-acid. The present study shows that both of these changes could be caused by small changes in the pH of human plasma which fall within the physiological range of pH found in blood. It has been shown that the amount of L-Tryptophan (tryp) bound to human albumin Fraction V is pH dependent, and that the change in binding was due to a change in the albumin (I). Aylward and Maddock found that when treatment of patients with Rheumatoid Arthritis (RA) was suspended there was less free tryp in their plasma than was present in plasma from normal subjects (2). They also demonstrated that these changes in the levels of free tryp could be correlated with changes in psychological behavicur (3). Tagliamonte et al (4) showed that there were daily changes in the free serum tryp concentrations, the levels being 45% higher at midnight than noon, and that these changes were independent of the total amount of serum tryp which remained constant. Since it has not been firmly established either why plasma from RA patients binds tryp excessively or why the daily fluctuations occur, it was of interest to plot the changes in free plasma tryp related to changes in plasma pH to see if within the physiological range of blood pH there was sufficient variation in tryp binding to account for the above observations. Materials and Methods Human plasma was obtained from the Red Cross Transfusion Centre in 200 ml plastic containers, each of which was produced from a single blood donation. L-Tryptophan was Sigma Grade from the Sigma Chemical Co., St. Louis, Mo. All other chemicals used were of Analytical Grade and glass distilled water was used exclusively. Three different methods were used to investigate the effect of pH on the binding of tryp to plasma proteins. In the 947

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first instance the pH of the plasma was adjusted to a series of levels ranging from pH 5.0 to 9.0 using either 3N HC1 or 3N NaOH. Ten ml of the plasma plus 3.0 ml of 0.9% NaC] were placed in a 17 ml Millipore ultrafiltration cell equipped with a PTI0000 nmwl membrane and filtered using 50 ps~ high purity nitrogen. The first 0.5 ml of filtrate was discarded and the next 5.0 ml were collected for analysis. This 5.0 ml sample was reduced to dryness by rotary evaporation under vacuum at 37°C and resuspended in 2.0 m] H20. The tryp was quantitated using a Beckman amino-acid autoanalyser and applying 0. 5 ml of the sample. The results of these experiments give the concentrations of endogenous free tryp in normal p]asma over the pH range being investigated. The second method involved clearing the plasma of all n~nhydrin positive small molecules and then adding 100 mg/l tryp. Fifty m] samples of plasma were cleared by adjusting the pH to 3.0 using 3N HCI and applying to a 15 x 230 mm column of Dowex 50W-4X cation exchange resin that had previously been equilibrated with H20 adjusted to pH 3.0 using 3N HCI. When it was apparent that the plasma was being eluted, 40 ml were collected, using H20 at pH 3.0 as the f~nal eluant. Samples of plasma were also cleared of small mo]ecules by dialysing 20 ml of plasma against 5 changes of 1.0 1 of 0.9% NaCI at 2°C over a period of seventy-two hours. Ten ml aliquots of the cleared plasma with added tryp were adjusted to a pH range from 6.0 to 8.0 using 3N NaOH or 3N HC1 and filtered as in the first metho4. The first ml was discarded and the second ml was kept for analysis. These samples were quant~tated by adding 0.2 ml of a solution containing 250 ml 4N sodium acetate pH 5.51, 750 ml methyl cellosolve, 20 g ninhydrin and 0.4 g stannous chloride. The mixture was heated in a boiling water bath for 5.0 min, diluted with 3.0 ml H20 and read on a spectrophotometer at 570 nm. Plasma cleared by both methods but without added tryp was also filtered and no ninhydrin reaction was detected. Dialysis was used in the third method b y 2 1 a c i n g 20 ml cleared p ] a s m a raised to either pH 6.5 or 8.0 in 3/4" diameter dialysing tubing and dialysing against a 40 ml solution of 100 mg/l tryp, 5.0 g/l sodium citrate and 8.0 g/l sodium chloride at either pH 6.5 or 8.0. The dialysis was carried out at 4°C for 24 hrs with constant stirring. One ml aliquots of the externa] solution were analysed for tryp as in the second method. The pH of both the internal and external solutions were reversed (pH 6.5 to 8.0 and pH 8.0 to 6.5) and dialysed for a further 24 hrs. One ml samples were again analysed for tryp content. Filtration and dialysis experiments w e r e also done using sodium salicylate to see if a similar pH effect could be detected. Ten ml of plasma plus one ml 20 mM sodium salicylate adjusted to either pH 6.5, 7.25 or 8.0 were placed in the filtration cell and the second ml of filtrate retained and diluted with 25 ml H20. These samples were measured on a spectrophotofluorometer (excitation 300 nm and emission 400 nm). Dialysis experiments were done at the same three levels of pH, using 2.0 ml plasma in I/4" dialysis tubing against 4.0 ml of a solution containing 320 mg/1 sodium salicylate, 5 g/1 sodium citrate and 8.0 g/1 sodium chloride. After dialysis for 24 hrs, I ml samples of the external solutions were analysed as above.

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Results Figure I shows the change in the concentration of endogenous free tryp in human plasma through a pH range from 5.0 to 9.0, and that the major change occurs between pH 7.0 and 7.5. Since the average total concentration of tryp in adult plasma is approximately 10 mg/l (5), the range in percentage free tryp in Figure I is 2% to 40%.

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FIG. 1 Change in concentration of endogenous free tryp in human plasma over a range of pH values from 5.0 to 9.0. When human plasma was cleared of ninhydrin positive small molecules and 100 mg/l tryp added to the plasma, the amount bound ranged from 28 mg/l at pH 6.0 to 60 mg/l at pH 8.0 as shown in Figure 2. Thus in spite of the concentration of tryp being ten times the normal and about equal molar concentration to albumin, a similar change to that found with endogenous tryp was demonstrated. The results given in Figure 2 are those obtained from plasma cleared of small ninhydrin positive molecules on Dowex resin. The corresponding levels of bound tryp, using plasma cleared of small molecules by dialysis, were, pH 6.0, 27 ± 31 pH 7.0, 38 ± 2, pH 7.5, 53 ± 5: and pH 8.0, 60 ± 7, which are not significantly different from the levels in Figure 2. The two methods are therefore equivalent for the purpose of these experiments, but the Dowex method is preferred because of the time element.

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60

~-

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FIG. ? Change ~ th~ bound e~ncentratic:z ef ~rs,p vJhen JO0 ~g/? tryp was added tc cleared human plasma (see text) over a range of pH values from 6.0 to 8.0. The results of the binding experiments using the d~alysis procedures with tryp confirmed the change in binding with change in pH and also showed that the changes were reversible well beyond the normal physiological range of blood pH. At pH 6.5, 23 ± ~ mg/1 tryp were bound and when t h e ~ H was raised to 8.0, 51 ± 7 mg/l were bound. At pH 8.0, 55 ± 9 mg/1 were bound and after changing to pH 6.5, 27 ± 6 mg/l were bound. Each figure is the mean ± s.d. of four determinations. The binding of sodium salicylate to plasma proteins showed no variation with altered pH either by filtration or dialysis. Ninety-one per cent was bound at the three levels of pH (6.5, 7.25 and 8.0) when measured by filtration and 83% was bound when dialysis was used. Discussion It has been proposed that RA is caused by a relative deficiency of free anti-inflammatory peptides in blood due to excessive binding to plasma proteins and that the clinically effective drugs act by binding and displacing some of the bound peptides (6). Aylward and Maddock found that RA patients d~d bird

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tryp excessively (2) and that the difference was sufficient to cause changes in the severity of depression (3). The predicted anti-inflammatory substance has been isolated (7) and found to be extremely potent when compared to sodium sallcylate. The difference reported in the concentration of free tryp between RA patients and controls was 0.17 mg/1. It can be calculated from Figure 1 that if t h e p H is raised from 7.0 to 7.5 the free tryp is lowered from 3.0 mgfl to 0.75 mg/l or 0.45 mg/l for 0.1 pH units. From these results it is possible for a rise in pH of 0.04 away from normal to be sufficient to account for the difference between the binding of plasma from RA patients and controls. If the binding parameters of the anti-inflammatory substances are found to be similar to tryp, then the lowered resistance to inflammation in RA patients could be accounted for by the same pH shift as mentioned above. Tagliamonte et al (4) found an increase of 45% in the levels of free tryp at 24 hrs when compared to 12 hrs. They reported total levels of the amino-acid as 22 mg/1 compared to about 10 mg/1 from various sources (2,5,6) and an average free le~,~! ~f 1.57 mg/1 compared to about 0.85 mg/1 (2,6). It is therefore reasonable to assume that due either to methods or calculations their figures for both free and total are doubled. This does not alter their reported percentage increase but does change the absolute mg/1 increase in free tryp from 0.58 to 0.29 mg/1. Calculations show that a pH change of 0.064 would have to occur to account for this increase. The 95% range of pH in arterial whole blood is 7.386 to 7.462 or a variation of 0.074 (5) which is greater than the change needed to cause the daily cycle. Since the range of free tryp levels in the daily cycle of normal subjects is not unlike the difference between RA and normal subjects, it would be of interest to determine whether RA patients have a similar daily cycle at a lower overall level or the cycle is suspended close to the 12 hr level of normal subjects. Acknowledgements The author wishes to thank the Canadian Red Cross Transfusion Service for supplying plasma and Dean Alexander of the College of Pharmacy, University of Toronto, for his co-operation in making laboratory facilities available. Re ference s I.

R.H. McMENAMY and J.L. ONCLEY, J. Biol. Chem. 233, 1436-1447

2.

M. AYLWARD and J. MADDOCK, J. Pharm. Pharmac. 25, 570-572 (1973). M. AYLWARD and J. MADDOCK, Lancet !, 936 (1973). A. TAGLIAMONTE, R. GESSA, G. BIGGIO, L. VARGIU and G.L. GESSA, Life Sciences 14, 349-354 (1974). K. DIEM and C. LENTNER (Eds.), Documenta Gei~v, Scientific Tables, Seventh Edition, J.R. Geigy S.A., Basle (1970). J.N. McARTHUR, P.D. DAWKINS, M.J.H. SMITH and K.B.D. HAMILTON, Br. Med. J. 2, 677-679 (1971). J.N. McARTHUR, M.J.H. SMITH and P.C. FREEMAN, J. Pharm. P h a r ~ c 24, 669-670 (1972).

3. 4. 5. 6. 7.

(1958).

Possible relationship between pH of plasma and rheumatoid arthritis.

L i f e S c i e n c e s V o l . 1 8 , pp. P r i n t e d i n t h e U.S.A. 947-952 Pergamon P r e s s POSSIBLE RELATIONSHIP BETWEEN pH OF PLASMA AND...
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