Maternal Hyperglycemia and its Effect on the Placental Transport of Ascorbic acid (37,38)

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1 Peditr. Res. 16: (1982) Mternl Hyperglycemi nd its Effect on the Plcentl Trnsport of Ascorbic cid (37,38) EDWARD P. NORKUS,'~~,~~' JORGE A. BASS, AND PEDRO ROSS0 nstitute of Humn Nutrition nd Deprtment of Peditrics, Bbies Hospitl, College of Physicins nd Surgeons, Columbi University, New York, New York U.S.A. Summry This report explored the possibility tht mternl hyperglycemi during pregnncy might produce competition between glucose (G) nd scorbic cid (AA) nd reduce the trnsfer of AA to the fetus using n in vivo single-pss plcentl perfusion technique in the guine pig. Levels of G in mternl plsm incresed from mg/dl (ii. f S.E.) before AA + G infusion to mg/dl t the end of ech study (P < 0.001). Preinfusion levels of AA in mternl nd fetl plsm were 0.29 & 0.02 mg/dl nd 0.46 f 0.03 mg/dl, respectively. Mternl infusion of AA or AA + G produced significnt increses in mternl plsm AA (P < 0.025) nd fetl plsm AA (P < 0.001); however, fetl plsm AA from dms infused with AA + G were significntly below fetl plsm vlues obtined when only AA ws infused (P < 0.005) suggesting tht when mternl plsm G is incresed, the plcentl trnsport of AA to the fetus is reduced. Preinfusion levels of AA in plcentl tissue were mg/dl nd infusion of AA or AA + G produced significnt increses in AA in this orgn (P < 0.001). Comprison of the AA in plcentl tissue from fetuses of dms infused with either solution demonstrtes tht significntly less AA ws present fter AA + G infusion (P < 0.005). Additionlly, when AA + G ws infused significnt decrese in AA trnsferred cross the plcent ws found (P < 0.001) s mternl plsm G incresed beyond 200 mg/dl. The dt suggest tht in the guine pig glucose my compete with scorbic cid trnsfer cross the plcent nd tht mternl hyperglycemi my reduce the biovilbility of AA to the fetus. Specultion An cute episode of mternl hyperglycemi interferes with the plcentl trnsfer of scorbic cid. Therefore, mternl hyperglycemi during gesttionl dibetes my result in decresed supply of scorbic cid to the fetus. Ascorbic cid is present in ll tissues of the body (3, 9). Among those tissues tht hve been studied, the uptke of this compound displys sturtion kinetics (4,7, 10, ll,20,24,26,28-33). Becuse most of these tissues lso ccumulte scorbic cid ginst concentrtion grdient, trnsport is believed to be crrier medited nd energy dependent. Recent reports indicte tht glucose inhibits the uptke of scorbic cid by red blood cells nd hert endothelil cells in vitro (17, 34) nd suggest tht due to structurl similrities, glucose nd scorbic cid compete for crrier sites on the cell surfce. f this occurs in vivo, the hyperglycemi tht ccompnies overt dibetes could reduce the tissue uptke of scorbic cid. Previous reports from this lbortory indicte tht scorbic cid is trnsported cross the plcent by crrier-medited mechnism in mn nd the guine pig (20, 33). Becuse both species require scorbic cid s vitmin (3) the fetus relies totlly on the plcentl trnsfer of scorbic cid to supply its developmentl nd metbolic needs. Therefore, mternl hyperglycemi during preg- nncy might produce competition between glucose nd scorbic cid for crrier sites on the plcentl membrne nd reduce the trnsfer of scorbic cid to the fetus. This report ws designed to explore this possibility using n in vivo single-pss plcentl perfusion technique in the guine pig. MATERALS.AND METHODS Outbred, Hrtley strin guine pigs (Cmm Reserch nst., Wyne, NJ) were ob~ined ner term (68-72 dys) of their second gesttion. Upon rrivl, the nimls were cged individully in room with 50% humidity, constnt temperture (22 C) nd 12 h light-drk cycle. A stndrd guine pig diet nd wter were provided d libitum. At the time of study the gesttionl ge of the nimls, extrpolted from fetl weights (12, 13), ws estimted to be between 60 nd 67 dys. The technique to mesure scorbic cid (AA) trnsfer in n in situ preprtion hs been described in detil elsewhere (20). ~riefl~, cnnule were inserted into the mternl jugulr vein nd crotid rtery nd venous blood smple ws obtined to estblish bse-line AA nd glucose (G) levels. The uterus ws exposed through midline bdominl incision nd fetus, whose plcent ws positioned to llow esy ccess to ll vessels, ws chosen. The umbilicl vein nd n umbilicl rtery were cnnulted. The fetus ws removed nd the plcent perfused (1 ml/min) with clf serum nd sline (l:l, v/v) vi the umbilicl rtery. Perfusion fluid ws wrmed to 38OC before entering the plcent by pssge through coil submerged in the sline bth in which the lower hlf of the guine pig ws immersed. Blood levels of AA nd G then were incresed by infusion (4.3 ml/h) into the mternl jugulr vein. This infusion fluid contined either AA lone or AA nd G dissolved in lctted Ringer's solution. Both solutions were prepred immeditely before use with L-scorbic cid nd D- glucose (36). Perfuste leving the umbilicl vein ws not recirculted but collected t 3-min intervls yielding 3-ml smples. After n initil 10 min of plcentl perfusion, mternl infusion begn into the jugulr vein. Mternl blood smples were tken before the plcentl perfusion period nd t 15-min intervls throughout the infusion period vi the crotid rtery. Similrly, blood smples were obtined whenever possible from the umbilicl vein of fetuses left in utero before nd fter mternl infusion. At the conclusion of ech experiment (90 min), plcents were retined for nlysis. Mternl blood pressure nd hert rte were monitored throughout ech experiment by trnsducer (Stthm, Hto Rey, PR) ttched to tubing from the crotid rtery nd connected to polygrph (Grss nstrument Co., Quincy, MA). All smples of plsm, plcentl homogente nd perfuste either remined on ice for prompt ssy or were deproteinized nd stored (-20 C) for ssy within 3 dys (20). Mesurements of AA were performed using the Bessey et l. (1) modifiction of the 2,4 dinitrophenylhydrzine method. Assys of AA nd blood G from dms infused with AA nd G were performed immeditely upon completion of n experiment. Glucose levels in plsm nd perfuste were determined by glucose oxidse method (Gluco-Stt, Generl Dignostics, Morris Plins, NJ).

2 Differences in mens between vlues obtined fter AA infusion with nd without glucose were tested using the Student's t test. The reltionship between mternl nd fetl plsm levels before nd fter AA infusion ws nlyzed using simple regression techniques. To determine whether there ws difference in the reltionship between mternl nd fetl levels in the group exposed to AA nd G nd the group exposed to AA lone, n F test ws used. Multiple regression nlyses were used to estimte the reltionship between the mount of AA crossing the plcent per min per g of plcent nd mternl plsm AA concentrtion holding mternl plsm G levels constnt. Anlysis of covrince ws used to determine if sttisticlly significnt difference occurred between the G nd the non-g experiments (8, 14). HYPERGLYCEMA AND ASCORBC ACD RESULTS At the beginning of surgery, blood pressure for ll nimls ws within the norml rnge for guine pigs (19), systolic 80 f 7 mmhg nd distolic mmhg (% +_ S.E.). Upon opening the bdomen the systolic nd distolic blood pressure dropped to mmhg nd 26 f 4 mmhg, respectively. This lowered blood pressure remined stble throughout the reminder of ech experiment. Hert rte ws stble nd norml throughout ech study, 218 f 8 (Z + S.E.) bets per min (19). Significnt differences in these prmeters were not found mong the 14 nimls infused with AA lone nd the four dms infused with the combintion AA nd G solution (AA + G). The perfused plcents nd the fetuses from the perfused plcents did not differ significntly in weight from the nonperfused plcents nd fetuses mong the dms infused with either AA or AA + G. Ech dm ws infused with single solution of AA or AA + G. Preinfusion levels of AA in mternl plsm were (S.E.) mg/dl nd mternl infusion of either AA or AA Time (minutes Fig.. Chnges in mternl plsm scorbic cid concentrtion mong nine nimls of nerly identicl body weight (36) tht were infused with either scorbic cid lone (0, A) or scorbic cid nd glucose (0, A). At the end of ech experiment, mternl plsm vlues mong dms infused with scorbic cid t 0.7 mg/dl (A, A) nd 1.0 mg/dl (0, 0) re both significntly different from preinfusion levels (P < 0.025). The group infused with 1.0 mg/dl scorbic cid (0) hd three nimls wheres the other groups hd two nimls ech. The results re expressed s mens e S.E. Time (minutes) Fig. 2. Chnges in mternl plsm glucose concentrtion mong four dms infused with scorbic cid nd glucose (0) nd five dms infused with only scorbic cid (0). Zero time mternl plsm glucose levels re computed from ll nine nimls. At the end of ech experiment the plsm glucose concentrtion mong dms infused with scorbic cid nd glucose were significntly different from both the zero time vlues (P < 0.001) nd the postinfusion glucose vlues mong dms infused with only scorbic cid (P < 0.001). Ech point represents the men S.E. + G produced significnt increses in mternl plsm AA levels (P < 0.025, Fig. 1). Similrly, the levels of G in mternl plsm incresed from 104 _+ 4 (S.E.) mg/dl before AA + G infusion to (S.E.) mg/dl t the end of ech study (P < 0.001, Fig. 2). Plsm G levels mong five dms infused with only AA were 93 +_ 6 (S.E.) mg/dl fter the infusion period. Before the mternl infusion, AA levels in fetl plsm, (S.E.) mg/dl, were significntly higher thn mternl plsm vlues (P < 0.001). Mternl infusion of either AA or AA + G significntly incresed the AA concentrtion in 24 fetl plsm smples to 0.92 f 0.06 (S.E.) mg/dl (P < 0.001); however, during AA + G infusion lower fetl plsm levels were observed thn during mternl infusion of AA lone (P < 0.005, Fig. 3). These results suggest tht when mternl plsm G concentrtion is incresed the trnsport of AA to the fetus is reduced. Preinfusion levels of AA in plcentl tissue were _ 0.5 (S.E.) mg/100 g of tissue nd infusion of either AA or AA + G produced significnt increses in the AA content of ll plcents tht were exmined, (S.E.) mg/100 g of tissue (P < 0.001). Further, comprison of the AA in the plcentl tissue from dms infused with either AA or AA + G demonstrtes tht significntly less AA ws present in this orgn fter mternl infusion of AA + G (P < 0.005, Fig. 4). Becuse the concentrtion of AA in the perfuste leving the plcent ws mesured nd the rte of plcentl perfusion nd the plcentl weight were known, it ws possible to clculte the nmoles of AA tht crossed the plcent per min, per g of plcent. Figure 5 shows these dt plotted ginst mternl plsm AA concentrtion when either AA or AA -b G were infused. Anlysis of these dt reveled significnt decrese in AA trnsferred, expressed s nmoles of AA per min, per g of tissue when AA + G ws infused (Fig. 5). For ll mternl plsm AA levels mong dms infused with AA + G corresponding mternl plsm G levels were known. Accordingly, multiple regression nlysis ws used to estimte the reltionship between mternl plsm AA nd the mount of AA crossing the plcent holding mternl plsm G levels constnt. This nlysis determined tht plcentl trnsport of AA ws decresed significntly (P < 0.001) when AA + G ws dministered. Finlly, nlysis of covrince ws used to determine if sttisticlly significnt difference in AA trnsport occurred between dms infused with AA nd those given AA + G (Tble 1). Plcentl trnsport of AA ws not ffected t

3 NORKUS ET AL. Post - infusion oscorbic ocid in moternol plosmo (mg/dl) Fig. 3. The reltionship between mternl nd fetl plsm scorbic cid concentrtions before nd fter mternl infusion of either scorbic cid lone (0) or scorbic cid nd glucose (0). The estimted regression equtions re: preinfusion, y = ~; fter scorbic cid infusion, y = ~; fter scorbic cid nd glucose infusion, y = ~. Fetl scorbic cid levels fter scorbic cid nd glucose infusion were significntly below fetl levels found fter infusion of scorbic cid lone (P < 0.005). Ech point represents single observtion. 80 Pre-infusion Post - infusion 60 - r.0.12 pz oscorbic ocid in moternol plosmo (mg/dl Fig. 4. The reltionship between mternl plsm scorbic cid levels nd plcentl tissue scorbic cid concentrtions before nd fter mternl infusion of either scorbic cid lone (0) or scorbic cid nd glucose (0). The estimted regression equtions re: preinfusion, y = ~; fter scorbic cid infusion, y = ~; fter scorbic cid nd glucose infusion, y = ~. All postinfusion vlues re significntly different from preinfusion levels (P < 0.001). Plcentl tissue scorbic cid levels fter scorbic cid nd glucose infusion were significntly lower thn plcentl scorbic cid concentrtions found when only scorbic cid ws infused (P < 0.005). Ech point represents single observtion. mternl plsm glucose levels below 200 mg/dl; however, t mternl plsm glucose levels between mg/dl significntly less AA crossed the plcent per min, per g of plcent (P < 0.01). At mternl plsm G levels bove 250 mg/dl, the significnce level of this decresed AA trnsport incresed (P < 0.001, Tble 1). DSCUSSON Previous reports from this lbortory hve demonstrted tht AA is trnsported cross the plcent by crrier-medited mechnism in the rt, guine pig, nd mn (20, 24, 33). Present dt suggest tht in the guine pig, glucose my compete with AA for the plcentl crrier nd decrese the biovilbility of AA to the fetus. Therefore, mternl hyperglycemi during pregnncy my ffect the plcentl trnsfer of AA in mn. n both the depncretized dog (22) nd the streptozotocindibetic rt (39, the serum level of AA is decresed reltive to the nondibetic niml wheres urinry excretion of the vitmin is incresed. Lower serum AA levels hve lso been reported mong dibetic humns (2, 3, 5, 27). Although these studies did not ttempt to correlte these findings with blood G levels, they re consistent with n hypothesis tht the biovilbility of AA is reduced in dibetes mellitus. Two studies hve estimted the tissue AA sttus of the dibetic individul by mesuring the concentrtion of AA in leucocytes. One group of 127 dibetic subjects ws found to hve lower leucocyte AA levels thn the nondibetic controls (5) wheres second group of 44 dibetic ptients hd leucocyte AA levels tht were similr to control subjects (27). Other studies tht hve exmined the tissue levels of AA in dibetes indicte tht pltelets from dibetic humns (25) nd liver tissue from lloxn-dibetic rts (23) lso hve lower AA content thn control specimens. n ddition, the present study suggests tht plcentl tissue AA levels my be ffected by mternl hyperglycemi. A comprison of the plcentl tissue AA levels mong nimls infused with either AA lone or AA + G demonstrtes tht significntly less AA is present in plcentl tissue from dms infused with AA + G. Vitmin C exists in blood nd tissues in two biologiclly ctive forms, either the reduced L-scorbic cid (LAA), or the oxidized dehydroscorbic cid (DHA). Both forms possess 100% biologic ctivity (6); however, it ppers tht DHA is the only form of the

4 HYPERGLYCEMA AE' JD ASCORBC ACD 749 vitmin tht is trnsported by the crrier-medited process (10, 11, 18, 20, 21). This my explin why dibetic subjects hve been reported to hve higher levels of DHA in their plsm thn nondibetic individuls (2, 3, 27). The present study indictes tht n cute episode of mternl hyperglycemi (>200 mg/dl) cn reduce mrkedly the AA tht is trnsferred by the guine pig plcent. These experiments hve not explored the effect of chronic mternl hyperglycemi during pregnncy. Still, the possible implictions of the present finding for the gesttionl dibetic deserve further ttention prticulrly becuse mny similrities hve been suggested between the vrious ngiopthies ssocited with dibetes mellitus nd the degenertion of vsculr tissue tht ccompnies scorbic cid deficiency (16, 17, 33) Ascorbic Acid n Moternol Plsrno (rng/dl Fig. 5. The effect of mternl plsm scorbic cid concentrtions on the trnsfer of scorbic cid by the guine pig plcent (nmoles per min per g plcent) when either scorbic cid lone (e) or scorbic cid nd glucose (0) were infused into the dm. Ech point represents one observtion. Multiple regression nlysis ws used to estimte the reltionship between mternl plsm scorbic cid nd the nmoles of scorbic cid crossing the plcent per min per g plcent holding mternl plsm glucose levels constnt (y = ~ x glucose). When scorbic cid nd glucose were infused, the nmoles of scorbic cid crossing the plcent per g of plcent were significntly less thn when scorbic cid lone ws infused (P < 0.001). Tble 1. Sign$cnce levels nd t vlues for the nlyses of covrince ' ndependent vribles Mternl plsm glucose (mddl) x x2 Glucose For the nlyses, the dependent vrible equled the nmoles of scorbic cid crossing the plcent per min per g of plcent. Regressions were run for six different levels of mternl plsm glucose; x = mternl plsm scorbic cid concentrtion; glucose = one if dms were infused with AA + G nd glucose = zero if dms were infused with AA only. t vlues re shown in the prenthesis. REFERENCES AND NOTES 1. Bessey, O., Lowry, O., nd Brock, M.: The quntittive determintion of scorbic cid in smll mounts of white blood cells nd pltelets. J. Biol. Chem., 168: 197 (1947). 2. Chttejee,. B. nd Bnejee, A.: Estimtion of dehydroscorbic cid in the blood of dibetic ptients. Anl. Biochem., 98: 368 (1979). 3. Chttejee,. B., Mjunder, A. K., Nndi, B. K., nd Subrmin, N.: Synthesis nd some mjor functions of vitmin C in nimls. Ann. N. Y. Sci., 258: 24 (1975). 4. Finn, F. M. nd Johns, P. A.: Ascorbic cid trnsport by isolted bovine drenl corticl cells. Endocrinology, 106: 811 (1980). 5. Ginter, E., Zdichznec, B., Holzerov, O., Tich, E., Kobz, R., Koikov, M., Cerr, O., Ozdin, L., Hmb, F., Novkov, V., Ssko, E., nd Gher, M.: Hypocholestrolemic effects of scorbic cid in mturity-onset dibetes mellitus. nternt. J. Vit. Nutr. Res., 48: 369 (1978). 6. Hms, R. E.: Active compounds nd scorbic cid ntgonists. n: W. 4. Sebrell nd R. S. Hrris, Ed.: The Vitmins, Chemistry, Physiology, Pthology, Methods, Vol. 1, second ed., pp (Acdemic Press, New York, NY, 1967). 7. Heth, H. nd Fiddick, R.: The ctive trnsport of scorbic cid by the rt retin. Explt. Eye Res., 5: 156 (1966). 8. Hoel, P.: n: ntroduction to Mthemticl Sttistics, fourth ed., (John Wiley nd Sons, nc., New York, NY, 1971). 9. Hornig, D., Weber, F., nd Wiss, 0.: Autordiogrphic distribution of [-4C] scorbic cid nd [-4C] dehydroscorbic cid in mle guine pigs fter intrvenous injection. nternt. J. Vit. Nutr. Res., 42: 223 (1972). 10. Hornig, D., Weber, F., nd Wiss, 0.: Uptke nd relese of [1-'4C] scorbic cid nd [-4C] dehydroscorbic cid by erythrocytes of guine pigs. Clin. Chim. Act, 31: 25 (1971).. Hornig, D., Weiser. H., Weber, F., nd Wiss, 0.: Uptke nd relese of [- "C] scorbic cid nd [-14C] dehydroscorbic cid by leucocytes of guine pigs. Clin. Chim. Act, 32: 33 (1971). 12. Hugget, A. nd Widds, W.: The reltionship between fetl weight nd conceptus ge. J. Physiol., 114: 306 (1951). 13. bsen, H.: Prentl growth in guine pigs with specil reference to environmentl fctors ffecting weight t birth. J. Exptl. Zool., 51: 51 (1928). 14. Johnston, J.: n: Econometric Methods. second ed., (McGrw-Hill Book Co., New York, NY, 1972). 15. Linner, E. nd Nordstrom, K.: Trnsfer of D-isoscorbic cid nd L-scorbic cid into guine pig eyes. Document Ophthl. (Den Hg), 26: 164 (1969). 16. M~M, G. V.: Hypothesis: the role of vitmin C in dibetic ngiopthy. Perspect. Biol. Med., 17: 210 (1973). 17. Mnn, G. V. nd Newton, P.: The membrne trnsport of scorbic cid. Ann. N. Y. Acd. Sci., 258: 234 (1975). 18. Mrtin, G. R. nd Mecc, C. E.: Studies on the distribution of L-scorbic cid in the rt. Arch. Biochem. Biophys., 93: 110 (1961). 19. Mrshll, L. H. nd Hnn, C. H.: Direct mesurement of rteril blood pressure in the guine pig. Proc. Soc. Exp. Biol. Med., 92: 31 (1956). 20. Norkus, E. P., Bssi, J., nd Rosso, P.: Mternl-fetl trnsfer of scorbic cid in the guine pig. J. Nutr., 109: 2205 (1979). 21. Rih, N.: On the plcentl trnsfer of vitmin C: n experimentl study on guine pigs nd humn subjects. Act Physiol. Scnd., 45 (suppl. 155): 1 (1958). 22. Rlli, E. P. nd Sherry, S.: Effect of insulin on plsm level nd excretion of vitmin C. Proc. Soc. Exp. Biol. Med., 43: 669 (1940). 23. Rikns, L. E.: Effect of lloxn dibetes on rt liver scorbic cid. Horm. Metb. Res., 13: 123 (1981). 24. Rosso, P. nd Norkus, E.: Prentl spects of scorbic cid metbolism in the lbino rt. J. Nutr., 106: 767 (1976). 25. Sji, K. E., Kleinfeller, J., Brewington, P., Gonzlez, J., Hempling, H., nd Colwell, J. A.: Decresed pltelet vitmin C in dibetes mellitus: possible role in hyperggregtion. Thrombosis Res., 15: 639 (1979). 26. Shrm, S., Johnstone, R., nd Qustel, J.: Active trnsport of scorbic cid in drenl cortex nd brin cortex in virro, nd the effects of ACTH nd steroids. Cn. J. Biochem. Physiol., 41: 597 (1963). 27. Som, S., Bsu, S., Mukhejee, D., Deb, S., Roy Choudhury, P., Mukhejee, S., Chtte jee, S. N., nd Chtte jee,. B.: Ascorbic cid metbolism in dibetes mellitus. Metbolism, 30: 572 (1981). 28. Spector, R. nd Greene, L.: Ascorbic cid trnsport by clonl line of pheochromcytom cells. Brin Res., 136: 131 (1977).

5 750 NORKUS ET AL 29. Spector, R. nd Lorenzo, A,: Ascorbic cid homeostsis in the centrl nervous system. Am. J. Physiol., 225: 757 (1973). 30. Spector, R. nd Lorenzo, A.: Specificity of scorbic cid trnsport of the centrl nervous system. Am. J. Physiol., 226: 1468 (1974). 31. Stevenson, N.: Active trnsport of L-scorbic cid in the humn ileum. Gstroenterology, 67: 952 (1974). 32. Stevenson, N. nd Brush, M.: Existence nd chrcteristics of sodium-dependent ctive trnsport of scorbic cid in the guine pig. Am. J. Clin. Nutr., 22: 308 (1969). 33. Streeter, M. nd Rosso, P.: Trnsport mechnisms for scorbic cid in the humn plcent. Am. J. Clin. Nutr., 34: 1706 (1981). 34. Verlngieri, A. J. nd Sestito, J.: Effect of insulin on scorbic cid uptke by hert endothelil cells: possible reltionship to retinl therogenesis. Life Sci., 29: 5 (1981). 35. Zebrowski, E. J. nd Bhtnger, P. K.: Urinry excretion pttern of scorbic cid in streptozotocin dibetic nd insulin treted rts. Phrm. Res. Commun., /: 95 (1979). 36. Dms used in this study (n = 18) weighed 1342 k 110 g (S.D.) nd thus their Copyright nterntionl Peditric Reserch Foundtion, nc /82/ $02.00/0 plsm volumes vried [ ml (S.E.)/100 g body weight, Sisk, D.: Physiology. n: J. E. Wgner nd P. J. Mnning, ed., The Biology of the Guine pig, pp 63-92, (Acdemic Press, New York, NY, (1976)) n n ttempt to stndrdize the doses of scorbic cid nd glucose tht were dministered to ll nimls per ml of plsm volume, the mternl infusion solutions contined L-scorbic cid nd D-glucose t concentrtions of M nd M, respectively. 37. This reserch ws prtilly supported by grnts HD10167 nd HD10016 (Creer Development Awrd). 38. The work reported in this pper ws submitted by E. P. Norkus s prt of his Ph.D. disserttion, Columbi University, Present ddress: Dept of Biochemistry nd Drug Metbolism, Hoffmnn-L Roche nc., Nutley, New Jersey Requests for reprints should be ddressed to: Dr. E. P. Norkus, Deprtment of Biochemistry nd Drug Metbolism, Hoffmm-L Roche nc., Nutley, NJ Received for publiction My 7, Accepted for publiction Mrch 10, Printed in U. S. A.