THE EFFECTS OF ANTICHOLINESTERASE AGENTS ON THE BLOOD CHOLINESTERASES LEVELS OF NORMAL AND GLAUCOMA SUBJECTS* BY Irving H. Leopold, M.D. AND (BY INVITATION) JVarendra Krishna, B.SC., M.B.B.S.,** AND Robert A. Lehman, PH.D.*** ANTICHOLINESTERASE AGENTS (cholinesterase inhibitors) are used ex- tensively in the-management of the glaucomas, in the form of local application to the eyes. In primary glaucomas, these drugs are used to lower intraocular pressure because of their effects on the iris, the ciliary body, and the vascular system of the eye. In closed angle or angle closure glaucoma, they act by freeing the angle of the obstructive effect of the iris, by producing miosis. In open angle or wide angle glaucoma, it is not known whether they exert their beneficial effect primarily by their action on the ciliary body musculature or by their action on the blood vessels, but it is known that they improve the facility of aqueous outflow. Inhibition of cholinesterases (ChE) results in the %preservation of acetylcholine from hydrolysis so that it can exert its muscarinic action on the iris, the ciliary body, and the blood vessels. These tissues, like the erythrocytes of the normal human being, possess Group I cholines- terases or acetylcholinesterases (AChE) (other equivalent terms pro- posed: specific ChE, true ChE, e-ChE, aceto-ChE), which are con- cerned in the neurohumeral function of acetylcholine. The blood plasma contains Group II cholinesterases (various terms designated: non-specific ChE, unspecific ChE, pseudo-ChE, s-ChE, butyro or butyryl-ChE, propiono-ChE, X-ChE) which are not concerned with neurohumeral transmission (1, 2, 3, 4). No reliable reports are avail- able on the cholinesterase content of the iris and ciliary body of glaucomatous eyes because of the obvious difficulty of obtaining the *From the Department of Ophthalmology, Graduate School of Medicine of the University of Pennsylvania, and the Research and Clinical Departments of the Wills Eye Hospital, Philadelphia, Pa. **"Fight for Sight" Research Fellow of the National Council to Combat Blind- ness, Inc., New York, N.Y. ***Director of Research, Campbell Pharmaceuticals, Inc., New York, N.Y. TR. AM. OPHTH. Soc., vol. 57, 1959 64 Leopold, Krishna and Lehman material in the early stages of the disease. The cholinesterases levels in the whole blood of glaucoma patients have been shown to lie within the normal range (5). In clinical use, anticholinesterase drugs are instilled repeatedly and' for an indefinite period into glaucomatous eyest It is reasonable to expect that some absorption may take place from the conjunctival sac, the nasal mucosa, and even the gastrointestinal tract into the general blood stream. That this is indeed so has been shown for DFP and Humorsol, with which the blood cholinesterases levels were depressed a few hours following a single instillation into the eyes (6, 7, 8). The short- acting cholinesterase inhibitors, physostigmine and prostigmine, ap- proximately equally inhibit Group I ChE or AChE and Group II ChE; whereas among the long-acting cholinesterase inhibitors DFP inhibits mainly group II ChE, while Phospholine and Humorsol in- hibit chiefly AChE (9, 10, 11, 12). The present investigation has been undertaken to determine the effects of these anticholinesterase agents on the red blood cells and plasma cholinesterases levels of normal human subjects and glaucoma patients when administered locally into the eyes over periods from a few hours to two months. METHODS Cholinesterases determinations were carried out by the method of continuous titration at constant pH. To a reaction vessel bearing calomel and glass electrodes is added 100 c.c. of a solution having the following composition: acetylcholine bromide, recrystallized from alcohol (0.012M), 0.27 percent; NaCl, 0.9 percent; KC1, 0.03 percent; CaC12 * 2H20, 0.034 percent; q.s. with glass distilled water, 100 percent. The vessel is inserted into a water bath at 37.5° + 0.010 C. and con- nection made to apparatus for automatic titration with 0.02N sodium hydroxide solution with simultaneous plotting of volume of base added against time. After the reaction vessel has reached the bath tempera- ture, sodium hydroxide is run in until the pH is 7.4. Then, 1 to 2 c.c. of sample is added and the acetic acid, liberated by hydrolysis of the substrate, is titrated continuously at a pH of 7.4 + 0.05. After 20 min. the reaction' is stopped. The slope of the line is estimated graphically from the chart and the rate is expressed in milliequivalents of acetyl- choline hydrolyzed per hour per cubic centimeter of sample. The titration equipment has been described previously (13). This study was carried out on out-patients at Wills Eye Hospital, Philadelphia. "Normal subjects" were patients with no ocular pathology Anticholinestierase Agents 65 other than refractive error. Venous blood samples were used in all cases and clotting was prevented by adding 1 mg. of heparin per 10 c.c. All samples were refrigerated until analyses were completed. Hematocrits were determined within 24 hours and whole blood and plasma were analysed for cholinesterases within five days. Packed red cell values were obtained by calculation. Drugs used in this study were physostigmine salicylate (eserine salicylate); Prostigmine methylbromide (neostigmine methylbromide); DFP (Floropryl, Isoflurophate) Phospholine iodide (echothiophate iodide, 217-MI); Humorsol (demecarium bromide, BC-48). BASE LINE CHOLINESTERASES LEVELS OF THE BLOOD OF NORMAL HUMAN SUBJECTS AND GLAUCOMA PATIENTS Twenty-two normal subjects and 19 patients with primary glaucoma were selected. An attempt was made in all cases to exclude any other disease which would influence the blood cholinesterases levels, such as liver diseases, blood dyscrasias, etc. (14, 15). Only those patients with glaucoma were included who had been on pilocarpine therapy at WHOLE BLOOD PLASMA R.B.C. NORMAL GLAUCOMA NORMAL GLAUCOMA NORMAL GLAUCOMA .6- *0.0 0 - E .5-. I.- 0 0 ,4-. 40 000 _J 0) * 0 .3- 0 0 . wC.) -~ *00 _J - ow' .2+ 0 *4t 0 .1- 00 0 - FIGURE 1 Comuparison of base line blood, plasma, and red blood cells cholinesterases levels in normal and glaucoma patients, 66 Leopold, Krishna and Lehman TABLE 1. BASE LINE BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF NORMAL HUMAN SUBJECTS Cholinesterase activity (mM acetylcholinc hydrolyzed,ljhr.1'c.c.) Patient Whole blood Plasma Red blood cells 1 .309 .182 .493 2 .334 .193 .521 3 .430 .220 .620 4 .269 .124 .500 5 .379 .255 .550 6 .303 .173 .505 7 .394 .309 .507 8 .295 .169 .448 9 .309 .136 .617 10 .240 .109 .483 11 .283 .164 .448 12 .415 .307 .571 13 .227 .098 .420 14 .326 .154 .518 15 .281 .136 .481 16 .352 .167 .577 17 .302 .164 .510 18 .378 .312 .476 19 .385 .260 .567 20 .19:3 .120 .314 21 .328 .169) .486 22 .224 .087 .438 the time of this study and in whom no cholinesterase inhibitor had been used for a period of at least two months prior to the collection of blood samples. One blood sample from each patient was subjected to analysis and the results are given in Tables 1 and 2. The mean and standard deviations for normals and glaucoma patients were computed from these data and compared with those of Sawitsky et al. (16), who employed a similar procedure in their determinations. These have been summarized in Table 3. Our normals are quite consistent with the normals reported by Sawitsky et al. However, significantly lower red blood cell cholinesterases levels were found in glaucoma patients. Rados (5) failed to find a change in whole blood cholinesterases in glaucoma which is not surprising since the unchanged plasma would obscure changes occurring in the erythrocytes. Studies to elucidate this point further are in progress and wvill be reported subsequently. It Anticholinesterase Agents 67 TABLE 2. BASE LINE BLOOD PLASMA.AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF GLAUCOMA PATIENTS Cholinesterase activity (mM acetylcholine hydrolyzed/hr./c.c.) Paticnt Whole blood Plasma Red blood cells 23 .307 .149 .510 24 .326 .211 .485 25 .285 .206 .403 26 .366 .252 .544 27 .212 .082 .352 28 .316 .181 .485 29 .316 .195 .453 30 .292 .223 .400 31 .292 .170 .468 32 .219 .052 .440 33 .269 .161 .400 34 .172 .072 .323 35 .323 .172 .542 36 .224 .146 .293 37 .248 .145 .430 38 .238 .114 .425 39 .354 .166 .549 40 .302 .184 .487 41 .251 .143 .421 TABLE 3. MEAN AND STANDARD DEVIATIONS OF BLOOD PLASMA AND RED BLOOD CELLS CHOLINESTERASES LEVELS OF NORMAL HUMAN SUBJECTS AND GLAUCOMA PATIENTS Cholinesterase activity (mM acetylcho- line hydrolyzed/hr./c.c.) No. of pts. Plasma Red blood cells Normals (Wills Eye Hospital) 22 0.182 ±0.068 0.502 A0.068 Normals ( Sawitsky et al.)* 15 0.199 ±0.060 0.525 ±0.053 Glaucoma patients (Wills Eye Hospital) 19 0.159 ±0.052 0.443 ±0.072 *Figures obtainied by multiplyinig the values by 0.06 for activity in mM/hr./c.c. of sample (16). 68 Leopold, Krishna and Lehman 0.5 % ESERINE (0.1cc 02) 5.0% PROSTIGMINE (Olccc02) WHOLE PWLHSMA ROLEB.C | D L O C. BLOOD PLSA RBC BLOOD PLSA RBC .6- .5~~2 4 V ~ ~ ~ ~~~~V` ,,i'~~\,,A.N \/':. 3 : *..2 'NAN~~~~~~~~~~~D I.~ ~~H BEFORE 35 1 3 5 135 1 3 5 1 3 5 1 3 5 INSTILL.4) - HOURS A F T E R I NSTILLATION - FIGURE 2 Hourly influence of eserine and prostigmine on blood, plasma, and red blood cells cholinesterases levels. seems pertinent to mention here that the cholinesterases activity may vary widely from individual to individual, but tends to remain fairly constant in one individual. Age, sex, diet, physical exertion, heart rate, and blood pressure are without influence on it (16, 17).