Document 14Nxzkw3XaLnY3QLvvvXGXm

/' * / Reprinted from Clinics Chimica Acta Elsevier Publishing Company, Amsterdam - Printed in The Netherlands THE EFFECT OF EXPERIMENTAL LEAD POISONING ON SOME ENZY MATIC ACTIVITIES OF THE KIDNEY G. C. SECCHI, L. ALESSIO a n d A. CIRLA with the technical assistance of M, SCHWEIZER Laboratory of Enzymology, Institute of Occupational Health, University of Milan (Italy)* (Received October 13, 1969) -. : i 1 j * ; I i ; I : SUMMARY The alterations of lactate dehydrogenase and its isoenzyme fractions, of glucose6-phosphate dehydrogenase, and of glutamate dehydrogenase were studied in the kidneys of 12 guinea pigs poisoned with a daily dose of 120 mg/kg body weight of lead nitrate given by means of an oesophageal catheter, and in the kidneys of 12 guinea pigs made ischemic by partial constriction of the renal artery. The investigation was made on homogenates of kidneys in toto and, on homogenates of cortex and medulla separately. In the kidneys of lead-poisoned guinea pigs and in the kidneys with ischemia as a result of stenosis of the renal artery, similar metabolic alterations were observed. In both experimental situations a marked increase in type M isoenzymes of lactate dehydrogenase (LDH4 and LDH6), an increase in glucose-6-phosphate dehydrogenase up to values twice those found in normal kidneys, and a marked reduction in gluta mate dehydrogenase activity were observed. The increase of glucose-6-phosphate . dehydrogenase activity was only moderately prevalent in the cortical zone, and the other enzymes examined did not undergo alteration of their percentage distribution between cortex and medulla. These results are interpreted as indicating a metabolic adaptation of the kidney to a condition of tissue hypoxia, in both experimental situations. * Requests for reprints be addressed to: Clinica del Lavcro, via S. Barnaba 8, Milano (Italy), The following abbreviations are used: G6PD: Glucose-6-phosphate dehydrogenase {D-Glutose-6-phosphate: NADP oxidoreductase E.C. i.i.E.49). 6PGD: Phosphogtuconate dehydrogenase (6-Phospho-D-gluconate:NADP oxidoreductase E.C. 1.1.1.44). LDH: Lactate dehydrogenase (u-Lactate-.NAD oxidoreductase E.C.1.1.1.27). LDH,, LDHS, LDHj, LI>Ht. LDHS: Lactate dehydrogenase isoenzymes. G1DH: Gtutamate dehydrogenase (L-Glutamate:NADP oxidoreductase E.C.1.4.1.4). Clin. Chim. Acta, 27 (1970) 467-474 N36907 468 SECCHl et al. INTRODUCTION Renal involvement in human and experimental lead poisoning is clearly estab lished by pathological and clinical findings. In the acute stage of lead poisoning, the onset of renal damage has been attri buted to acute ischemia1-3, with transitory and reversible reduction of the renal blood flow and of the glomerular filtration rate. Moreover, the presence of amino aciduria is an indication of early tubular.damage, which has also been demonstrated by micro scopic and ultramicroscopic studies-12. The prevalence of renal insufficiency in sub jects exposed for many years to lead is proof that lead-nephropathy can evolve towards an irreversible nephroangiosclerosis8'*8"'-13*16. In the last few years, several studies carried out on kidneys made ischemic by partial constriction of the renal artery, have drawn attention to alterations of enzy matic activities related to the metabolism of pyruvate and glucose-6-phosphate. Hess and Pearse10 showed, by histochemical methods, an increase in G6PD and 6PGD activities in the macula densa of ischemic kidneys. This finding has been confirmed by Gross and Hess", Fisher18, Hess and Regoli19, Peart20 and Vander21*. A positive correlation between an increase in G6PD activity in the macula densa, hypertrophy of the juxta-glomerular apparatus, increase in juxta-glomerular granularity, and a rise in renin synthesis have been clearly demonstrated in the ischemic kidney20.22-". More recently, in clamped kidneys a significant increase was demonstrated in the M-type isoenzymes of LDH (LDH, and LDH5)28.22. Therefore, in the kidneys of guinea pigs poisoned with lead nitrate, we have examined the'activities of those enzymes known to be modified by renal ischemia. We compared the LDH isoenzymatic pattern and G6PD activity in guinea pig kidneys made ischemic by constriction of the renal artery and in the kidneys of guinea pig poisoned with lead nitrate. At the same time we studied the activity of G1DH, an enzyme localized in the mitochondria, which are the site of the main cellular oxidoreductive processes. Since, at least as far as G6PD is concerned, the alteration in activity has been observed previously only in the macula densa, our investigation was performed not only on whole kidney, but also on cortical and medullar homogenates of the same organs. MATERIALS AND METHODS Male guinea pigs, weighing 400-500 g were divided in three groups of 12 each. The first was used as control. The second was subjected to ligature of the right renal artery as described by Schroeder et al.s0. The degree of stenosis was standardized, as advised by these authors, by inserting a 0.5-mm diameter metal filament between the renal artery and the silk thread, which was gently removed after the knot had been tied. The animals were sacrificed by decapitation after 20 days, and only those animals exhibiting a weight reduction of clamped kidney below 35-40% in respect to the contralateral kidney were utilized for our experiments. This result was obtained in 80% of the animals treated. * Dae to the low specificity of histochemical methods used the true identity of these enzymatic activities is uncertain: in fact, Capelli et alpropose a more generic denomination, i.e. "NADPH tetrazolium reductase". Clin. Chim. Acta, 27 (1970) 467-474 LEAD POISONING AND KII The third group wi with a daily dose of 12c oesophageal catheter. Al The kidneys were used for the studies on 1 were separated. The salt 1 dipped in isotonic NH,C I washed in Tris-bnffered <t volumesof buffered saliu were twice frozen and dr Enzyme determinations Lactate dehydroget tical test according to t as micromoles NADH/i made by electrophoresi enzyme fractions were zolium salts according f obtained by reading on Ghtcose-6-phosphc the method of Lohr and mg protein. Glutamate dehydt Schmidt's method*6. ' protein. EXPERIMENTAL RESULT ' Lactate dehydrogenase l The amount of ' TABLE I QUANTITATIVE DETERMINj POISONED GUINEA PIGS, Al ARTERY The values of lactate dt NABH/min/rag protein, of the total activity, are Groups Normal SB Lead-poisoned SB Ischemic SD ____________' ' Statistical evaluation: Normal/Lead-pois Normal/Ischemic . t DUP050312381 SECCHI el al. loisoning is clearly estab- .f damage has been attrituction of the renal blood ssence of amino aciduria demonstrated by micromal insufficiency in subaephropathy can evolve dneys made ischemic by n to alterations of enzyacose-6-phosphate. Hess se in G6PD and 6PGD ing has been confirmed d Vander41*. A positive mla densa, hypertrophy ular granularity, and a ! ischemic kidney40'44-27, as demonstrated in the h lead nitrate, we have ifted by renal ischemia, ty in guinea pig kidneys } kidneys of guinea pig 3 activity of G1DH, an he main cellular oxidomed, the alteration in i, our investigation was medullar homogenates nee groups of 12 each, to ligature of.the right losis was standardized, letal filament between ed after the knot had j days, and only those >w 35-40% in respect ris result was obtained mtity of these enzymatic omination, i.e. "NADPH . . t LEAD POISONING AND KIDNEY ENZYMES 469 The third group was poisoned, in accordance with the method of Pernis et al.31, with a daily dose of 120 mg/kg body weight of lead nitrate given by means of an oesophageal catheter. All animals were decapitated after 40-50 days of treatment. The kidneys were dissected longitudinally into equal parts, one of which was used for the studies on the kidney in toto. In the other half, the cortex and medulla were separated. The samples of tissue were cut into thin slices which were then twice dipped in isotonic NH4C1 at 40 in order to hemolyze the red cells and were afterwards washed in Tris-buffered saline. The organs were finally homogenized at 4 with 9 volumes of buffered saline (v/v) in a Potter-Elvejhem tissue grinder. The homogenates were twice frozen and dried. The protein content was determined by Lowry's method. Enzyme determinations Lactate dehydrogenase (E.C.1.1.1.27--LDH) was determined by Warburg's op tica] test according to the method of Bergmeyer el al?3. The values were expressed as micromoles NADH/min/mg protein. The identification of LDH isoenzymes was made by electrophoresis on Cellogel, using Veronal 0.06 M, pH 8.6 buffer. The iso enzyme fractions were identified with a method based on the reduction of tetrazolium salts according to Barnett08. The quantitative evaluation of the fractions was obtained by reading on Chromoscan photodensimeter (Joyce, Loeb). Glucose-6-phospkate dehydrogenase (E.C.1.1.1.49--G6PD) was determined by the method of L8hr and Waller04. The values were expressed as ^moles NADPH/min/ mg protein. Glutamate dehydrogenase (E.C.1.4.1.4--G1DH) was determined according to Schmidt's method00. The values were expressed as micromoles NADH/min/mg protein. EXPERIMENTAL RESULTS Lactate dehydrogenase The amount of LDH activity was found to be raised in the kidneys of both TABLE I QUANTITATIVE DETERMINATION OF LACTATE DEHYDROGENASE IN KIDNEYS OF NORMAL AND LEADPOISONED GUINEA PIGS, AND IN KIDNEYS MADE ISCHEMIC BY PARTIAL CONSTRICTION OF THE RENAL ARTERY The values of lactate dehydrogenase of kidney homogenate in toto. expressed as micromoles NADH/min/mg protein, and of the cortical and medullar fractions, expressed as a percentage of the total activity, are given. Groups Kidney in toto Cortex . (%) Normal SD Lead-poisoned SD Ischemic SD 6.8 O.64 9-5 2.1 7-4 . 3-2 41.5 *4 39-7 2.6 43-6 1-5 Statistical evaluation: Normal/Lead-poisoned p < 0.001. Normal/Ischemic p > 0.05. Medulla . (%) 38.5 I-4. 60.3 z.6 56.4 1-5 C/fa. Chim. Acta, 27 (1970) 467--474 M DUP050312382 47P s e c c h i ei a!. TABLE II CHARACTERIZATION OF LDH ISOENZYMES IN KIDNEYS OF GUINEA PIGS Normal and lead-poisoned guinea pig's kidneys and kidneys made ischemic by partial constriction of the renal artery were examined. Values are expressed as percentage of total activity. Groups LDH, (H H H H) LDH, (H HUM) LDH, (HUM M) LDH, (H M M M) LDH, (M M M M) Normal Kidney in toto Cortex Medulla Lead-poisoned Kidney in toto Cortex Medulla Ischemic Kidney in toto Cortex Medulla 37 (SD 4-2) 37 (SD 6.7) 37 (SD 10.5) 26 (SD 3.4) 25 (SD 1.9) 24 (SD 4.9) 26 (SD 3.7) 28 (SD 3.7) ?7 (SD 4-4) 24 (SD + 1.9} 24 (SD 2.8) 23 (SD 3.0) 22 (SD 2.6) 22 (SD 2.6) 25 (SD 3.8) 22 (SD 3.7) 23 (SD 2.0) 22 (SD 2.3) 20 (SD 2.0) 19 (SD 4.5) 20 (SD 4.4) 21 (SD 0.6) 21 (SD 1.9) 22 (SD 2.7) 22 (SD 0.9) 22 (SD r 1.3) 23 (SD 2.6) 14 (SD 3.0) 14 (SD 3.5) 14 (SD 3.0) 19 (SD 2.5) ig (SD i.2) 18 (SD 2.5) 19 (SD 3.3) I7(SD 1.7) 18 (SD 3.0) 5 (SD 2.3) 6 (SD 3.0) 6 (SD 2.6) 12 (SD 3-4) ` 13 (SD -t- 2.6) 11 (SD 4.3) II (SD 3.7) 10 (SD 3.5) .10 (SD 2.4) experimental groups. However, a greater increase was observed in the lead-treated animals than in guinea pigs subjected to partial constriction of the renal artery (Table I). The average values obtained were 6.8 pmoles NADH/mm/mg protein in the nor mal kidneys; 9.5 jttmoles NADH/rain/mg protein in the kidneys of the lead-poisoned guinea'pigs; and 7.4 /mioles NADH/min/mg protein in the ischemic kidneys. The percentage distribution of this enzymatic activity in the cortex and medulla (Table I), was not modified with respect to controls. ' In normal guinea pigs, the electrophoretic characterization of LDH isoenzyme fractions showed a prevalence of H-type isoenzymes (LDHj and LDH,.) in the kidney tissue in toto: in fact the fractions LDH! and LDHa accounted respectively for 37.2% and 24.2% of total activity; the LDHS fraction corresponded to 19.6% of total, while the fractions LDH* and LDH,S, were present in percentages of 13.5% and 5.1% respectively (Table II and Fig. 1). The distribution of isoenzymes of LDH in the LEAD POISONING AND KID LDH 5 s 15,2/* 4 = 22 5*/o 3 s 20.8/ 2 = 16.9<V 1 s24.6 it 311 kidney in toto Fig. 2. Electrophoretic charac poisoned guinea pig. cortex and medulla was si in toto. In the kidneys of th in the activity of the M-t; 18.5% and the LDHa actr of M-type LDH isoenzyme and Fig. 2). In the clamped kidn could be recognized, with a of animals LDH4 activity : LDH5 to 10.7% of total a and LDHa was of the sam Fig. 1. Electrophoretic characterization of lactate dehydrogenase isoenzymes in kidneys of normal guinea pigs. , Clin. Chim. Acta, 27 (1970) 467-474 Fig. 3. Electrophoretic charact neys made ischemic by constri DUP050312383 s e c c h i el al. LEAD POISONING AND KIDNEY ENZYMES 471 c by partial constriction of the renal LDHi (H M M M) LDHt (M M M M) 14 (SD 3.0) *4 (SD 3-5) 14 (SD 3-) 19 (SD 2.5) 19 (SD 1.2) 18 (SD 2-5) 19 (SD 3.3) 17 (SD 1.7) 18 (SD 3.0) 5 (SD 2.3) 6 (SD 3.0) 6 (SD 2.6) (SD 3.4) 13 (SD 2.6) I r (SD 4.3} II (SD 3.7) 10 (SD 3.5) io (SD 2.4) ) 1 irved in the lead-treated of the renal artery {Table n/mg protein in the norleys of the lead-poisoned e ischemic kidneys. The ;x and medulla (Table I), ation of LDH isoenzyme tnd LDH2) in the kidney d respectively for 37.2% . to x q .6% of total, while ;es of 13.3% and 5.1% :nzymes of IJDII in the cortex and medulla was substantially the same as that observed in the kidney tissue in toto. \ In the kidneys of the lead-poisoned guinea pigs we observed a marked increase in the activity of the M-type isoenzymes (LDH* and LDH5); the LDH* activity was 18.3% and the LDH* activity was 12% of total activity. The increase in the activity , of M-type LDH isoenzymes was of the same order in the cortex and medulla (Table II and Fig. 2). ; ..... In the clamped kidneys, a similar alteration in the pattern of LDH isoenzymes could be recognized, witli a significant rise of M-type isoenzymes. In fact, in this group of animals LDH* activity in the kidney tissue in toto amounted to 18.8%'and that of LDH5 to 16.7% of total activity. As in lead-treated animals,.the increase of LDH, and LDH* was of the same amount in cortex and in medulla (Table II and Fig. 3). neys made ischemic by constriction of the renal artery. Clin. Chiai. Ada, 27 ((970) 467-474 ' ivY. I I DUP050312384 472 s e c c h i el al. TABLE III QUANTITATIVE DETERMINATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN KIDNEYS OF NOR MAL AND LEAD-POISONED GUINEA PIGS, AND IN KIDNEYS MADE ISCHEMIC BY PARTIAL CONSTRICTION OF THE RENAL ARTERY The values of glucose-6-phosphate dehydrogenase of whole kidney homogenate expressed as micromoles NADPH/min/mg protein, and of the cortical and medullar fractions, expressed as a percentage of total activity, are given. Groups Normal SD I-ead-poisoned SD Ischemic SD Kidney in toto 51 5-4 107 12.7 76 n.4 Carle# (%) 43 1-3 .48 0.8 50 2 Medulla /%) 57 3-3 5* 0.8 50 2 Statistical evaluation: Normal/Lead-poisoned Normal/Ischemic p < o.ooi. p < 0.001. Glucose-6-phosphate dehydrogenase G6PD evaluation in kidney tissue in toto showed a marked increase of this enzymatic activity in the kidneys of lead-poisoned guinea pig and in the kidneys made ischemic by constriction, of the renal artery. The values obtained were 50-io-8 //moles NADPH/min/mg protein in normal kidneys, 107.2-io-3 //moles NADPH/ min/mg protein in kidneys of lead-poisoned guinea pigs, and 75 io~3 //molesNADPH/ min/mg protein in ischemic kidneys (Table III). As can be seen from Table III, in both experimental groups the rise in activity was present in the cortex and in medulla; however the increase was clearly higher in the cortex. Glutamate dehydrogenase G1DH evaluation in the kidney tissue in toto showed a significant reduction of this enzymatic activity both in the kidneys of lead-poisoned guinea pigs and in the kidneys made ischemic by arterial stenosis. The values obtained were 0.631 //moles NAD/min/mg protein in normal kidneys, 0.484 //moles NADH/min/mg protein in TABLE IV QUANTITATIVE DETERMINATION OF GLUTAMATE DEHYDROGENASE IN NORMAL AND LEAD-POISONED GUINEA PIGS, AND IN KIDNEYS MADE ISCHEMIC BY PARTIAL CONSTRICTION OF THE RENAL ARTERY The values of glutamate dehydrogenase of whole kidney homogenate, expressed as micromoles NADH /min/mg protein, and of the cortical and medullar fractions, expressed as percentage of total activity, are given. Groups Kidney in toto Cortex (%) Medulla (Vo) Normal SD Lead-poisoned SD Ischemic SD 0.63 0.19 0.48 0.15 0.43 0.14 57-2 5-5 60.5 3-5 64.2 4-5 43 5-5 40 3-5 36 4-5 Statistical evaluation: Normal/Lead-poisoned p <: 0.05. Normal/Ischemic p < 0.05. * Clin, CAii. Acta, 27 (1970) 467-474 I LEAD POISONING AND KIDNE' kidneys of lead-treated gui: ischemic kidneys. The perce: tical and medullar fractions < in the medullar fraction in k in activity is however also o' DISCUSSION The experiments hen guinea pigs and in the kidne same kind of alteration in e: G6PD and LDH4, LDHE at both experimental groups. In clamped kidneys c histochemic&Hy in the maci with an increase in renin sy Recently, Ambrosi et < apparatus and an increase i: of lead-poisoned rabbits. In these experiments G6PD activity is not exclus apparatus is localized, but groups. Since G6PD is the e interpret this finding as an the conspicuous increase in of renin synthesis but also a: by hypoxia. The increase in M-tj treated and in ischemic kic the M-type UDH isoenzymt optimum activity occurs ii these isoenzymes in the k made ischemic by constrk expression of metabolic adj tion, because of the reduce pyruvate in the tissue, whi can adequately metabolize The suggestion that i matic alterations are relat the behaviour of G1DH. A the kidneys of lead-poison of renal artery, and sugge experimental situations. In conclusion, as re behaviour was observed in made ischemic by constri indicate that renal hypoxi: DUP050312385 s e c c h i et al. ROGENASE IN KIDNEYS OF NOR:EMIC BY PARTIAL CONSTRICTION Incy homogenate expressed as dullar fractions, expressed as a LEAD POISONING AND KIDNEY ENZYMES 473 kidneys of lead-treated guinea pigs and 0.482 /imoles NADH/min/mg protein in ischemic kidneys. The percentage distribution of this enzymatic activity in the cor tical and medullar fractions of the kidneys shows a more marked reduction in activity in the medullar fraction in kidneys of both experimental groups; a distinct reduction in activity is however also observed in the cc'tical zone (Table IV). ilia DISCUSSION a marked increase of this ea pig and in the kidneys !ues obtained were 50 io~3 7.2 -io-8 mmoles NADPH/ d 75 io~3 ^molesNADPH/ be seen from Table III, in the cortex and in medulla; 1 a significant reduction of led guinea pigs and in the >tained were. 0.631 /imoles 'JADH/min/mg protein in NORMAL AND LEAD-POISONED. 1CTION OF THE RENAL ARTERY ate, expressed as micromoles .s, expressed as percentage of la The experiments here described show that in the kidneys of lead-poisoned guinea pigs and in the kidneys made ischemic by constriction of the renal artery, the same kind of alteration in enzymatic activities occurs. There is a marked increase in G6PD and LDHlt LDH5 activities and an evident reduction in G1DH activity, in both experimental'groups. In clamped kidneys of rats, an increase in G6PD activity was demonstrated histochemically in the macula densa by several authors16-"-31, and has been related with an increase in renin synthesis. Recently, Ambrosi et of.38-5' demonstrated a hyperplasia of the juxta-glomerular apparatus and an increase in G6PD activity of the macula densa, also in the kidneys of lead-poisoned rabbits. In these experiments we have been able to demonstrate that the increase in G6PD activity is not exclusively found in the kidney cortex, where juxta-glomerular apparatus is localized, but can be shown also in the medulla, in both experimental groups. Since G6PD is the enzyme leading into the hexose monophosphate shunt, we interpret this finding as an expression of kidney adaptation to hypoxia. Therefore the conspicuous increase in G6PD activity might be considered not only as an index of renin synthesis but also as an indication of a more general metabolic disorder caused by hypoxia. The increase in M-type isoenzymes of LDH that we have observed in leadtreated and in ischemic kidneys is consistent with, this hypothesis. It is known that the M-type LDH isoenzymes catalyze the pyruvate - lactate reaction and that their optimum activity occurs in conditions of low Oa tension. The increased activity of these isoenzymes in the kidneys of lead-poisoned guinea pigs and in the kidneys made ischemic by constriction of the renal artery, can also be considered as an expression of metabolic adjustment of the tissue to cellular hypoxia. In such a condi tion, because of the reduced activity of the Krebs cycle, there is an accumulation of pyruvate in the tissue, which only an increased activity of M-type LDH isoenzymes can adequately metabolize to lactate. The suggestion that in the kidney tissue of lead-poisoned guinea pigs the enzy matic alterations are related to a condition of hypoxia, is further demonstrated by the behaviour of G1DH. A marked reduction in this activity was observed in both the kidneys of lead-poisoned animals and in kidneys made ischemic by constriction of renal artery, and suggests the existence of a mitochondrial involvement in both experimental situations. In conclusion, as regards the enzymatic activities investigated, an identical behaviour was observed in the kidneys of lead-poisoned guinea pigs and in kidneys made ischemic by constriction of the renal artery. Therefore, these experiments indicate that renal hypoxia occurs in experimental lead poisoning. Clift. Chim. Acta, 27 {1970) 467-474 t 1 it ri1.; DUP050312386 ACKNOWLEDGEMENTS The authors express their appreciation to Prof. E. C. Vigliani and Prof. B. Pemis for helpful discussions. REFERENCES 1 A. M. Fis h b e r g . Hypertension and Nephritis, Lea & Febiger. Philadelphia. 1964. 2 M. CREPET a n d F. Go b ba t o , Med. Lavoro, 45 (1954) 532. 3 M. Cr e p e t , F. Go b ba t o , P. Ma r t in o a n d F. Sir a c u s a , Minerva Nefrol., 3 (1956) 69. 4 I. C. Ra d u l e s c u , G. T. Din is c h io t u , C. Ma u g s c h , C. Io n e s c u a n d J. Te o d o r e s c u -Ex a r c u , Arch. Maladies Profess., 18 (1957) 125. 5 Z. Ra d e s o v ic , M.'Sa r ic , T. Bb r it ic a n d J. Kn e z e v ic , Brit. J. Ind. Med., iS (1961) 222. 6 G. Ric h e t , C. Al b a h a r y , R. Ar d a il l o u , C. Su l t a n a n d A. Mo r e l -Ma r o j e r , Rev. Franf. Etudes Clin. Biol., g (1964) 188. 7 B. T. Emme r s o n , Metals and the Kidney in Renal Disease, Blackwell Scientific Publications, Oxford, 1967, p. 561. 8 F. Go b ba t o a n d P. Ch ie s u r a , Minerva Nefrol., 15 (1968) 12. 9 V. K. Wil s o n , M. L. Th o ms o n a n d C. E. De n t , Lancet, ii (1953) 66. 10 S. Co h e n , A. Y. Sw e e t , W. Ma u t n e r , J. Ch u r g a n d E. Gr is h ma n .J 1mi\ J. Diseases Children, 100 (i960) 559. 11 J. J. Ch is h o l m, J. Pediat., 60 (1962) 1. 12 R. A. Go y e r , Lab. Invest., 19 (1968) 71. 13 A. Ma z z o l e n i, Med. Lavoro, 45 (1954) 482. 14 V. Da n il o v ic , Brit. Med. J., i (1958) 27. 15 R. Lil is , N. Gr a v il e s c u , B. Ne s t o r e s c u , C. Du mit r iu a n d A. Ro v e n t a, Brit. J. Ind. Med., 25 {1968) 196. 16 R. He s s a n d A. G. E. Pe a r s e, Brit. J. Exptl. Pathol., 40 (1958) 243. 17 F. Gr o s s a n d R. He s s , Pros. Soc. Exptl. Biol. Med., 104 (i960) 509. 18 E. R. Fis h e r , Lab. Invest., to (1961) 707. 19 R. He s s a n d D. Re g o l i, Brit. J. Exptl. Pathol., 45 (1964) 666. .20 W. S. Pe a r t , Pharmacol. Rev., 17 (1965) 143. 21 A. J. Va n d e r , Physiol. Rev.. 47 (1967) 35922 D. Re g o l i, R. He s s , H. Br u n n e r , G. Pe t e r s a n d F. Gr o s s , Arch. Intern. Phannacodyn., 140 (1962) 416. 23 A. J. Ma r x a n d H. W. De a n e , Endocrinology, 73 (1963) 317-' 24 Y. J. Ka t z, P. R. Pa t e x a n d S. Be r n ic k . Circulation Res., 11 (1962) 955. 25 M. Ko h l h ar d t a n d D. Vo t h , Z. Ges. Exptl. Med., 136 (1963) 361. 26 J. Ca p e l l i, L. G. We s s o n a n d G. E. Ap o n t e, Lab. Invest., 16 (1967) 925. 27 J. Ca p e l l i, L. G. We s s o n a n d G. E. Ap o n t e, Nephron, 5 (1968) 106. 28 G. Ema n u e l l i, L. Al e s s io , G. Fio r e l l i a n d G. Pe r p ig n a n o , Boll. Soc. Ited. Biol. Sper., 44 (1967)130. 29 K. Ko b aYASHI, K. Ka t o , K. Ma e d a a n d T. Ima i, Clin. Chim. Ada, 25 (1969) 5930 M. A...Sc h r o e d e r , A. P. Na s o n , R. E. Pr io r , J. B. Re e d a n d W. T. Ha e s s l e r , Amer. J. Physiol-., 214 (1968) 469. 31 B. Pe r n is , S. De Pe t r is , R. R. Be a r d a n d G. Ka r l s b a d , Med. Lavoro, 55 (1964) 81. 32 H. U. Be r g me y e r , E. Be r n e t t a n d B. He s s , in Methods of Enzymatic Analysis, Academic Press, New York, 1965. p. 736. 33 H. Ba r n e t t , Biochem. J., 84 (1962) 83P. 34 , G. W. Lo h r a n d H. D. Wa l l er , in Methods of Enzymalic Analysis, Academic Press, New York, 1965, p- 74435 E. Sc h mid t , in Methods of Enzymatic Analysis, Academic Press, New York, 1965., p. 732. 36 L. Amb r o s i, F. Vime r c a t i, C. Dl Nu n n o a n d G. De St a s io . Med, Lavoro, 60 (1969) 118. 37 C. Di Nu n n o , F. Vime r c a t i, G. De St a s io . L. Amb r o s i a n d L. St r a d a , Med. Lavoro, bo (1969) 343- Clin. Chim. Acta, 27 (1970) 467-474 I- I DUP050312387