Urinary lysosomal glycosidases after renal allotransplantation: Correlation of enzyme excretion with allograft rejection and ischemia

Clinica Chimica Ada, 45 (1973) 349-359 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands CCA

349

5660

URINARY

LYSOSOMAL GLYCOSIDASES

ALLOTRANSPLANTATION: WITH ALLOGRAFT

ROBERT

SANDMAN,

CORRELATION

REJECTION

RICHARD

AFTER

RENAL

OF ENZYME

M. MARGULES

AND SAMUEL

L. KOUNTZ

Pediatric Clinical Research Center, Depavtment of Pediatrics and Department of California, San Francisco, San Francisco, Calif. 94122 (U.S.A.) (Received

EXCRETION

AND ISCHEMIA

of Surgery,

University

February 2, 1973)

SUMMARY

Urinary ,$galactosidase, ,%glucuronidase and N-acetyl-j3-glucosaminidase were measured in patients with renal allotransplants and compared with normal controls. Increased excretion of all three enzymes was noted in the transplant patients resulting possibly from mild chronic rejection. A second part of the investigation correlated renal function with daily N-acetylb-glucosaminidase excretion by the patients. In acute rejection, enzyme levels rose sharply from a baseline then decreased following successful treatment. With cadaveric grafts and initially good urinary flow, N-acetyl-t?I-glucosaminidase levels were high and decreased as creatinine clearance improved; however, with initial oliguria, levels were low and rose as diuresis began then decreased to a baseline. This was attributed to a washing out of enzyme released during the unavoidable ischemic period involved in handling cadaver kidneys. Because it reflects physiological changes in the kidney, daily monitoring of urinary N-acetyl-/3-glucosaminidase should be helpful in the diagnosis of renal damage caused by rejection and ischemia.

INTRODUCTION

Increased urinary acid phosphatase, alkaline phosphatase and #I-glucuronidase in a patient with a cadaveric renal transplant was reported in 1968 by Ballantyne ct al.‘. Kallet and Lapcoa studied urinary t!I-glucuronidase excretion in three patients with renal transplants. They found that, although only one of the patients showed an increase in absolute urinary enzyme concentration, all three had elevated enzyme/ creatinine ratios. In 1970, Price et al.3 reported increased urinary /I-galactosidase and N-acetyl-/&glucosaminidase in patients with renal trauma caused by surgery; the greater the degree of trauma, the greater the level of urinary enzymes. Dance et aL4 later suggested that measurement of urinary hydrolases might be useful in following the course of renal transplant patients.

SANDMAN

350

t?t d.

The investigation reported here is divided into two parts. In the first, urinary levels of B-D-galactoside galactohydrolase (EC 3.2.1.23, B-galactosidase), /I-P-glucuronide glucuronohydrolase (EC 3.2.1.31, #l-glucuronidase) and b-z-acetamido-z-deoxyn-glucoside acetamidodeoxyglucohydrolase (EC 3.2.x.30, ~?-acetyl-~-glucosaminidase) were assessed in 16 patients at random times after renal allotransplantation. No correlation between quantity of enzyme excreted and graft function was made. The second part of the investigation compared the daily excretion of a lysosomal enzyme with parameters of renal function. /%alactosidase and ~-glucuronidase were omitted from this study because the former enzyme is somewhat unstable and its substrate is difficult to dissolve and maintain in solution while low urinary concentration and high blank values in the assay argued against use of the latter enzyme. N-Acetyl-Bglucosaminidase, which is present in relatively high amounts in urine and which is stable in frozen urine, was used for the daily assessment of lysosomal enzyme excretion in 19 patients after renal allotransplantation. During the study period, intervals of stable graft function as well as episodes of reversible and irreversible graft rejection occurred. Our results indicate that frequent measurement of the enzyme can be a useful adjunct for monitoring graft function and for early detection of graft rejection. MATERIALS

AND

METHODS

Patients Excretion of fi-galactosidase, @-glucuronidase and N-acetyl-@-glucosaminidase by recipients of renal transplants was compared to the excretion of these enzymes by normal controls. No attempt was made to correlate excretion with the functional state of the organ. Enzyme determinations were performed on urines from 16 patients (nine male, seven female) at random times after transplantation. Aliquots of a z4-hour urine collection were used for 13 assays and randomly voided samples for the remaining three. Normal control urines were aliquots of first morning voidings from 16 normal adults (nine male, seven female). The second part of the study consisted of a daily investigation of N-acetyl-Pglucosaminid~e excretion by 19 recipients of renal transplants (eight male, II female). In this experiment, enzyme excretion was compared with the clinical condition of the kidney. An aliquot of a 24-h urine collection stored at 4O between voidings was used for these determinations. Prior to assay all urine samples were centrifuged at rzoooxg for 15 min. Cadaveric kidneys which had been maintained from 24 to 50 h on the Belzer perfusion apparatus6 were used in 22 recipients; the remaining patients received kidneys from living related donors. Immunosuppression and treatment of rejection were achieved with standard procedures adopted by the renal transplant uni%‘. Effective renal plasma flow8 was measured in recipients of cadaver organs. Clinical laboratory tests and clinical courses were closely monitored. Rejection was diagnosed by changes in serum creatinine, creatinine clearance, urinary sodium, body weight, renal plasma flow, renal scans and clinical criteria. Enzyme assay P-Glycosidase levels were determined by a moditication of the methods of Leaback and Walker* and Mead et at [email protected] of urine samples were diluted with

URINARY

LYSOSOMAL

351

GLYCOSIDASES

a solution of bovine albumin. The assay was performed by adding 0.4 ml of appropriate buffer-substrate solution (see reagents below) to each of two IO x 75 mm culture tubes. After equilibration at 37”, 0.1 ml of diluted urine was added to the first tube of each set. All tubes were stoppered and incubated at 37” for I h. After incubation, 2.0 ml of 0.4 M, glycine-sodium hydroxide buffer, pH 10.3, was added to both tubes of the set to terminate the enzymatic reaction and enhance the fluorescence of liberated 4-methylumbelliferone. After adding the glycine buffer, 0.1 ml of diluted urine was added to the second tube of each pair which served as a blank. Hydrolysis of substrate was estimated from a standard curve prepared with solutions of 4methylumbelliferone in glycine buffer. Fluorescence was measured with a Model SPF 125 spectrophotofluorimeter (American Instrument Co., Silver Spring, Md.). Excitation and emission wave lengths were 368 and 448 nm, respectively. Because of the convenience of measuring and comparing enzyme content in random samples, enzyme activity was expressed as nmoles 4-methylumbelliferone released per hour of incubation per milligram of urinary creatinine. Although creatinine excretion rates are not always constant in renal transplant patients, we found, in agreement with Price et uZ.~, that similar results were obtained whether excretion was expressed as units of activity per day or per milligram of creatinine. Creatinine and electrolytes lyzer@ and standard methodology.

were determined

with

the Technicon

AutoAna-

Reagents +Methylumbelliferone stock solution (0.15 mmole/l). 26.42 mg (Eastman Organic Chemicals, Rochester, N.Y.) was dissolved in I 1 of 0.01 N sulfuric acid and kept under refrigeration in an amber bottle. The working solution (3 pmoles/l) was prepared fresh daily by diluting the stock solution with water. To verify stability, standard 4-methylumbelliferone solutions were compared fluorimetrically with a standard quinine sulfate solution before each assay. Substrates Substrates (0.05 mmole/l), obtained from Koch-Light Laboratories, brook, Bucks, England, were dissolved in buffers as follows: 4-Methylumbellife~yl-/%wgalactopyranoside: 16.9 mg/roo ml 0.1 acetate buffer, pH 4.50. 4-Methylumbellifeeryl-B_D-glucuronide.H,O: 20.3 mg/roo ml 0.1 acetate buffer, pH 4.75. ~-Methylunabellife~yl-~-acetamido-~-deoxy-~-~-gluco~y~anos~de: 18.95 citrate-phosphate buffer, pH 4.60, ionic strength 0.21 (ref. II).

Ltd., ColnM, sodium M, sodium mg/roo ml

Bovine albumin solution Bovine albumin solution (I mg/ml) : Bovine albumin Fraction V (Pentex Inc., Kankakee, Ill.) or crystallized bovine plasma albumin (Armour Pharmaceutical Co., Kankakee, Ill.) was dissolved in water. Before it was used it was ascertained to be iree of the specific glycosidase to be determined. RESULTS

Our initial

work

on the enzyme

assays

demonstrated

that

the addition

of

352

SANDMAN

Ct al.

IIO90IOO80-

9070-

60-

50-

AO-

30-

INCUBATION

TIME IN HOURS

INCUBATION

TIME IN HOURS

Fig. r. /I-Galactosidase activity (nmoles substrate hydrolyzed/h incubation) per ml undiluted urme when sample is diluted with albumin solution or water. o---o, albumin; A--- -A, water. Fig. 2. ,!I-Glucuronidase activity (nmoles substrate hydrolyzed/h incubation) per ml undiluted urine when sample is diluted with albumin solution or water. O--O, albumin; A----A, water.

INCUBATION

TIME

IN HOURS

Fig. 3. N-Acetyl-/I-glucosaminidase activity (nmoles substrate hydrolyzed/h undiluted urine when sample is diluted with albumin solution or water. A----A, Water.

incubation) per ml G-----O, albumin;

albumin to the enzyme reaction mixture is essential. Figs. 1-3 show the activities of /!-galactosidase, /?-glucuronidase and N-acetyl-,4-glucosaminidase in urines diluted with water or with an albumin solution. All enzymes were more or less unstable during the one-hour incubation period when water was the diluent. With an albumin solu-

353

URINARY LYSOSOMAL GLYCOSIDASES

tion, activities of B-glucuronidase and N-acetyl-p-glucosaminidase were stable up to at least 4 h; the stability of B-galactosidase was greatly increased. Part r

Tables I and II give the urinary excretions of ,!I-galactosidase, /?-glucuronidase and N-acetyl-p-glucosaminidase by 16 renal transplant patients and 16 normal controls. Highly significant differences were observed in ,9-glucuronidase and N-acetyl/Sglucosaminidase excretion between the two groups (Table I). The difference for /Igalactosidase was somewhat less significant. The observed instability of B-galactosidase even in the presence of albumin might be partially responsible for the wide ranges of the enzyme that were found. TABLE

I

URINARY GLYCOSIDASE

LEVELS

IN

16 NORMAL

Enzyme levels are expressed as activity urinary creatinine. ( ) = range.

CONTROLSAND

16 KIDNEY

(nmoles substrate

TRANSPLANT

hydrolyzed/h

Enzyme

Normal

Transfilant

/I-Galactosidase ,!-Glucuronidase N-Acetyl-B-glucosaminidase

74 zt 38 (25-162)~ I9 f I3 (6- 59) 90 f 49 (43-186)

I89 zk I70 52 + 32 682 * 472

(24(I2-

703) 130)

(114-1718)

P obtained from Student’s “1” test and represents the significance the normal controls and the transplant patients. b Standard deviation calculated using the formula: _qa-x)2 J N-I

URINARY

per mg Pa

a

TABLE

PATIENTS

incubation)

0.01--0.02

0.001 O.OOI

of the difference between

II GLYCOSIDASES

EXCRETED

BY

PATIENTS

WITH

KIDNEY

TRANSPLANTS

Patient

Donor

Days after transplant

#?-Galactosidasea

p-Glucuronida&

N-Acatyl-/Iglucosaminidasea

P.J. J.L.B. D.K. A.B. M.J. SC. G.R. G.Y. C.G. J.R. D.B. L.H.L. SM. B.C. L.S. A.G.

Living Living Cadaver Cadaver Cadaver Cadaver Living Cadaver Cadaver Cadaver Living Living Cadaver Living Living Cadaver

3 3 5 5 5 5 7 8 8 IO I5 I7 22 50 I48 I75

209 217 24 46 703 116 4II 65 IO9 I72 324 86

I30 44

985 582 278 I39 1718 581 II43 423 1158 666 529 I429 524 333 303 II4

a Enzyme levels are expressed urinary creatinine.

as activity

I50 160 128 IO9

2: 47 70 II4 I9 43 52 74 35 54 40 36 12

(nmoles substrate

hydrolyzed/h

incubation)

per mg

Table II shows individual values for the excretion of the three enzymes by each of the patients studied. It may be noted that at the time when the analyses were performed there was no correlation between the amount of enzymes that were excreted and the living or cadaveric origin of the transplanted kidney. There seems

SANDMAN

354

et al.

to be a tendency

toward lower excretion of N-acetyl-/3-glucosaminidase with increasing post-operative time; a similar trend, however, is questionable for ,!I-galactosidase and B-glucuronidase. The level of enzyme excreted by an individual recipient does not seem to reflect the future course of recovery.Of the four patients (A.B., G.Y., C.G. and D.B.) who later required transplant nephrectomies, one excreted relatively low amounts of the hydrolases, two excreted intermediate quantities and one patient excreted high amounts. Another patients (L.H.L.) with a large urinary enzyme excretion at 17 days post-transplant recovered normal renal function. Two other patients with high enzyme excretions died: patient M. J. died IO days post-operatively from acute hypoxemia due to congestive heart failure. Patient G.R. died 83 days after the operation from cardiovascular collapse and respiratory arrest due to gram-negative sepsis. Part 2

The lack of correlation between the level of urinary enzymes in isolated samples and the course of recovery suggests that each graft may establish its own baseline of enzyme output and that deviations from this baseline may parallel physiological changes in the transplanted organ. To study this, the daily excretion of one lysosomal enzyme, N-acetyl$-glucosaminidase, was determined in rg patients with grafts from

OL

G

R

“=

8

I

0 DAYS

I 5

AFTER

z’i

s I IO

I

3 I 15

TRANSPLANT

I

20

I 0

I I

I 10

DAYS

AFTER

I 16

I 20

I 25

I 30

TRANSPLANT

Fig. 4. Urinary N-acetyl-,%glucosaminidase excretion and creatinine clearance in female patient Ischemia and acute tubular necrosis were present immediately after transplantation. No clinical diagnosis of rejection. Numbers in parentheses show values for effective renal plasma flow (ml/min). O---O. urinary enzyme activity (nmoles substrate hydrolyzed/h incubation) per mg urinary creatinine; O-----O, creatinine clearance (mljmin). urine Fig 5. Urinary N-acetyl-/?-glucosaminidase excretion, creatinine clearance and q-hour volume in female patient (MAB) with cadaver allotransplant. Oliguria and severely depressed renal function were evident following transplant. No clinical diagnosis of rejection. Numbers in parentheses show values for effective renal plasma flow (mljmin). O-O, urinary enzyme activity (nmoles substrate hydrolyzed/h incubation) per mg urinary creatinine; l - - - - l , creatinine clearance (ml/min) ; A-A, 24 hour urine volume (ml).

(J J) with cadaver allotransplant.

355

URINARY LYSOSOMAL GLYCOSIDASES

I 5

I 0

I 10

I 25

I 20

I 15

DAYS

AFTER

I 30

I 40

I 35

I 45

I

so

TRANSPLANT

Fig. 6. Urinary N-acetyl-/3-glucosaminidase excretion and creatinine clearance in male patient (DW) with cadaver renal allotransplant. Pseudomonas infection treated by GentamycinB therapy 40 days after transplantation. Treatment of clinically diagnosed rejection episode at arrows. Numbers in parentheses show values for effective renal plasma flow (ml/min). O---O, urinary enzyme activity (nmoles substrate hydrolyzed/h incubation) per mg urinary creatinine; O-----O, creatinine clearance (ml/min).

b

I

Ib

A

io

is

i

is

io

is

DAYS AFTER TRANSPLANT

Fig. 7. Urinary N-acetyl-/%-glucosaminidase excretion and creatinine clearance in male patient (RR) with cadaver allotransplant. Treatment of clinically diagnosed rejection episodes at arrows. Numbers in parentheses show values for effective renal plasma flow (ml/min). o---o, urinary enzyme activity (nmoles substrate hydrolyzed/h incubation) per mg urinary creatinine; e-----o, creatinine clearance (ml/min).

13 cadaver donors and six living donors. Six of the 13 recipients of cadaver grafts experienced no rejections during this study. The other seven as well as the six recipients of grafts from living related donors had a total of 15 rejection episodes. Eight of these episodes were reversed by therapy, one reversed spontaneously and six required transplant nephrectomies. Initially, creatinine clearance was depressed in all 13 patients who had received cadaver kidneys. This was attributed to tubular damage occasioned by ischemia

SANDMAN

356

t?t d.

-150

-125

,

-IO0

i u' z s 4 "

-75

-0

I

0

I 5

I 10

I I5

I

I 20

25

DAYS AFTER TRANSPLANT

Fig. 8. Urinary N-acetyl-/I-glucosaminidase excretion and creatinine clearance in male patient (RF) with living related (parent) allotransplant. Possible rejection with spontaneous reversal may urinary enzyme activity (nmoles have occurred on days 7-g after transplantation. O---O, substrate hydrolyzed/h incubation) per mg urinary creatinine; l - -- - -0, creatinine clearance (ml/min).

I

I

I

0

5

10

I 15

I

20

I

15

I

JO

/

35

I

40

DAYS AFTER TRANSPLANT

Fig. 9. Urinary N-acetyl-,!?-glucosaminidase excretion, creatinine clearance and 21 hour urine volume in female patient (AB) with cadaver allotransplant. Oliguria and severely depressed renal function were evident after day 26. Transplant nephrectomy was performed 37 days after transplantation Clinical diagnosis of rejection episodes at arrows. Numbers in parentheses show values for effective renal plasma flow (ml/min). O---O, urinary enzyme activity (nmoles substrate hydrolyzed/h incubation) per mg urinary creatinine; O-----O, creatinine clearance (ml/mm) ; n---a, q hour urine volume (ml).

during removal of the kidney and maintenance on the perfusion apparatus, and by ischemia during vascular anastomosis. Along with the depressed creatinine clearance, N-acetyl$-glucosaminidase was initially elevated, but decreased to a stable, though still elevated, baseline as creatinine clearance returned toward normal values (Fig. 4).

URINARY

LYSOSOMAL

GLYCOSIDASES

357

In some cadaver graft recipients whose renal function was severely depressed to the point of olignria, enzyme levels tended to be depressed until diuresis occurred. At this point, accumulated ~-acetyl-~-glucosam~idase appeared to be washed out, inasmuch as activity rose to a high value and then returned to a stable baseline as creatinine clearance returned to normal (Fig. 5). Neither acute tubular necrosis nor initial enzyme elevation occurred in the recipients of living related kidneys. After a stable baseline was achieved, elevation of ~-acetyl-~-glucosaminidase was noted only once in the seven patients without documented rejection. This occurred during Gentamycin@ therapy for a Pseudomonas urinary tract infection (Fig. 6). During the eight rejection episodes which were reversed therapeutically, the average peak elevation of ~-acetyl-~-glucosaminidase was 3.5 times baseline value. Enzyme elevation correlated roughly with deterioration of other parameters of renal function and returned to baseline at about the same time as recovery of these parameters to normal (Fig. 7). One possible rejection episode seemed to reverse spontaneously. In this patient, the enzyme increased to 5 times that of baseline then returned to normal after 2 days; this was paralleled by a decay and return to normal of creatinine clearance (Fig. 8). Of the six graft rejections which resulted in transplant nephrectomies, the maximum increase in the enzyme was an average of 8 times base level. If transplant nephrectomy was delayed, enzyme levels decreased when oliguria occurred (Fig. 9). DISCUSSION

The results reported here have been approximately corrected for urinary flow rate changes by means of the creatinine index (ratio of urinary enzyme level to urinary creatinine). The validity of this index in the case of /?-glucuronidase has been discussed by Bank and Bailine 12.They report a flow dependent variation in excretion, but believe that a correction for differences in urinary flow rates can be made by use of the enzyme to creatinine ratio. Kallet and Lapco2 also used this ratio to measure b-glucuronidase levels in random samples of urine from renal transplant patients. Price et ~1.3 further substantiated the validity of this index in that they found no diurnal variation in the excretion of @-galactosidase and N-acetyl-/?-glucosaminidase in normal subjects. Evidence that the renal tubule is primarily involved in allotransplant rejection has been presented by Darmady et a&l3 who described a characteristic malformation of this structure during rejection, and by Kountz et aAx4who showed that rejection resulted in tubular necrosis occasioned by an initial attack on the peritubular blood vessels. Hume et aLI also related rejection to tubular rather than to glomerular damage. The lysosomal glycosidases studied here are particularly abundant in the proximal tubule of the kidneyx6-1*. Tubular destruction may increase the amount of enzymes in urine, as occurs in kidney damage induced in rats by tubule-specific nephrotoxic agentsZO+. Because of their high molecular weights, these enzymes are thought not to pass through the glomerulus and only enzymes released by renal damage should appear in the urine. Detection of allograft rejection is a major problem in renal transplantation. No simple and reliable criteria for early diagnosis are available. Clinical tests now in use include creatinine clearance, urinary creatinine and electrolytes, serum creatinine and electrolytes, blood urea nitrogen, renal scans and effective renal plasma flow. Sig-

358

SANDMAN

et ad.

nificant trends in the results of these tests, together with considerable clinical acumen and intuition, help to make the diagnosis of rejection. The findings reported here confirm that IeveIs of ~-galactosidase, ~-glucuronidase and ~-acetyl-~-glucosaminid~e are high in the urine of patients who have undergone renal trauma, such as experienced by recipients of renal transplants. Results indicate, however, that deviations from a stable baseline established by each transplanted kidney are closely related to actual changes in the physiological state of the kidney. ~-acetyl-~-glucosaminidase is particularly well adapted to daily determinations of the excretion of a lysosomal enzyme. This enzyme can be measured by a rapid and sensitive fluorimetric technic; it is a stable protein which can be stored in frozen urine and urinary IeveIs of this enzyme increase in renal trauma due to surgery and disease3+. The present study establishes two more conditions in which the enzyme is elevated and in which its serial determination is of prognostic significance, i.e., in ischemia and rejection of an allotransplanted kidney. Ischemia associated with cardiac arrest in the donor, organ preservation and reimplantation could explain the initially high levels of urinary enzyme found in patients with cadaver grafts. In the patients with acute tubular necrosis and oliguria, there may be a delay of several days before high urinary levels of enzymes are observed. This phenomenon may be due to a washout of cellular residue as diuresis begins. Low grade chronic rejection of transplanted kidneys may be a common condition which could account for the elevated levels of ~-acetyl-~-glucosaminidasefound in the urine of most of the recipients reported here. In an acute rejection crisis, however, enzyme levels rise rapidly and return to baseline if treatment is successful. Because of the ease and sensitivity of this assay method, as well as the stability of ~-acetyl-~-glucosaminidase and its reflection of physiological alterations, daily measurement will be a helpful addition to tests now in use to diagnose rejection. ACKNOWLEDGEMENTS

This investigation was supported with funds provided by the Division of Research Resources, RR-79, U.S. Public Health Service. The authors wish to thank Mrs. Uta Birnbaum for her assistance in carrying out some of the analyses of enzymatic activity. REFERENCES I B. BALLANTYWE, W.G. WOODAND P.M.MEFFAN, Brit.iWed.J., ii (1968)667. 2 H. A. KALLETAND L.LAPCO,j. ~702.,97(1967) 352. 3 R. G. PRICE, N. DANCE, B. RICHARDS AND W. R. CATTELL,CE~~. China. Acta, 27 (1970) 65. 4 N. DANCE, R. G. PRICE, W. R. CATTELL, .J.LANSDELL AND B. RICHARDS, Clin.Chi,m. Acta, 27 (1974 87. 5 F. 0. BELZER AND S. L. KOUNTZ, Ann. Surg., 172 (1970) 394. 6 S.L.KOUNTZ,R, PAYNE, K. K.KIDD,H. A.PERKINSAND F.O.BELZER, AM.SOC, Artificial Interna Organs, Trans., in press. 7 S. L. KOUNTZ AND F. 0. BELZER, Ann. Surg., 176 (1973) 509. 8 S. KOUNTZ. Prof. Nucl. Med., 2 (1~~1 21% 9 D. H. LEA~ACK‘AND P. G. WA&&, b&%em.,J., 78 (1961) ISI. IO 1. A. R. MEAD, 1. N. SMITH AND R. T. WILLIAMS, Biochem. I., 61 (1~55) 569 II 5. J. ELVING, J.-M. MARKOWITZ AND I. ROSENTHAL, AnaE.Chem., 28 (1956) 1179, 12 N. BANK AND S. H. BAILINE,N~~ Engl.J. M8d., z?yz (1965) 70.

URINARY

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GLYCOSIDASES

359

13 E. M. DARMADY, W. J. DEMPSTER AND F. STRANACK, J. Pathol. Bacterial., 70 (1955) 225. 14 S. L. KOUNTZ, M. A. WILLIAMS, P. L. WILLIAMS, C. KAPROS AND W. J. DEMPSTER, Nature, 199 (1963) 257. 15 D. M. HUME, J. P. MERRILL, B. F. MILLER AND G. W. THORN, J. C&z. Invest., 34 (1955) 327. 16 A. M. RUTENBURG, S. H. RUTENBURG, B. MONIS, R. TEAGUE AND A. M. SELIGMAN, J. Histothem., Cytochem., 6 (1958) 122. 17 M. WACHSTEIN, J. Histochem. Cytochem., 3 (1955) 246. 18 T. SONODA AND T. KUSUNOKI, Med. J. Osaka Univ., IO (1959) 119. 19 D. G. TAYLOR, R. G. PRICE AND D. ROBINSON, Biochem. J.. 122 (1971) 641. 20 D. ROBINSOX, R. G. PRICE AND N. DANCE, Biochem. J., 102 (1967) 533. 21 D. COOXROD AND P. 1’. PATERSON, J. Lab. Clin. Med., 73 (1969) 6.