Plasma ammonia levels in newborn infants admitted

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1 rchives of Disese in Childhood, 198, 55, Plsm mmoni levels in newborn infnts dmitted to n intensive cre bby unit R BDDS, HUGHS, ROSSR, ND J C B FNTON Deprtment ofpeditrics nd Neontl Medicine, Hmmersmith Hospitl, nd Deprtment of Chemicl Pthology, St Brtholomew's Hospitl, London summry ftl cse ssocited with severe hypermmonemi is described in which no ure cycle enzyme deficiency could be found. This prompted further investigtion of blood mmoni levels in neontes dmitted to the premture bby unit t Hmmersmith Hospitl. 12 specimens were tken from 42 bbies within the first 3 weeks of life; the bbies hd vriety of clinicl conditions. The men mmoni level ws 94.5,mol/l (132.3 ±g/ 1 ml) (rnge ,umol/l ( jig/ 1 ml), SD ± 41.). These results, lthough higher thn the rnge for older bbies in hospitl, were not s high s in the bby with severe hypermmonemi. Seril levels in 1 bbies suggested tht the rnge of blood mmoni levels ws gretest in the first 2 weeks of life nd nrrowed considerbly fter this period. Gret cre is needed in collecting blood smples nd mesuring them if ccurte results re to be obtined. n 1977 ftl cse ssocited with severe hypermmonemi ws seen t Hmmersmith Hospitl. plsm mmoni level tken 3 hours before deth ws 3,umol/l (42,ug/1 ml) nd cerebrospinl fluid tken shortly before deth showed level of 4 tumol/l (56 Fg/1 ml). liver biopsy ws tken immeditely fter deth, but no enzyme defect of the ure cycle could be found, nd investigtions for orgnic ciduri did not show n inborn error in brnch-chin mino-cid metbolism. The clinicl detils re given below. Three cses of severe hypermmonemi in the neontl period were reported by Pollck et l.l nd 5 more were reported by Bllrd et l.2 n none of these bbies ws n enzyme defect observed, nd no explntion for the cuse could be found, but it ws suspected tht hypermmonemi in the neontl period might not be rre occurrence. Cse history boy ws born normlly to 22-yer-old primigrvid t 35 weeks' gesttion fter spontneous onset of lbour. The pgr score t one minute ws 9. The birthweight ws 2-58 kg (1th centile) nd, prt from slight grunting, no bnormlities were noted initilly. Milk feeds were strted vi nsogstric tube t 2 hours. The grunting continued for 14 hours nd t this stge the bby ws noted to be irritble but no cuse for this ws pprent. t 24 hours his skin becme mottled nd he developed gsping respirtions; blood sugr, ure nd electrolytes, clcium, mgnesium, nd cerebrospinl fluid were ll norml. The only bnorml finding ws metbolic cidosis. t 26 hours the bby hd generlised convulsions which could not be controlled stisfctorily. n seeking n explntion for the deteriorting condition, metbolic disorder ws considered nd plsm mmoni level estimted with the result lredy mentioned bove. However, the ure cycle enzyme ctivities mesured in the liver biopsy (tken shortly fter deth) by the method of Brown nd Cohen,3 s described by Levin,4 did not show ny defect. The nlysis of urine for orgnic cids by gs chromtogrphic nd mss spectrometric methods of Chlmers et l.5 lso filed to show n inborn error of orgnic cid metbolism. The mino-cids were quntittively exmined in plsm, urine, nd cerebrospinl fluid using n ion-exchnge chromtogrphic LKB mino-cid nlyser.6 The moderte increses in different mino-cids in the different specimens were typicl of the sickest bbies exmined nd did not suggest ny specific metbolic disorder of mino-cid metbolism. Despite ventiltory ssistnce, the condition of the bby stedily deteriorted nd he died t 39 hours. t necropsy there ws smll subdurl hemorrhge over the left prieto-occipitl region, blood clot in the cistern mgn, smll bilterl 516 rch Dis Child: first published s /dc on 1 July 198. Downloded from on 28 pril 218 by guest. Protected by copyright.

2 Plsm mmoni levels in newborn infnts dmitted to n intensive cre bby unit 517 germinl lyer hemorrhges, smll blood clot in the 4th ventricle, nd bilterl necrosis of the periventriculr tissue. n the lungs, there ws evidence of inhled mteril (epithelil squmes nd grnulr, fomy debris), nd extensive hyline membrne formtion in the respirtory bronchioles. The cse prompted this investigtion of plsm mmoni levels in neontes with vriety of clinicl conditions nd the results re now presented. The project ws pproved by the Royl Postgrdute Medicl School ethics committee. Ptients nd methods 42 bbies dmitted to the premture bby unit t Hmmersmith Hospitl were investigted. Their birthweights rnged from 74 to 388 (men 1823) g, nd gesttionl ges (s ssessed by Dubowitz Score7) were between 27 nd 41 (men 32 4) weeks. 35 bbies were preterm (less thn 37 weeks) with 4 smll for gesttionl ge (below 1th centile); 3 of the 7 term bbies were lso smll for gesttionl ge. Of the 42 bbies, 14 died in the neontl period. 27 hd birth sphyxi (defined s n pgr score of less thn four within the first 5 minutes of life). n this group, 12 hd respirtory distress syndrome (9 requiring ventiltion), 1 hd respirtory distress syndrome nd convulsions, 3 recurrent pnoe nd 1 pnoe nd convulsions (these 4 were ventilted), 1 meconium spirtion nd 1 meconium spirtion nd convulsions, 1 pneumoni, 2 birth sphyxi ssocited with mternl sedtion, 2 hd convulsions, nd 1 severe birth trum. The remining 2 hd no problems other thn birth sphyxi. Of the 15 who did not hve birth sphyxi, 11 hd respirtory distress syndrome (5 required ventilting), 2 hd pnoe (both ventilted), 1 convulsions secondry to hypoglycemi, nd 1 hd no problems other thn being preterm. Blood smples were tken from indwelling umbilicl rtery ctheters, vi rdil rtery stbs or venepuncture when blood ws required for tests connected with the mngement of the infnts. 15 ml blood ws collected in syringes contining heprin free of chlorocresol. The specimens were centrifuged nd the plsm stored in liquid nitrogen until they were sent to St Brtholomew's Hospitl for mmoni levels to be mesured. The specimens were sent in continers pcked with dry ice. Mesurements were mde using n mmoni electrode s described by Prk nd Fenton.8 nitil smples were tken on the dy the bby ws dmitted to the unit nd further smples were tken during the first 3 weeks of life. ltogether 15 mesurements were mde, 3 of the results hve been excluded becuse of gross hemolysis of the blood smples. Results 12 plsm mmoni levels were mesured between dy 1 nd dy 19 on the 42 bbies. The men level ws 94 5 Fmol/l (rnge ,umol/l, SD + 41 ). Fig. 1 shows the plsm mmoni levels plotted ginst postntl ges. 8 results were obtined on 28 surviving bbies nd 22 on 14 bbies who died in the neontl period. The men for the survivors ws 89 1,Lmol/l (rnge V-mol/l, SD ± 36 5) nd the men for those who died ws 114,umol/l (rnge mumol/l, SD ± 36.7). Figs 2 nd 3 show the initil plsm mmoni levels for ech bby (tken between dys 1 nd 4) plotted ginst gesttionl ges nd birthweights. 1 surviving bbies hd t lest 4 blood mmoni mesurements tken during the first 19 dys of life 28- o c * t Died l v Survivors 2 4 6' Postntl ge (dys) t Fig. 1 Plsm mmoni levels plotted ginst postntl ges. - 3 U, h v Died * Survivors. 4- *-- i- t Gesttionl ge (weeks) Fig. 2 Plsm mmoni levels plotted ginst gesttionl ges. (Smples tken between dys nd 4). 9---" rch Dis Child: first published s /dc on 1 July 198. Downloded from on 28 pril 218 by guest. Protected by copyright.

3 518 Beddis, Hughes, Rosser, nd Fenton _ c tf 4- * * Died Survivors Birthweight (g) Fig. 3 Plsm mmoni levels plotted ginst birthweights. (Smples tken between dys 1 nd 4)...-% 2- z 16- S Postntl ge (dys) Fig. 4 Series ofplsm mmoni levels in 1 bbies in first 3 weeks of life. nd these levels re shown in Fig. 4. The 2 bbies with the highest levels (Cses 1 nd 2) ech hd prolonged fits fter severe birth sphyxi. n ll 1 bbies the rnge of results ws fr greter in the first week of life nd the rnge hd nrrowed considerbly 14 dys lter. Discussion Our studies suggest tht mmoni levels in sick neontes (32-255,umol/l) re well bove the rnge tht we ccept s norml for children nd dults (4-35,umol/l).9 We hve no experience of mesuring mmoni levels in helthy preterm nd term bbies. However, using microversion ofthe ction exchnge column, Oberholzer et l.1 found the plsm mmoni level for venous smples from neontes ws 6,umol/l (SD ± 2) nd for cpillry smples 86 Vmol/l (SD ± 2). Mesurements were mde on 15 neontes. Snchez et l.1 observed mmoni levels greter thn 9,mol/l in 5 of 9 infnts weighing less thn 1 g t birth, ll were receiving ssisted ventiltion. They thought tht these mmoni levels my hve been contributed to by the use of blood products with high mmoni content. However if the mount of mmoni dministered by this method is clculted, it is unlikely to ccount for considerbly rised mmoni level. There is some doubt bout the correct procedure for collecting nd preserving specimens. The methods dopted in this study re the result of work conducted over severl yers t St Brtholomew's Hospitl in dults. n norml dults there is little difference between mmoni levels mesured from rteril nd venous smples.12 However, differences cn occur under severl circumstnces-for exmple when rteril mmoni levels re incresing rpidly the peripherl tissues, muscles, nd brin ctively tke up mmoni, thereby cusing lower venous mmoni levels. Venous levels my rise bove the rteril levels when tissues liberte mmoni s occurs with exercising muscle or during the recovery phse in some cses of heptic com. t is therefore conceivble tht similr differences my occur in the neonte. Cpillry smples re less relible for plsm mmoni mesurements becuse contmintion is difficult to void nd levels my be flsely high.13 Cre is needed to void gross hemolysis s red cell dmge will lso result in misledingly high results. Blood should be collected into heprinised continers nd it is importnt tht the heprin is free from preservtives, such s chlorocresol, which my injure red blood cells nd liberte mmoni. Unless mesurements re to be mde t once, the blood should be centrifuged within 3 minutes of collection nd the plsm stored t -2C overnight, or -7C if longer storge time is necessry. Specimens my be kept for 3 weeks t -7C without dversely ffecting the mmoni level. There hve been numerous methods for mesuring mmoni in blood nd this fct lone bers witness to the difficulties tht hve been encountered in the pst. We prefer either n ion exchnge column or vpour phse 'ion electrode' for voltile bse (L or Orion mmoni probe). Prk nd Fenton8 showed good correltion between mesurements mde with these methods. We found tht ion exchnge resin in 'btched' shking methods gve unreproducible results. Methods using glutmte dehydrogense tended to lck sensitivity, nd the older lkli rch Dis Child: first published s /dc on 1 July 198. Downloded from on 28 pril 218 by guest. Protected by copyright.

4 Plsm mmoni levels in newborn infnts dmitted to n intensive cre bby unit 519 diffusion methods on which much of the older dt re bsed re recognised to suffer from some mmoni rtefct. mmoni is norml constituent of body fluids. n the dult, exogenous mmoni is formed in the lrge intestine s result of bcteril ction on ure nd other nitrogenous compounds. However, this cnnot ply significnt prt in the erly neontl period until bcteril colonistion of the intestine hs occurred. Therefore, in the neonte, the mjor contribution must result from the endogenous metbolism ofglutmine, glutmte, nd denylte.'4 n nimls, glutmine hs lso been shown to be tken up by the intestinl mucos nd hydrolysed, nd this my be significnt source of mmoni in the sterile intestinl trct of the newborn infnt.15 Under norml circumstnces excess mmoni is rpidly removed by the liver. Severe hypermmonemi in the neonte hs similr clinicl presenttion whtever the cuse. Principlly this consists of: refusl of feeds, vomiting, lethrgy, grunting respirtions, fits proceeding to com nd deth in severe cses. Severe hypermmonemi in the neontl period is generlly ssocited with inborn errors of metbolism. Since the report by Russell et l.16 of hypermmonemi due to ure cycle defect, there hve been mny reports of defects ssocited with this cycle. rrors in brnch-chin mino-cid metbolism (especilly methylmlonic cidemi nd propionic cidemi) my lso be ssocited with very high plsm mmoni level.14 Other cuses of hypermmonemi include liver filure, nd Reye's syndrome in the neontl period ws reported by Ppgeorgiou et l.17 Prenterl nutrition in preterm infnts my result in hypermmonemi However, whether this is due to the high concentrtion of mmoni in protein hydrolyste or to trnsient metbolic deficiencies in sick, preterm infnts is not certin.2 mmoni levels hve lso been found to vry with different types of milk feed. Rih et l.21 showed tht levels vried with the qulity nd quntity of protein in milk. t is known tht enzymes of the ure cycle re present in the humn fetl liver t lest by 16 weeks.22 However, the fetl liver is not ble to dpt very well to nitrogen lod23 nd this my lso pply to the preterm neonte nd ccount for rised mmoni levels. noxi is known to rise the mmoni level in isolted nervous tissue nd rised levels lso occur during shock sttes in nimls.24 Hypermmonemi hs lso been shown to occur fter perintl sphyxi5 nd so it is conceivble tht hypoxi due to respirtory problems in the neonte my contribute to the high levels. lthough hypoxi hs not been shown to result in rised levels in dults, the sme my not be true of neontes s they cn withstnd much greter degree of hypoxi thn dults nd direct comprison cnnot be mde. There my be number of fctors contributing to the rised level in the group of neontes tht we studied. They hd vriety of clinicl conditions nd it is not possible to sy whether there is reltionship between birthweight or gesttionl ge nd mmoni level. study on 'norml' preterm infnts would first be required nd then it my be possible to determine wht clinicl conditions, if ny, re relted to high mmoni levels. The seril mesurements in 1 bbies showed wider rnge over the first few dys of life which hd nrrowed considerbly by 14 dys; t tht time, ll but 1 of the bbies hd recovered from their cute illness (1 still required mechnicl ventiltion becuse of recurrent pnoe). Two of the bbies we studied hd prolonged fits fter severe birth sphyxi nd in both cses the mmoni levels were in excess of 15,umol/l but fell once the fits hd stopped. Both hypoxi nd incresed muscle ctivity26 my hve plyed prt. Hypermmonemi my result in the signs lredy mentioned but it is possible tht persistently rised levels my hve other side effects. t is interesting tht high level of plsm mmoni ws shown to increse intrcrnil pressure in rhesus monkeys.27 n erly increse in intrcrnil pressure in preterm infnts ws shown by Donn nd Philip28; nd this they relted to hypoxi. t my be tht mmoni hs prt to ply in the etiology of intrventriculr hemorrhge in very low birthweight infnts. However, whether these rised levels hve ny untowrd side effects is not esy to determine. We were unble to find nother bby with severe hypermmonemi similr to tht described t the beginning of this pper nd we hve been unble to ccount for such extreme increse in the level of plsm mmoni. Bllrd et l.2 treted 4 ptients successfully with exchnge trnsfusion nd peritonel dilysis. t is cler tht severe hypermmonemi without n obvious metbolic defect my be more common thn hd previously been relised. t is importnt tht clinicins should be wre of this syndrome nd lso tht they be wre of the pitflls in collecting specimens nd methods of mesurement if vlid results re to be obtined. We thnk Mr V Oberholzer, Deprtment of Biochemistry, Queen lizbeth Hospitl for Children, London, for the mesurement of the ure cycle enzymes, Dr R Chlmers, Division of nherited Metbolic Diseses, MRC Clinicl Reserch Centre, Northwick Prk Hospitl, for the urine orgnic cid nlysis, nd Dr Pmel Dvies for dvice. rch Dis Child: first published s /dc on 1 July 198. Downloded from on 28 pril 218 by guest. Protected by copyright.

5 52 Beddis, Hughes, Rosser, nd Fenton References Pollck L, Hnsen T, dms J, Jr, Beudet. Trnsient hypermmonemi in term nd preterm infnts (bstrct). PeditrRes 1978; 12: Bllrd R, Vinocur B, Reynolds J W, et l. Trnsient hypermmonemi of the preterm infnt. N ngl J Med 1978; 299: Brown G, Jr, Cohen P. Comprtive biochemistry of ure synthesis.. Methods for the quntittive ssy of ure cycle enzymes in liver. J Biol Chem 1959; 234: Levin B. Hereditry metbolic disorders of the ure cycle. dv Clin Chem 1971; 14: Chlmers R, Wtts R W, Lwson M. comprehensive screening method for detecting orgnic ciduris nd other metbolic diseses in cutely sick infnts nd children. nn Clin Biochem 1977; 14: Stein W H, Moore S. The free mino cids ofhumn blood plsm. JBiol Chem 1954; 211: Dubowitz L M S, Dubowitz V D, Goldberg C. Clinicl ssessment of gesttionl ge in the newborn infnt. J Peditr 197; 77: Prk N J, Fenton J C B. simple method for the estimtion of plsm mmoni using n ion specific electrode. J Clin Pthol 1973; 26: Fenton J C B, Willims H. mproved method for the estimtion of plsm mmoni by ion exchnge. J Clin Pthol 1968; 21: Oberholzer V G, Schwrz K B, Smith C H, Dietzler D N, Hnn T L. Microscle modifiction of ction-exchnge column procedure for plsm mmoni. Clin Chem 1976; 22: Snchez R, Ukrinski C, Perlin B, Frber S, Goldfinger D, Pomernde J. Hypermmonemi in low birthweight infnts (bstrct). Peditr Res 1978; 12: Tyor M P, Wilson W P. Peripherl biochemicl chnges ssocited with the intrvenous dministrtion of mmonium slts in norml subjects. JLb Clin Med 1958; 51: Bessmn S P. Blood mmoni. dv Clin Chem 1959; 2: Hsi Y. nherited hypermmonemic syndromes. Gstroenterology 1974; 67: Windmueller H G, Speth. Uptke nd metbolism of plsm glutmine by the smll intestine. J Biol Chem 1974; 249: Russell, Levin B, Oberholzer V G, Sinclir L. Hypermmonemi- new instnce of n inborn enzymtic defect ofthe biosynthesis ofure. Lncet 1962; ii: Ppgeorgiou, Wiglesworth F W, Schiff D, Stern L. Reye's syndrome in newborn infnt. Cn Med ssoc J 1973; 19: Johnson J D, lbritton W L, Sunshine P. Hypermmonemi ccompnying prenterl nutrition in newborn infnts. JPeditr 1972; 81: Heird W C, Nicholson J F, Driscoll J M, Jr, Schullinger J N, Winters R W. Hypermmonemi resulting from intrvenous limenttion using mixture of synthetic L-mino cids: preliminry report. J Peditr 1972; 81: Quinby G, Jr, Nowk M M, ndrews B F. Prenterl nutrition in the neonte. ClinPerintol 1975; 2: Riiih N C R, Heinonen K, Rssin D K, Gull G. Milk protein quntity nd qulity in low-birthweight infnts.. Metbolic responses nd effects on growth. Peditrics 1976; 57: Riihi N C R, Suihkonen J. Development of ure synthesising enzymes in humn liver. ct Peditr Scnd 1968; 57: Rihi N C R, Kekomki M. n: Ghdimi H, ed. Totl prenterl nutrition: premises nd promises. Chichester: Wiley, 1975; Nelson R M, Seligson D. Studies on blood mmoni in norml nd shock sttes. Surgery 1953; 34: Goldberg R N, Cbl L, Sintr F R, Hodgmn J. Hypermmonemi ssocited with perintl sphyxi (bstrct). Clin Res 1978; 26: Lowenstein J M. mmoni production in muscle nd other tissues. The purine nucleotide cycle. Physiol Rev 1972; 52: ltenu L L, Kindt G W, Chndler W F. ffect of elevted serum mmoni on intrcrnil pressure. Surg Forum 1976; 27: Donn S M, Philip G S. rly increse in intrcrnil pressure in preterm infnts. Peditrics 1978; 61: Correspondence to Dr Hughes, Deprtment of Peditrics, Hmmersmith Hospitl, Du Cne Rod, London W12 OHS. Received 1 July 1979 rch Dis Child: first published s /dc on 1 July 198. Downloded from on 28 pril 218 by guest. Protected by copyright.