512
`
`CHAPTER 26
`
`'!'hey
`Sphingolipids differ from l1ii:il.hi11 1111<1 ccphalin.
`orn phosphate miters of l\llhillf{09iuc bound to d1oli1rn m·
`et.hn11ol1\mi110 l\llll primarily nrc found in bruin t:issu1} ((,g,
`~phiugomyclin, i::alo1:t.olipin). Thu ratio of le<dthin /.o
`sphi11go111y1din (L/S) in nmniotic fluid or rc,:;uHcit-atcd uumi·
`<>t.ic fluid from t.hr. oral cavi1;y of Uw newborn is 1111 1w(:urotc
`nMses,;menl. of fot.ol mat.11rity 1)nd the i-oi;pirntory-di11t.re~s
`syndrome. Clmnges in phospholipid bioAynthcsis during
`gest.n(.ion reflect Ulll nginf( of the fct nl 111111{, 11i. I.he LIS rat.io
`nm·mull.v incrcm1t)s.
`'J'ay,S<U:h.~ di~1.mse is a lipi<i-Htol'llgc dil;(!n~o in which the
`central nervous syMtcm dcgenc rat.c:i becnuse of tho pro1~re~(cid:173)
`sive intrnnouroMI ucct111111l11tion of execs~ amount.I) of the
`1:1phinr,oli1>id g11nglioiiide GMi, 1'h1J necumult1tion of GMi in
`T11y-Sachs diacnse hos hccu shown lti be c11u~ed by II lnck nf
`the cni,vmc hexosaminid11ae A.
`'l'horeforc, t.hc mea1mre•
`mcnt of serum, WBC or amnio(.ic iluid h1!.tosami11idase A i!I
`imporumt in evaluating r.arriers 11nd iu dillg1ll)l!illf/ Tuy•
`Snchs di11em10 in t,he fetui;,
`Both hcxol;aminidilse A (hcol.-labih1) nnd hcxo11nminid11sc
`B (hcnt-i;tablc) 1:1111 cnt.oly?.c tlw conve rsion of 1-rnethylum•
`lmlliforyl-N-ucetylgolacto~111nine (11 !lynthet.ic subsl.rntc) to
`N-ncctylgal11ctosamino 11nd 4-met.hylumhcllifcrnnc.
`'l'hu
`cleovngn pro<lucL, 4-nwthylumbolliforone, fluoresces under
`ultraviolet, rnclint.ion 1111d (.he intensity of the lluorc!lccmc<l is
`n meosure of t.hc nct;ivity of U1e em1ymc. In no11c11rrien1, t>O
`to 75% of the tot.11I h<rxosaminidusc 11r.tivit,y is heat-labile
`(hr.xosnminidase A), 1111d in c111-riura 20 to ~5% of the total
`hcx0Rnrni11idl1Se oclivity i!I hc11t.-labil1\.
`The hlood fnt:t:v Acid~ occur in est.el'ified (El<'A) nnd nones(cid:173)
`t.erified (Nl<WA) forms.
`'J'rigf.yceri.dr. dcterminntfons 11re of
`value in differenti11t.in11 the hyperlipidr.mic states, i<i, es~en(cid:173)
`Linl (diet-il\dttced) hypo1·tri1ilycoridemin from fnmiliul hypo(cid:173)
`cholc11torolemin with or without triglycoridomin. Aft.er t.hc
`preliminary i;epnrnt.ion from l)hOSJ>holipids, triglyce1·idi;K
`most often orn dcl.c1·mincd in torm$ of thcfr glycerol moioty.
`Thci glycerol rclons<ld by snponificntion ia oxidized to fornwl (cid:173)
`dehvdc 1.md the lnUor determined hy lluorimetJ·ic or colori(cid:173)
`mot~ic ,,roccdures. Tri~lyceride:; ulm, can hr. dllt.<lrmined by
`couplinu the 1:lyct\rol lihiirnt:cd from lipaMe/n• r.hymotr.v1>si11
`t.1·ontmcnt of 1111nm, wit.Ii n glyccrnl kiul\se-pyruvat.c ltinase(cid:173)
`LDH iiygtcm nnd i;pcc(,romet.1·ic pstim11tfon ()f NADH. Nor(cid:173)
`mnl triglyceride levoh, llrc ll0 to 140 mri/1.00 mL. An in•
`creoso in t.ri1rlyceridcs will produce a milky np pet\runce ill
`scrum (lipemic). E:FA analy6Cll 11re b,rned ohm on t.he rMC·
`tion of alk11linc hydroxylamine with ester$ of foU.y 11dds to
`form hydroxnmic ncids which produce t\ 1·cd colc>r with fonic
`d1loriclr,.
`0118 chromut-0graphie procedmcs huve been used to q11a11•
`iit.ntc tlw various fatt.Y "cids; le, p11lrnitic, ~tMric, olcic, liu•
`oleic and linolenic acids. Mono-, di- Llll<l triglycoridr.i; 1111,0
`c,111 be l!CJ)llrated into clussos and qonnt.it.nt~d by eohnnn or
`thin .. lnyer chromo.togrnph:,,, t1nd i11fr11recl i;pcct!'omctl'y.
`'l'hc total fnt.ty :idd~ ofplosmn rnnge from 200 t,o 460 mg/100
`mL in the fllst.ing st.ntc; th<ly nre derived from glycoridCll,
`chole9terol el!Wl'$ ui1d phospholipids.
`All t.lw lipi<l11 in plo!lnlll circulnl;<\ in r-0mbin11tion with
`'l'he free fatty ucids ure bound to albumiu oncl the
`pl'Owin.
`lipids ngg1·eg11t.c with other prolein11 to form lipopmleins.
`l~l<lctroph(lreRi11 ond t1ltmcentl'if111{at.ion nra t.he principnl
`meLhoda u~ccl to soporat.<! nnd identify lipopro(.(lin fumiliefi .
`Chytomimms (Sr >400), pro•fl-iipl)proteins (Sr 20- 100), ft·
`lipopmteim (Sr 0 ... 20) nnd <•-lipoprntein.~ Me U1e four mnjor
`clnsscs ht order of incrm1sin!( density ond migrution on ccllu•
`lose oc<~t.ot.e electrophoresis. Chylomi<:rons nre represt1nt.1.1-
`tive priminil:v of dieLllry or 1ixoircmot1s l,riglycc1·ides, pro-(:/·
`lipoprot.eins of c:mdog<•n<ms glyceridc!s, {1-lipoprot.ein~ c1f cho(cid:173)
`lesterol 1111d its cisl.crs nnd <r•lipoprolC!ini; of c:hol<1stcrol and
`phoRJ>holipid~. Ahno1·1MI li1>01,rot.<lin!I thot mny nppOIII' in
`pln~mo include flonLinll f~-lipoproteini;, li1>01)1'(>toi11 X nnd
`
`Mmplexes of normal lipoprot.cine with lg/\ 11nd lgG myclomn
`prnt.eim1 (nut.oimmune hypcrliJ)()l>roteinemii,). Ag,i, ~ox
`diot., fnHting, po~t.urr. drnngcs nnd tr1111me1 cnn nit.er u,~ lipi<I
`prnfile.
`'l'ho li))oprotcin ch18scs uaunll~• arc sl!pnrated by pnper,
`,1r,nrollo m· ccllnlos!!. acet:,te electrophoresis. 1'he s{.l'ips ,11,0
`Ht.Dilrnd wit,h fnt-f!Ol\tble dyes (Sudcm Bl11cl1 m· Oil Hud O)
`and qu11ntitl\tecl by clcrn1iomet.ric sc1111nit11r. P1·im11ry liy1)or(cid:173)
`lipo1>rotcincmins arc clnssificd into normnl und l'ivc uli11or(cid:173)
`mnl typ<i~ ha:1cd on cholest<:rol and trigly{:(lt·ido levels and
`lipoprotr:in analysis. Hypcm:hyl<lmkronomia {Typ1i I),
`hyJlcr-/j-lipoproteincmio ('l'ypc II), broad P-hand ('l'ype Ill),
`hwcr-prn•/1-lipoprot:fiiMmin ('I'ypc IV) ond hyp<ll'· t>ro-#-li(cid:173)
`po1>rot.ei11cmi11 and chylomicrc>1rnmi11 ('l'ype V) m·c the major
`d11~Mo~. Carbohydrate and fi1t-tolorancN-;1,11dics, posl.-h<1pu(cid:173)
`ri11 lipnMi act.ivity und clinicnl symp\,omotol111:Y also arn inti.,.
`grntod into 1.h(i cliu1:m>,.~is of the vnriou, subdns~cs. The
`prcse1tcC1 or prcdisposit.ion I.Cl coronary artery diacusc und
`othcl' disense stntes i~ nsaodatecl wi th the various t.:,,pus.''1
`Storoid!i nnd Othor Hormonos-- 'l'ht• ~t.oroids 1>os.~c~" a
`common Kt:rud,urn, t.hc Jl<lrhydrncyclopent1111iJphennnthrc11e
`l)ttelc\lla, und inclucle cholusl.t:rol, bile odd$, 1\lldrogons 1111<1
`t:hc ad1·cnocorticnl, ndrcnomcdullor.Y, oHt;o,,:1,nie nnd J>l'Ol{{il:I•
`tntional hortno11e,:;,
`Andrn~tcronc, d11hyd rocpinndror;(,erone, ctiocholan-311·•
`ol-17-oirn, J J ,.keto1111dro8t1.mmc, 11-ket,oct:ior.holnnolonc,
`11/:1-hydroxyandro~toronll mid 11 ~-hy<l1·oxyctiocholnl)O)o11c
`are the pl'i11d1>al 11rim1ry 17- lwt.ostemids (17/{8).
`'l'hMe
`nnd1·011cnic hormonc6 rue rh1rived from the 11drMnl and, in
`moles, t.us1.iculm· funct.ion. The pri11cip11I minory 11teroicl
`met-0,bolhcs in this r,roup of umlrogcms nrc fou nd hot.h in Utt1
`rree form , 1111d as conjugnl.cfi of glucuronideM, !!ulfol:(\$ <ir
`acetate~~. 1'lwir clct.crminntion in mine involves the ncid
`hydrolysi~ of ilrn conjugates, oxtn\cUon wil:h ol'gnnic imlvcnt.,
`renction with lllknlinc m.-dinit,ro bonicne (7,immm·rnan 1·cac:•
`tion) 1111d colorimotric csl.imntion of t:he chromor;en.
`'l'he
`indiviclunl 171{8 can ho sepnrntccl by 'l'LC prior t.o nnnlysis
`l:o obtain further i11formnti<m on tho individuul !ltcroidi;.
`'fhc ncmnnl ndult urine vuhtCll> arc; mnlc, !) t.o 2-1 mg/d11~;
`fomnle, 5 t.o 17 mg/day. Decl'cu1sed cxcrn t:ion ii; seen in
`hyponc:tive d isenRc of (.be pituit.11ry, 1,ona<fa nnd ad1·e1111h1.
`IncrMfit1d (!>U:rct.ion is SC!Cn in hy1Jerplt1~i11, cancet· m· 1:umor11
`uf t.Jw adrnnak
`Test.osl'cmme i11 the mot.I. pot.t,nt andrnge11 in blood. The,
`mMsurt,m<lnt. of uri111u·y or sc,rum 1.est0Ht()1·onl~ is uset\11 in
`distingui1;hing 1101·mnl t;nd hypogonndul maim; 1rnd in l.rent.(cid:173)
`ing hil'1mtism in the fcmftlci,
`'l'hi~ hormone iH detcl'mincd by
`glls chromol.oi;raphy, compeW,ivc pl'Ot1,in-bi11ding, iaot.01>·e
`dilut.ion 1>1' RI A 1>rocedurm1. Normal so1·11m l.estostorone i~
`0.2 Lo 1.1 µg/100 mL in tl1e mole nnd <0.J µg/'JO0 mL in the
`fcmnlo.
`'l'lm 11111:urnl estr11!(enic hormcmes ore estradiol, est1·011u
`nnd eHtriol, produced in the go11ndR, odronuhi till() pluc:e1\lv..
`T ho rli!lat.ivo umount.~ of the Lhreo e1.trogcmii rise 11nd foll
`concomitnntly duril1u tho meni:;t.rual cyckt Matel'llal, uri•
`unry totul-cstrogr.n 11xcrcition, cspuciully est.riot, i~ nn indi•
`rect index of U1c int:c:1rrity und viability of t he fol.otJlm:cntnl
`unit:. Analysis involvllH ncid 01· gluc11ronid111,c-11rylsulfotnsc
`hyclrolyi:;i~ of the conjugntc$, l'llmovnl of 11rin11ry ,:h1c:o!IC if
`prcsm1l, cxtrnction und colorim~tric or fl11ol'i11wtric nMlvsin.
`In (.ht! dctcrminnt.ion, after ncid hydrolyHi~ 1111d eU1er extrac(cid:173)
`tion of th<l 11rirm, t.hc:i e,ilrol{ens 11rn mcl,hyl11tt~d with dimethyl
`sulfnl.a 11nd chromnlogrnphi<:ully ~c1)nrnted prior to rnaction
`with phenohmlfuric acid l.o yield a rnd r.hromo1,:tll1 for colori(cid:173)
`moti·ir. nnalyi;is. The normal (!sl.ro1~011 m1t.1>ul is 4 to 60 µg/24
`hr in the female and up t.o 25 11g in thr. male, Estro,~en
`deficiency cnn he 1·elnt.ell to (1vnl'in11 fuihn·e nnd pit.uitnry
`dcficienc:y.
`Pl'OJ?<rst111·c11rn is a pror,estut:ional hol'l)1onc which is ~r.<irnt(cid:173)
`lld by the corpus luuiom of t:hc: ovnry mid 1111'10 hy t.he 1.1<l rcmol
`
`FRESENIUS EXHIBIT 1013
`Page 126 of 408
`
`

`

`r.ort.Hx. S1m1111 J>l'Of/Cktcron(• dot<:nninotfon is of v11l.ll(.: in l,hn
`dcl~J()Lion of ovuk,t.ion and is a nll)l\fitlf<: of the ,wcrntMy.
`nctivit.y of the ploccnt.ll durin1: prtlf{lloncy. Prog<•r.terotHl itt
`ck1lllrmi11ed in sc1·um by RIA, doohlc-isotopc dnrivatiwt:ion.
`,;11s-liquid <:hromat.oi,irnphy or comp1ll,it.ive prot!lin-hi11di11g
`tochniqU(!!;. Norm,11, me. nii!.runl-cyde sonun prol{C~lerotie
`Jowlr. vnry holwe(m O and l .6 ,,g/ 100 mL.
`f're1:1w1wdio/. i~ Urn 1)rind1>al mcl.i1holitc o/' progci;(.(:!ronc.
`' l'h<l urinary dctcrminl\t;ion of ))rer,n111wdiol cxcretfon i1: ;111
`indirect. ind«x of r>i:ogustcront• levels but i11 auhjcct. f'.o va riil•
`tion due to indivi<luol difforen<:rn, in lwpnt.ic nrntaboli:nn of
`t,hi~ hMmone 1md iA not, rnp1·crnent.al.ive of to(.til 1md0Kcnous
`prof(('..Sl.ot·om, pr<1d11d.ion.
`Adrcmul l:(ll'lt)X ~1.croids includt, gh.1c<1corl,i<:oids, ondro•
`genR, c~t.rn,rcn8, pfOff!!Stm·on<i imd mint!rulo<:orlfooidll, Glu(cid:173)
`toc:orti<:uids c:1111 bti dr.tcrmined os plnsma c:ort.isr,I (pli1sm11
`17 .. 0H (:(Wticosteroid11), udnnry-fl'cc (umionju1(nrod cmti$(il)
`or total-urinary 17-0H c,,rticn.~tor1Jicls. 'rh<1 lllt.t.l'r ilrc de•
`wrmiocd in uriM ns 17-hetogenic .~t,miic!H (l'll<GS). The
`171{8 in urine Ol'<l reduced with borohvdride t.o alcohols; the
`J '7-0H steroids nre <>xidi:1.cd with i;(1dil1m bismuthat~ or pori(cid:173)
`odnlo 10 l'll<S and quaol.ilat.ed hy the ,illrnline dinit rob(lll·
`wne met.hod.
`'l'hc J'l .. ()[! !lt<iroids cnn be ql\llnl.itated di(cid:173)
`rectly by the ph<,nylhy<lrai.ine .. sulfuric acid rNtction after
`hydrolysis of ~lucurouide conjugul:cs and chromnlo1:i·aphic
`'l'h<i 17-0H r.teroid analysis only dct11rmine$
`p11l'ificatio,J,
`compounds with t.ho dihydroxyncot~mc side ch11in, imch ns
`tetmhydrocortiHOI or t.et.rnhydrocorLisonc; tlui l7l<GS nnal(cid:173)
`y;;is indudeR the 17-0l·l •corticosl:eroid$ with the dihydroxy(cid:173)
`oc(it.one side chuin nnd Urn p1·ul:(nune1.riol t.ypc of compouncl.
`Normal 171((}$ duily urinary excret.ion iu 5 to 23 mg in llw
`male and :~ to l5 mg in the fomalo. They nre reduced Aignifi(cid:173)
`cantly in myxedem1111nd odrcnnl or 1111terior pil.uit!lr)' lnsuf(cid:173)
`l 11asmn corti~ol usufllly i~ measured hy f1 uorime\ .•
`ficicmcy.
`rk or wii.-chromul.ogrnphic procodure!l.
`Atdost.e1·011e is the most. 1wtivc member of the\ min()rn\o.
`corticoid group. The d~t.ormination of uri11a1·y nldo$Ul1·011e
`is of value in differen\.iatini; b1rnign essential hy1,c:irt.1msicm
`from primary l)ldostc:ronii;m (Conn's syndrome), whil;h i$
`c1111~cd by nn adnmnl nden()mn ond ii; u0c:omf)1\nied by hy•
`pcrtmision, A dm.able•isot,ope derivntiial.ion hichniqur, il,
`usnd.
`l h-innry 11l<lo~tNOlll! i8 acctylotocl with :<H ,acet.ic nn•
`hydride; aldoi;teron<l• 1•1C•diaceuite sl,tndord is added <iarly
`in Urn procedure. T he: :IJ:1 /HC !lpccifiti activll,y oft.he finnl
`product: ii; moa~llrC?d 11ft.er chrnmtll{>flrOphic purification nnd
`i~ ,1 dil'ect measuromenl of ,11do~t.eronr.. T lw normal uldc)•
`sler()nc levoli; of ahout. 10 µg/doy are eknint.ecl in Conn•~
`disM~e and \11;1.111lly nro osfloci,,t~d wit.h low imrnm potni;si(cid:173)
`um, ;;odillll\ rntcnUon nnd low-concmll.riltion a lk11line urine.
`'l'ho 11111:el'ior pit.uit,nry 8ecretcs thrc~ !lubRtanc!ls (J1011ado(cid:173)
`lropills) whidt regulate gonadnl nctivity: / ollidt>.-sti:mulat.•
`i:n.11 lwrmrme (PSH), luteinizi'.11,: lrormom! or i11t.er.~tit.i.al eel.I
`hormone (]Cf:H, Ul) and l«t<•otropin (J,'l'Jl). 'l'he go1w !o(cid:173)
`l,ro1>ilrn ur<i 1rl.vco1>rotr.irni. Biooaa11y mnt.hods c1111 be usr.cl lo
`dotcrm inc gonodc1t.rophic not.ivit.y. Afl.<;r froct.ionut.ion und
`i~olalic,n t.hc mill<~ extl'ltcl ia t1Rs11vcd in l;ellt.:inimt\h; flfl t~> l,hc
`folliculnr 1;rowt.h of lhc: rwarie~ in hypophySC!Ctomiz.11d 1111i(cid:173)
`mnls or incro11sll in (.C'!1,tic:ul1n, 0\•11ri1111 or ut.1:n-inc woi{lhl, in
`vmi<)u:; animul modols. RIA tcchniquus hove been d1wcl·
`oped for f:he~e 1:onndotrnpios and r<ipt·cscnt. Lhe most. s<m:1i(cid:173)
`t.ive ond precise tnC!tu:uremcmt. m,,1.hocl.
`Analysi$ of scrum or urinnry placcnt.11.l lac:lo1w11 (HP!,)
`and ,~horio11ic //OTl<idc)trnpin (HCO), u J)l11c:f!nb1l-dtarivcd
`p,otoin hormone, iB usoful in tho ditt(lllOi;i$ of th rc.>ot:oned
`ohort.ion, hydttliform mole and choriocm·c:inom!l, HCG,
`pregnnncd iol and progcst.er()llC as woll :rn lotal /llld fr.iction•
`at.od !l~trn1:<ms £tro useful in tmit.in!l for pre,1111111<:y. HCG and
`HPL readily uro mou11urod by RlA find lc,w vohw:i nm seen in
`thront.one.d abort.ion rrnd intl'lluterinc fetal de11U1.
`'J'IHl incrum;e in HCG in Lhr. ~r.rum 01· uri1ll\ of (.ho ))rn1;1111nt
`
`CLINICAL AN/IL YSIS
`
`513
`
`fornt1lc i11 the bar,is of n roll I.inn J)l'l!J;111m<:y 1.,,.~f. T~sl. eom!)n•
`ncrnt.s c<msi~t. of 11n anl.i1t1m in Uw form of HCU latex p,nti,
`dci~ and an HCC: 1m(.ii;<>rnm. W hcln rmti:;111·11m h; mi,rnd with
`urine cunr.oinintr II clrit<:<:tuhlc; 11:vd of BCG, it i~ nr.uf.rnliied
`and no n1{1d11Lina1.io11 of l11t.ex-ant.it,1m purt.icJe,., 11c<:11r fom:lu(cid:173)
`l i11(lt.io11 i.nhi/1i/.i<Jn teill) . Th<:\ 1:omm1irdnl applic11tio11 of
`1.h1) HCG nssay f~ives li1horaLorfo~ ll rnpicl, a,~eu1·11w 1mt1;nnn·
`,:y lest by t.okin1111dv,rnt111:(1 of monoclonal nn l:ihody ,ipocifi<: ..
`ity and scnsitJvit.)1•
`i\ m<H)Oi:l<mnl slid(: fHOtci:dur<:\ on urine,,
`Duodon (Or1:<111<1n lJici/]l!(l/ll.i<·s), U6~S t.wo di!'forn11\. mono(cid:173)
`doncil ant.iboclicm, 11n<, 01~ni11sl, HCG mid one 111tainst the
`HCGn ~ubu nil. fol' maximum :;pecificit.y. Agglutt1rnt.ion ill·
`di<:oles Ii po~itiw l,c,st with a scmsitivity lovd of 500 mil!
`HCG/ ml,, <lehictin(t p1·cg1uilli:~ 11 fow duy,:; nf1.1!r conccJpl.ion.
`llum nn t/rncvth lwtm(ilW nnd i.11su/i11 Mc prntciins whkh
`:mi of diagnoatic vnluc in 1:rowth-rnt.11 st.11dic,:1 and di11lwki,.
`T h11~ nm best. <111ant.it.11t.1id hy RI A.
`Bpineph1·ine mid mirr.pine1,hri11e are biolo!fic11lly 11ctivc
`colc,c:hol11miMs detivcid from the: ndr1mal mcd111!1., uncl ;;ym(cid:173)
`pnt.het.ic norv1i elldillr,s. Catticholamina~ al'c: nwnsurnd in
`t.ho bJood nnd uri ne afler frit<:lionatfon on :1lumin11 or ion ..
`c:xclrnnge columns, oxida tion al. pH :J.!i 01· 6.0 and ~~1hS1\(cid:173)
`quenL nuori1m,1.ric 111111lyAis. Urine <!at.c,dwli.11nh1cs ar(: in •
`crcosccl t:o > 31}0 ,,i;/2'1 hr in adrmwl med111lory l,i;;sue tunH,rn
`(J)hMc:hr(1mor.yl.oma). The normal pfognrn lnvd i:~ 2.l to (L:.i
`,,r,/L with abo1ll. 80% (HI noreplnoplu·i!H'.
`\!011U(1'imanddit add (VMA) is t.h n 11rinc nwtaboliui of
`the$C two 1:at.cd1ol111nincs. It.~ qunn(.i\.y in urine rclkd .. ~ thl,
`1mdogm1orn; sccrntion Iii' catcc:holaminC\s. VMA rnn lw dt\·
`wrmi ncd colorirnctricolly, nfh)r oxt.rnc:tfon of' Lhe urine wit.h
`ethyl acetn\.c nnd diawt.izntion with p • 11il.rn1111iline and ci.h11-
`nolami1w i11 Uw 1>ro1'cnce of carbcmute i<>11.
`\/MA ahi11 can he
`1ne11:iun1d npect.romr.t.rically follcnvin1~ poriodatu oxichct.ion tu
`vunillin und solvent: ext.m<:\.ion. T he nm111:il otit.(rnt i:1 o to
`12 mg/24 hr.
`lfomc,v,milli<: ac:id (H VA) i:i not. i1 mcn.abolil:c, of i,pinc,ph •
`1·inc or norcpinephrine, hut ii, prodm:ed from a common
`prc1!UnJ()r, dopnmilw, Elevaf.ed HVA (!Jwre lfoll is dia1rno!;t.ic
`in c 1ts(!S of neurobl/.18!.oma.
`']'hr. hio~.vnt,hc~h; or ser,)l.011i.n (5 .. hydmxyt. r,\'J)t.i11uin(1) nnd
`urinory e.~(:l'()\.ion of it~ met:1bolitR., li-hy<lrnxyindolf,11cetk
`acid (ii•lfl/\A) , nn, incrc,niicd i11 argcmtaffine !.umorn.
`'l'hC8(!
`have a vc,ry large <:J)))(ldt.y (,() mctoboli'l.O t.ryprnph1,1w sl.or(sK
`t.o 8erot.onin. Urina1·y 5-HJA/\ incrc:-,i:es limn I to'/ m1:/21t
`hr to a!\ much :is 1 1(/24 hr in thi~ type of tumor.
`HiUru.bin, ,i \,r.t:rapyrrolr. whi1:h h; dc,rivecl from ,;(!llc,~c,mt
`rcd•c<ill do1;rnd11Uo11, nornrnlly occurx in low nl!lc<mt1·nt.io11
`in t.he blO()O.
`l n bile, it is p1·1•i;(;n(. as 1.hu wat<•r-:;olu blc
`conju1:,otod 11cyl<ligl11c11l'Ot1ldc,. In hlood, l>iliruhin is (:i::htly
`bound to plai;mo albumin. 'l'hc, reduct.ion ofbilirnhin in {.h<:
`int.cstin<~ yields uro hili1)0J~(;I) which is, in I.urn, oxidiicd to a
`brown pig11Hmt-.. -urobilin.
`$omni hiliruhin is dot.c1·mi1H,d hy coupling wiLh diozotizud
`sulfonilic ocid to form awhiliruhin l'or colorimcu·ic analysis.
`'l'he <lil·r.cl. or c:1>11Jug11tMI bilirul>in lest i~ pel'l'ol'llic:d in (IQll-0·
`ous mmlia; U1e indil'i!CI or fri:<, bi/frubin ,rnnlysi!l is per•
`fonn0d in mc,t:lumol or c,nffcine .. godium b<mwate :;olut.ion.
`Nol'mol values in scrum arc: dirnct. O t.<) o.:J m1(/IOO mL:
`tot.ul, () to Ui mg/1()0 rnL.
`Clinic11I jaundice is o ycllowinl{ of tl11! \.is:me~ u~sociatcd
`witJ, hyperbiliruhinc?,mia; in hemolyt,k d11mni;11 1)1' the new·
`horn due t.o Rh and ABO i11cornp11tihilil.iM, i1111irnc:t. i,crnm
`hilirubin is elc,v(1t:cd, whCJrcmi; ucut.o lrnpatiti~ t'C'!Bull,~ in in•
`crooHc!I in 1.hc dirnG~ Lypn.
`l•~lcct:r olyt.1:i1 .... .'\"ht1 nonn11I pl11sma cd()(:t.rnlyl~, l1ivl:I is IM
`ml•:q/l, of cnLionfi nnd 1/i~ mRq/L of (lnion:i. 'l'hn o~mo\,k
`effect>.< of chloride, hicorhonot~,. 8c'>dium and 1)1)(,11s1;ium 111·e
`im})Ol'lim(. in I.he 11i.1iutc111mce of nomrnl m11$d e contract.ion
`ond w<1ter disl'.rib1tl.iou bntwe<;n c:nlb, 1>l11irnu1 n11cl inl.crnt.itfol
`fluid .
`
`FRESENIUS EXHIBIT 1013
`Page 127 of 408
`
`

`

`514
`
`CHAPTEI~ 21l
`
`l~lnmc 11hol,(>tnotry, 1itomic-uhsoq>t,ion spE,ct.romctry, 1H; ll(cid:173)
`tro1Hlcliv11tion 11m1lyr;i$, X-rny fhl()resccmcr., ion-i;pecific
`c;lei:t.rodc.1:11mcl colorimetric todrniQue11 11ro used in UI<! iden·
`tificatfon 1111d tlct.ermin1)l,ion of cation~ or unioni. in biolo1:i(cid:173)
`t11I n uida. Adv1mcc,; in technology h11vr. dcvdopud mult.i•
`f)h11so Ayfltem~ ca1>ablo of mHn1:1urini,: not 0111,v f!odium and
`1>otassium but uli;o chloride, carbon dioxide ,rnd t:olcium
`simulttmcously.
`Sodiwn um! potas$iWn scrum c-,oncontrutions am rnodily
`moMmcd by Jlnme 1>hot.mnctry or highly 1;c,ni,iti\•c und spe(cid:173)
`cific 111.omic-absorption l!poctromctry. The lntter \.echniqu<,
`is :iimilnr to emi!laion-flamc photometry, oxc<ipl, that, it mea(cid:173)
`sures energy oi; it Is ob!!orbod by atoms ralhcr tfom as il is
`omitted by aloms. 801.h tcdmiquos ore b11st1d on tho ehar•
`uctorh;tic a.bsor1)tion or emiasion wnvclcngt.hs of the c:nt:ions.
`Ion-specific 1(lo1i11 clectrodea nlso arc u~ocl for Na I und K 1
`clotenninotio111:1, climintll:ing t.ho use of II fl111110 or combusti ..
`hie guH und cnn bo performed 011 whole blood, 1•li111m11 or
`SOl'llll\.
`Chloride levels in 801'\llll or ut'ine nrc dotermincd by Lit:ra•
`tion with acid mercuric nit rote solution in the prm;cn1:c of 8-
`diphenylcnrboionc indiMf.()r. They also moy btt dct.crmincd
`1>otent:iomotrically with n ~ilvcr-;,iilvcr chlorid(l pH elcct.rndC\
`'l'he normal Hemm voluM ure tali t1) J 55 mE(l
`O$i!Cmbly.
`Nn/1,, 3.9 to 5.G mgq l</L and 95 to 106 ml~1 GI/L; orine
`lovols are L60 l,o 197 mEq Na/duy, 20 u> U-1 mJ•:q l{/d11y and
`180 to 270 mli4 Cl/day.
`Scrum socliwn, pc,f,a.~sium, d,./w·i.<le and bicarbom1te dCl(cid:173)
`tcrminutions aro uAeful indicnw.r" in udrennl coi·t.i<:ol in»uffi•
`ciency, rcnul ond (:nrdinc fnilurc, nnuria, tlehydrotfon, ali·
`mcntary tract di11ooaos associated wit.h cliarrhcn nnd vomit(cid:173)
`ing ond incrcnscd reno) elect-rolyto CX(\rction (diu1·ot,it:
`thernpy).
`'J'ho dctcrminnt.ion of exccsi; chloridi~ (>50 mli,;q/L) in the
`portJpirution of J)ll l,ients wilh pom:roolic (')'&lie fibrosi:J i,; nn
`nccurntc diagnostic t.ool. Pcrspimtion iutim11latocl by plac·
`ing tho pntient'11 hnnd in a plastic hag for 15 to 20 min or,
`profernbly, by an iontophoresis tech11iq110 in which pilocnr(cid:173)
`plne nit.rato ions nrc transported thrnugh i;mnH nrcns of the
`s kin to produce locnl perspir11Lion.
`'l'hc chloride content
`may be qu1mtit11ted wilh ;;ilvcr niiri)l.o---1>ot.ussi11m dHomt•tli·
`imprognnted papers or wit:Ji ion-sclectivo oh!ctroclcs.
`Bicarbonal-0, phosphauis, sodium, potasiiium mid chlol'idc
`C(ll\Cen1.rat,ions orci relawd to mniutemmce of ndd-base hnl.
`unco in tho hody, The pH of l,hc blood reflects t-he st11io of
`the acid-busc bnlonco and ia reluted muthr.mntirn1lly \.o
`HCO:1 .. concentrntion tmd 1,11rtinl prMsuro of CO2 (pCO?.) in
`blood by the Hendcrson-HM9olb11ch equation.
`IH<X>:i-1
`pH"' 6.1 +log .. - -· ··,-·-···
`IHiCO:,I
`Bl<lod pll, M measured eluctrometricolly, ho!i II normnl
`r1111go of?.36 to 7AO for venoua a11mplm: nnd '7.!l8 io 7.-12 for
`artcdul snmfih!ij, 'J'hc pC02 fovel in blood is doi:ormined by
`measuring th<,, t>H of the blood ni thr(!O different 1>C0i coll·
`ccntral.ions,-..mw 1111.tive t.o tho blood nnd t,hli o(.her two ob(cid:173)
`tained by equilihrntion wit:h gatt mixtur(!I\ of known pCO?.,
`Blood bkorbonot.c levcla ulso may be de(:ennined by me11s111·(cid:173)
`ing the omo,mi of ncid neutrnlii.ed b.v 1>lnsmo or scrum nnd
`pCO:i cnlculated by Eq 2, The rclnt.ion:ihip betwcon 1>CO2
`t1nd ~rbonic 1rnid conc1111lr11tion is
`
`(2)
`
`mM pllr L
`torr
`'l'he rt)lo of oxygen rm<l hmnor,lobiu in rc:;pil'lltion hos hr.1111
`di11cussed prcviO\rnly. Mensm·em!!nl.s of bloocl pli nnd CO?.
`c:ontont 11re uRed in d ifforcntint-in~ respiratory 11cido:,iij (low
`1>H, hiKh CO2) from nwtabolk 11ddosis (low pH, low CO2) .
`Rlood ox:ygen (})01,) und p1irt.1mt. oxyl((!n M1l.11rntion 1wc
`mooiiurod by a polorogruphk method; the blood s,unple is
`
`1,lnct:d in II chamber nnd sepurated from ft combined pl11ti(cid:173)
`num and silvor-silver chlorido elect.rnde by a polypropylen<i
`membrune, By diffl1sion throui:h the mcmbrimc, e<1ui'Hbri(cid:173)
`um i11 CfllJ1bli~luid between Uw p02 of the blood 1111d u film of
`i,olution in t\ontuct wilh t:hc cleclrodc. A cmrenl, which is
`proport,i(1111\I (.<> blood pO2, is gt\llcrntcd uft~r the n1)plicaUon
`of n polarizing voltnue.
`Caldum and plwsplwrus !ll'C\ imporlunt min(m1l1; i11 the,,
`procor;stis of hone calcifical.ion, norve irritability, mu8cle
`contrnction and blood congulnt.ion. Cnlchun is pre!lont. in
`1>lasmu u6 un ultrafiltcruhle (ionic und nonionic) form and n
`prolcin .. b0tmd fract.ion. Blood })hosphorui; consisl!i ofinor(cid:173)
`i,:nnic phosphonis, orglmic pho!1ph11t<, eKtor (G6P, ATP) nncl
`pho11pl1olipicl11.
`Sorum and urine calcium levelK nrc determined l'Qt1tincly
`by Utrntion with EOTA 01· li:G'l'A using a nuorcsceni. c11lcein
`or cakichrome indic11tor. Othm· methods ore basccl on the
`colorimot,·ic anolyllifl of colcium-ll\(!t.hylthym(>I blue com(cid:173)
`plex in the prosencc of 8-11uinolinol. to 1>rcvont inwrforo111:e
`by m111incsi11m. Bi1;-(o-hydroxyphenylimino)cth11ne fonrn,
`o colorod complex with 1:8lcium and, in the pre11enco of poly(cid:173)
`vinyl11yrrolidone to inhibit 1>ho~phate jntel'feronce, is n Ben•
`sitiw and Bpocific method for cnlcium. C11lcium is deter•
`mhw<l h1mt by 111;omic-uh11or11tion spcd.rom11t1·y, Aa wit:h all
`cations, 1:alci11m C!ll> be dcterminod by emi11~ion- or absorp(cid:173)
`l ion-flnme photometry or ion-Holoct.iv<i clecLrndus.
`Inorganic 1,hoaphon1s levels aro det.ormined by l'Mction
`with acid molyhdatc rengmit to form ph(1SJ>h<1molyhdic ocid
`which, in turn, is rcducod with uminonophtholsulfonic ncid
`or p-dimothylnminophcnol sulfate lo give o bllll• complex
`which is e~timat.cd C<)lorimol.l'icnlly. Normnl i;erum levels
`ar<! 2.6 t-0 ,f,5 mg P/1()0 mJ., and 9 U:. 11 ll'lft Ca/100 ml,,
`Calcium lovoh1 ure decreased tmd phoflphoruR increaa()d in
`hy1>01n1mthyroidiHm; un op!)osil.o effect is sce.n in hypert1c(cid:173)
`t.ivity of this glnod. In rickot..~ nod oaleomolacin, tlici concen-
`1.rol-ions of both elements nre docres11ed.
`In eRl.nblishing
`primury hyr,erpnruthyroidism 1111d other co uses of hyporcol(cid:173)
`comia, tinily measur<imcnts for ioni1.cd culcium (Co2+) lll'<'(cid:173)
`replncing total Cu meos1u·c1,1euls w1ing ISJ~ technology.
`Co1>1>er, magnesium, zinc and iron ore 1.roco clemcnt.s in
`blood. 'J'hey are quunUt.ntecl n.iodily by flume 1>hotomet,ric,
`colMillHitl'ic or atomic-absorption tochniquoa,
`Organ Funtition 1'cim; ... .'J'hc 1moly6es of vurious blood or
`mim! con6t;itueni!I, clcttmninution of metobolic excretion
`rat.en of exogenous com1>ounds or endogmHRta tnet.ubolitos
`l)n<l effect of exogenous Mtimuli on these purnmetcnl ure used
`for o,•nltinUon of in sitii activiLy ond func tion of various
`orgnns. Oiwm fontition $f,udios ure porformcd in diael\Ses
`ni:11oci11tod wit.h the liver, kidney, purnthyroid, thyroid and
`pituitmy glimd, gaa1,rointestiiml tract, prrncrea:i, adrenals
`nnd gonad6, 'l'lrn princi ples und significnm:c of the nnuly:ifo
`u~ed in such cvah1ationi; have bcon dCllcrihed nlso in other
`sections of Uiis chapter.
`'l'csts for hepatic frmc:tion rne b1111cd lHl bilirnhin mewbo(cid:173)
`li!!m and excretion, curbohy<lrnte metubolisrn (galnct:ose tol(cid:173)
`ornnc:c,, t.ost), plnsma-protein clumgo11 (cophalin flocculntion
`l.1iRt and NG rnUo), nb>iormnl fat. mot11bolism, cleloxificntio1l
`mochuni11ms (hippuriC! ocid synt hc:;is), excrntion of injected
`1.1uh11t.unces IBSP), 1wot:hrombi11 fonnoLion ,1nd proviouiily
`discuHsed enzvme levoli;,
`DisoMO$ or' the liver ure due to cellulur ulturotiorn1 (hcp11-
`1.t•cclh1lll.r) or ob11trnctions to l,ho 11ow of bilo (ob11tructivo
`jou11clice).
`f:fopatC)(:ollular liver di~caae c1m b<1 Chl'Oni<:
`(postnccrofo: cirrho11i!!, cnrcinoma) or ucutc ( viral hc1>at:iti&,
`alcoholism, toxin- and 1:homicol-induccd).
`'J'he cr.plwlin /loc;cufat,ion tflst in ha1,(l(I on Lhe f1occulntion
`of ccphalin-emulsified cholesterol by -y-glol.mlin. In normal
`Herum 1111 ulbumin-likc p1·ot.oin will inhibit t,his renctfon; in
`ho1>11tic disll/lljll$, which produce nbnonunl -r·11lobulin or re(cid:173)
`duced albumin )(!vela, the flocculotion will occm·.
`The (li!t oxi{ic<1/:itm m,wlw11isms of t.hc, liver con be evulu-
`
`FRESENIUS EXHIBIT 1013
`Page 128 of 408
`
`

`

`11wd by int!'uvcnous administruLion of :iodium beiw.onte ancl
`estimation of the bonzoic ncid metabolite, hippuric add, in
`the urine:,. In he1)at~>pnrenchymnl diseose, a rccluce<l cuJ>nci·
`ty ofth<, liv«Jr to form hiJ)J)uric ncid by conjugation of 1{lycinc
`and bem.oic ncid ia ohimrvod.
`The obilil,y of U1c livor to excrete on injci:t.ed dye iH doter·
`mined in I.he JJSP test; tho sorum is a na ly~.ed for dye concen(cid:173)
`Lrotion nt n ~uitnble (,imc intorvnl oft.or rv adminis trot.ion of
`2 to 5 mg BSP/kg. Rodioiodinated (1'111) Ro8c Hcngol Sodi·
`um dye ohm hns been used in dye-excretion studiei; with
`isotopic estimut.ion of urin<i dye lev«Jls.
`l(id11e,v fu.nctio11 teSt!l nre bos<lcl on t:hc dctcrminnt.ion of
`blood nonprot.ein nitrogen (urco, uric acid nncl crentininc),
`elc<.:Lrolyt.eH, blood ucid-busc bnlunco, ro\ltine uri nnlysis nnd
`the c!Mranco of odminist.cre<l compounds in the tll'inf!.
`I\fost clearance studies 11.rn perfor1n<!d with imbstance,; tliut.
`ure not ro~orbt>cl or Hocroted by t.lw rnnul tubules:
`i11uli11,
`mnnnit.ol, Modium p-uminohippumto or J?.i;J-iotlmlamute $0·
`dium (aodium 5-ucietnmido -2,~.6-triiodo .. N•mcthyliao ..
`'l'lwso am ndminiswred intravenou~ly on<l
`phthulnmute).
`the rnto of ur ine -Oloomnce und glomel'Ulnr filtration iH cst.i(cid:173)
`mat.c:d by n11oly11i6 of lhe uriM.
`'!'he oxcrewry capacity of
`the 1·e1111l tubulnr opithellum cun he dct:ormincd by mcusur(cid:173)
`ing t.he r.luoranco rat.e of PSP.
`'l'he dy<J ii; injuctc,d IV anrl
`the rol.o of its cle(trmwc in urine ia delel'miu1id. PSP iM
`bound lootiely toaerum albumin and i~ rnmoved rapidly from
`the blood bv the reno! tubuk9.
`Sod ium iodohippurttt.e-( 11.1,n, which i8 eli.trncl..ed n.lmost
`c,)mpletoly from lhc blood on n ~inglc, Jlll6SOIIC through t.lic
`kidney, nlso h116 been t1111id in 1·ennl function studfos; 11 rerw(cid:173)
`gra.m or ie<>topic scan of both kidneys ia performed. 1'he
`te~t- providM data on renal 1.11hult1r ~01:rction, renal vuscular
`competence n11d reno I evocuntion ond is primarily uacfnl lls:.
`comp11riMn of individuol l<idnoy function. II. is imporlunt.
`t:o not e thot 50% of kidney funct.ion cnn bo cMnprnmised
`without nny significant clnrngo in the rout.inn renol function
`pnrnmewrs.
`Thyroid function lusts 1181u1lly me«sm·c the cil'culnting
`levelK oft.he thyroid hormone~, and not. the enct-,wg11n Mfod ..
`'l'h<i thyroid glnncl convert.s inorganit iodid<, to ihyro.ti1w
`{'l',1) nnd triie>doth)'l'Oll.ine r1:,). ·ra nnd 'I\ lll'C stored in (,he
`colloid part of t.h<i glnnd nR pnrl. ()f tho thyroglobulin m(1lo,
`culr. . Hypothalamic thyrolrnpin-relea.~in,: hormone
`('fHH) modiotes the rclomm or the 1>ituitnry thymLN11)ill
`(thyroi<l-siimuloti1111 lwrmcme., 'l'SH). gxCOHS levol$ of dr(cid:173)
`(011l11tin1~ T .1 depro8s, ond low lov<ili; ol' T.1 incroasc, 'l'SH
`'f'SH stimulotcR tho proteolytic dcgrndnt.ion of thy,
`relo11Ko.
`roglobulin to rnleaso 'f-1 and 'l':1, and incrcai;ois orgnnification
`of iodine. T 4 accounts for 90%of socrot11d thyroid hormone~
`nnd <!Xists in blood bound to thyro.thie-bi11rlin11 globulin
`(TB<l) or l hyroxine-bincii11}! p1·11albwnin ('l'BPA) 01· lo albu(cid:173)
`min. T:; is not protein-bound ond hn11 5 l.o 10 times tho
`hiolof,icnl 1>oto11<:y of 'I\ on o wei1:ht basis.
`'l'hernfore, '1'1
`re1>rcr;ents the major part of 1>rotein-bou11d ioclino (PBI).
`T he lev!!I of /re!<! th,yroxi,w (F'/'4), the oct.ivr. frnction in
`blood, itt regulau;d by 'I'◄ nnd T,1 releos<i and U1c lovols of
`binding proteini, in blood and tiMsues.
`'l'he uptake <>f orally odmini8tcrcd No 1:111 p1·epnrntione by
`the t hyroid gland con be ostimotud by isotopic 11cnnning of
`tho gland 24 hom·e nfto1· 1:111 ndmi11i1Jt1·otio11 nnd j9 nn indei,:
`of glunduliir fu11ctio11 (hyp<lrllctive, >fi0% uptnke; hyJ)O!lC·
`tive, <Hi%),
`PBJ determinul.ions ore b!l8<ld on t.he precipilat.ion of J>ro(cid:173)
`loin-bound thyroxine, removal of inorgonio iodiM by basic ..
`or onio11-ex<:hange chrmn11logri1J)hy, nlkolinu incinel'lllion t.o
`convert lhyrnxinc to ino1·gnnic iodide, ond, finally, (JunnUta•
`tion of iodidu by rcnct.ion wit.h nr11cmm1s t\cid nnd c(,ric am ..
`monium 8ulfo to, PHI is a g<1<ld ost.im11te of t utu.I cfrc11lt1Ung
`hormonol iodino, The norm11l rnnge is '1 to S µg/100 mL
`scrum.
`T.1 c:nn bu c.lcter:minod by column d1romnl.or,nlJlhy in
`
`CI.INICAL ANALYSIS
`615
`which it is scparoled t1nd iRolM.<id uy ivn-cxd1a11g11 chromil·
`tography, 11nrl then onnlyv.c<l <:olorimetricolly, Noni8ot(1pe
`thyroid RffBRYH havll been dc11c,Jop(!d 11Hing lhiol'ci,concc po·
`larir.ution methods fo1· 'l\ and frce-thy1·oxin index. h\ thc
`competitive f)Mtein -binding 118SllY fol' 'l',1t sc,nun 'l'.1 c:om·
`peleB with 1201·'1'~ for binding sil,<'.11 on o known unwu nt <Jf
`'l'ho rnUo of hound LO free l?.J,J is determi nocl !Jy
`'l'BG.
`11dsorption of 121;1 -'l'.1 not hound to 'fBG on an 1mio11-r.x•
`change re6il\ ombeddc,d in a polyur(!(,hnne sponge or n 1,orous
`de111.rnn gel, und i$ o clfrcct, index of 'l'.s lcvols. The f)rMencc
`of mcrcurinl11, inorgnnic iodide or iodiuotod rudiogrnphic
`compounds in IIOl'lllll interfere,:; wit:h thi:, '1'1 column and J>BI
`,,roco<l urr.~. 'J'hc cmnpetilivt-bindinfi procedure is affoc;l.od
`by the 1>rc6cnco of hit~hly protein-bound drog:; or <:hnnges in
`TBG leV<ils in scrum. The normal rnni:c of scmun 'r., is 2,9 t.o
`G.4 11g/lOO mL by t:olumn 1111d 3.0 t.o 7.0 µg/100 ml, by bind(cid:173)
`ing lllJ.~oy. T 4 oncl PB! nrCl incronscd in hypcrl,hyroiditm nnd
`the cnrly sl.ngcs of hcpnt.itis. 'I\ und PBI m·c d(ltrenscd in
`hyp{ll.hyl'<lidiflm 011d 11a1>hrosis.
`FT.1 olBo is det:crminocl in n compe1,it.ive p1·ot,oin-binding
`osHuy in which 12'•1'1'4 nnd !lo1·um nrci i11,:11bnwcl, and then
`dialysod to determine t,hc pcm:(,nt di11lyznbl1, JWJ.'I\. FT.1
`anulysi11 is uBcd in suapec,tod ubnorm111if;io9 in 1>rowin-bind·
`ing globulins. '}'4 binding cnpucil.y of 6ertun 'l'BG, 11lb11mi11
`and prenlbumin cnn be determined ofter clect.1·{1phornt.ic
`ijOpnrat.ion of thm;o p