Hereditary Disorders of Purine and Pyrimidine Metabolism: Identification of Their Biochemical Phenotypes in the Clinical Laboratory

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Hereditary Disorders of Purine and Pyrimidine Metabolism: Identification of Their Biochemical Phenotypes in the Clinical Laboratory D ALlIIOR VALlK, M.D., * AND J AM ES D. J ONES, PII.D.

• Objective: To describe a laboratory ap proa ch to th e diagno sis of hereditary diseases of purine a nd pyrimidine metabolism and emphas ize clinical sit ua tions ill whi ch these disorders should be con sid ered in the differenlial diagno sis. • Design: Disease-specific patterns were identified in random specime ns of ultrafiltered urin e by usin g gra d ient high-performance liquid ch ro ma tog r a phy with diode-array detection, a nd reference ra nges were esta blished for uri c acid, hypoxanth ine, xan thine, and uracil expr essed per creat inine in ran dom specimens of urine. • Material and Methods: Diagno stically significa nt purines and pyrimidines were sepa rated with use of a Supelco LC-Ill-S nucleoside column eluted with 25 mm ol/L ammoniu m acetate buffer and acetonitrilemethanol-water. Biologic nuids were prepared by ultrafiltration after addition of 3-m ethyluridine as inPurines and pyrimidines fulfill a wide range of diverse biologic functions. ranging from energy transport to storage and management of genetic informa tion .' Important physio logic roles such as signaling. transmitting. and modulating.i" reproduct ion•.5 and reactive oxygen species scavenging" have been described or postulated for many purines and pyrimidines. Some purines have been introd uced into cli nical practice as active pharmacologic agents.' Several features unique to purine and pyr imidine metabolism have been recognized. Their synthesis is tightly controlled in a cell-typespecific and cell-cycle-specific manner, and substantial recycl ing occurs in vivo in a tissue-dependent manner. Purines are salvaged primarily as free bases. and pyrimidines are usually salvaged as nuclco sides.!? Because extensive tumFrom the Di vision of Clinical Biochemistry and Immunology (D.V.) and Division of Laborat ory Genetics (1.DJ .), Mayo Clinic Rochester. Rochester, Minnesota. "Current address: J. Grego r Mendel Ch ildren ' s Hospit al. Bmo. The Cze ch Republic. . Add ress rcprinr requests 10 Dr. J. D. Jones, Division of Laboratory Genetics, Mayo Clinic Roche ster , 200 F irstStreet SW, Rochester, MN 55905.

140)'0 Clin Proc 1997;72:719-725

ternal sta nda rd. We used speci mens negative for scree ning of metabolic disorders to esta blish referenc e ranges. • Results: Disea se-specifi c patterns were identified in specime ns with purine a nd pyrimidine disorders and several urea cycle disorders cha racle r ized by increased production of pyrimidine. • Conclusion : The approach de scribed identified disease-specific pallerns of purine and pyrimidine disorders and seve ra l urea cycle disorders . We sugg est Ihat testing for purine and pyrimidine di sorders be don e in s peci mens evaluated in metabolic laboratories for "scree ning for inborn errors of metabolism." (Mayo Clin Proc 1997;72:719-725)

=

=

C V coefficient of variati on ; HPLC high-performance liquid ch ro ma tog raphy ; TLC = thin-layer chr om a tog rap hy; UV =

ultraviolet

over of purines and pyrimidines occurs in rapidly dividing cells. inhibition o f their synthes is de novo or recycling is a target of cytotoxic therapy.' Numero us genetic disorders of purine and pyrimidine metabolism (Table I) have been described in the past 40 years," :" with the exception of primary idiopathic gout (which was described ea rlier). Additional enzymatic de fects rema in to be characterized because some variants of purine and pyrimidine de novo synthesis defec ts proba bly lead to a lethal phenotype in utero. " T he laboratory diagno sis remains d ifficult because of generally nonspecific clinical manifestations crossing several medic al specia lties, ex istence o f disease variants, lack of physician awareness, and, to some extent , comp lexity and unavailability of necessary expe rtise and rapidly evo lving laboratory techniques." The determinations of serum uric acid and urinary uric acidl crea tinine ratio are simple and valuable tests that imply the d iagnostic possibility of purine metabolism-related disorders. Primary diagnostic procedures arc based on identification of metabo lites in urine and plasma by high-performance liquid chromatog raphy (HPLC)18 and two-dimensional thin7 19

co 199 7 Mayo Foundationfor Medical Education and Research

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l.ABORATORY DIAGNOSIS OF PURINE ANI> PYRIMIDINE DISORDERS

Ma,o Clio Proc, AuJ,tust 11197, Vol 72

Table 1.- Purine and Pyrimidine Disorders \ Vilh Recog nized Clinical Phen otypes -

Principalclinical manifestation

Compounds measured

Disorde r

Trea tmen t

HGPRT deficiency

Uric acid. hypoxanthine CU/p)IO If>

Neurologic, renal

rJH N

Uric acid (u/ p) tQ.Il'

Renal

PRPPS superacnvity

Uric acid (uJp)10- 16

Neurologic. renal

APRT deficiency

2.8-Dihydroxyadcninc (U)'(l.16

Renal

AMPDA deficiency

None

Muscular weakness

N Ol

XO deficiency

Xanthine. hypox anth ine (u)17 Xanthin e. S-sulfoc yslc inc (U)17

Renal

Diet

(and less se vere variants)

MoeI' deficiency ADA deficiency PNPdeficiency ASL deficiency ADA superactivity

Dco xyadenosine, adenosine {u/p) I()' 16 Inosine. guanosine. and their deoxya nalog ues (U!p)11). 16 SAl CA r, succ iny ladenosi ne (u/CS011 Low ATP in erythrocytes"

UMPS deficiency UMPH deficiency

Orotatc. orotid inc ( O) 17.IM UM P. e M P increased in

DPD deficiency DPH deficiency

DPD hetcrozygotes

6-TPMT deficiency or superactivity

Renal. neurologic

SCID. T-cell dysfunction

Neurologic, immunodeficiency

Neurologic

Anemia (D iamond-Hlack fan

syndrome) Ne urologic. hematologic

Anemia, basophilic spotting

erythrocytes" Thymine. uracil (U)11 Dihydrothymine, dihydrouracil (u)17

Neurologic Neurologic

None '

Adverse reaction to

5-fluorouracil N-6 substituted purine intolerance. nonrcsponsiveness

None"

Allnpurinolrenal symptoms Allopurinol-

renal symptoms Allopurinolrenal symptoms Allopurinol-

renal symptoms

avallable-criboseorally ( ?J1l) 16

Die' (? ) PEG·ADA. BMT Not available

Not available Not available

Undine Not available Not available Not available Dose adjustment Dose adjustment

•ADA = adenosine dcarninase: AMPDA = adenosine monophcsphate dearni nasc: APRT = adenine phosph oribosyhransferase; ASL =: adcn ylosucc inatc lyase; ATP = adenosine triph osph ate; RMT = bone marrow transplantation; eMP = cytidine monophosphate: csf =: cerebrospinal fluid; DPD =: dihydropyrimid ine dehyd rogen ase: DPH ;:;;: dih ydropyrirnidinasc; FJHN = familial juvenile hyperuricemic nephropath y; HGPRT = hypoxanthine. guanine phosph ori bosyhransfc rasc: MoCF = molybdenum cofactor; p =: plasma; PEG-ADA = polyethylene glycol-modified ADA: PNP ~ purine nucleoside phosphorylase: PRPPS ~ pbosphoribosytpyropbospbate synthase: SAICA r = succinoaminoimidazole-earboxamide riboside; SCIO:: severe combined immunodeficiency syndrome;TPMT= 6-thiopurinc mcth yltran sfcrase ; u :;:: urine; UMP :: uridine monopbcspharc: U M PH =uridine monophosphatc hydrolase; U M PS :: uridine monophosphate synthase; XO = xanthineoxidase. laycr chromatography (TLC) .'" Secon d-level testing to confirm a disease-specific pattern has used identical methods but involving prior frac tionation (mostly anion-exc hange tec hniqu es) 10 el iminate the ana lytic interferences invariab ly present in cli nical spt..ocimcns.20 22 An automated method requiring four sets of ultraviolet (U V) ab sorbance detectors and column swi tching has been applied eval uatio n of ultrafiltered urine for purine and pyrimidine disorders.P A so phistica ted HPLC procedure has bee n developed for iden tificarion of hypennodified nucleosidcs in hiologic flu ids by using phenylboronatc ge l for cova lent extraction of compo unds wit h vici na l cis -diol functionality, such a s ribo nucleosides." Herein we describe our ap proach to thc diagn osis of purine and pyrimidine disorders and emphasize cl inical situations in which physicians should consider these diseases as diagnostic possib ilities. Our goa l was to work with random urine specimens and avo id often-imposed restrict ions. Re-

'0

quiremcnts for a ..tow-purine" diet, 24· hour urine collec tion, and uno drugs" before sample co llec tion are impractical and often unachievable for patients, physici ans, and parents. We es tab lished a sing le two -mode re ve rse -phase HPLC proce d ure: a gradient mode I for pro filing of purine and pyrimidine compounds in unextracted biologic flu ids and an isocratic , short mode /I for ac hieving efficient uracil/pseudouridine and 2,8-dihydroxyadenine/ uric acid and orotic acid separation . The procedure analyzes native ultrafiltered materials and uses diode-army detect ion and in-line threewavelen gth quantifica tion. For ex tended testing, we used phcn ylboronic acid " and TLC frac tionation proced ures . Reference values have bee n published for no rmally occ urring metabolites (uraci l, hypoxant hine, xanthine, and uric acid) in random specimens." We demonstrate the diagnostic power of the method by usin g examples or purin e and pyrimidine de ficiency syndromes and urea cycle disorders characterized by lnterm inc nt pyrimidine overproduction.

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MaJo Clin Proc, AURusl

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Vol 71.

I. ABORATORY DI AGNOSIS OF PURINE AND PYRIMIDINE DISORDERS

M ATERIAL AN U M ETIIOUS

Instrum entation. -A Waters 600E gradient modul e was connected to a WISP 712 through a narro w-bore tub ing, Sampl es were injec ted on a Supelco LC- 18-S " nucleos ide" column (250 by 4.6 mm ) equipped with a Supelgua rd LC18-S precolum n" maintained at 24°C. The detection was done with a Waters 990 d iode-array detector and the 990 software (Waters). Chromatography.-Solvent A was 25 mmol/L amm onium acetate, pH adju sted to 4.02 ± 0.02 with glacial ace tic ac id (stable for I week ). prepared from 500 mmol/L of stock solution (stored refrigerated . stable for 3 months) and diluted with helium -degassed HP LC gradc water. Sol vent B was acetonitrile.methanol.wa ter (100:250:450) made fresh for each run (stable for about 30 hours). So lvent C was degassed HPLC grade wate r (fres h dai ly), and solvent D was methanol (a washing solve nt only - not used for separation). For purine and pyrim idine profi ling (mode I), the grad ient sell ing was as fo llo ws (in percent, programmed as "table I " in the instrumen t): 0 108 minules isocratic A50:B3:C47, 8to 35 min utes lincar to A50 :B20:C30, 35 to 38 minutes linear to A50 :B47:C3, 38 to 44 minutes isocratie A50 :B47:C3. 44 to 45 minut es linear to A50:B 3:C47, and 45 10 70 min utes isocrati c co lumn equi libration A50:B3:C47 . Ana lysis was recorded at 235 to 400 nm from 2 to 45 m inutes. For mode II. the sen ing was as follows (instrument " table 2"): 0 to 15 minu tes isocratic A30 :C70, 15 10 16 minu tes linear A30:B67 :C3. 16 to 24 m inutes isocratic A30:B67 :C3 , 24 to 25 minut es linear A30 :C70, and 25 to 40 minut es isocrati c co lumn equ ilibrati on A30:C70. In strum ent Catibration.s-Su xs: solutions (I rnmol/L) in water (alkalinized with sa turated Li,CO" if necessary ) of orotic acid. uracil. uric acid (d issolved in sa tura ted Li,CO ,), 2.8-
121

(ce rebrospinal fluid , amni otic fluid ), higher volumes at a 4: I ratio were processed , and 50 10 80 ~L o f plasma (and up to 180 ~L of amniotic fluid, ce rebrospinal fluid , or other low co nce ntration material) was injccled onto a co lumn. Ur ine With C rea li nine.-For samples with less than 10 mgfdL, before measurement the urine was warmed for about 10 minules to 55 °C to di ssolve precipitated material. Preparation was there after identica l 10 that for plasm a; the injcction volume was 20 ~L. For samples of IO to 100 mgfdL, (50 ~ L of urine + 50 ~L of water) + 25 ~L of internal standa rd were pipetted into Ihe Ultrafree unit. and 20 ~L was injected . For 100 to 150 mgfdL , (25 ~L of urine + 75 ~L of water) + 25 ~L of internal slandard were pipe tted into the unit. and 20 ~L was injected . For samples greater than 150 mgfdL , the procedures were the same as for 100 to 150 mgfdL, but I0 ~ L was injected . For mode II. sample preparation and inject ion vo lumes were identical, but no internal standard was added. Ur ine Fractionation for Extended Testing. -For fractionation, I00 ~ L of urine was mixed with 25 ~L of intern al standard. and 20 ~L was ap plied per 15-mm lane as a narrow streak on thc HPL C plate (Merck catalog no. 5628/5. with a fluorescence indicator to enable visual identifica tion of zones ). Three lanes were app lied per specime n. and a fourth lane was a standard of 3-melhyluridine. thymine. and guanosine. Th e plate (up to the end) was deve loped once in butanol :glacial acet ic acid .water (8; I : I). After migrat ion, the plate was inspected undcr UV light (254 nm ). Three fraction s were isolated (one fracti on from each lane); thus, an interna l standard cou ld be included in each fraction: ( I) from the internal standa rd band (included) to th ymine (includ ed); (2) from the intern al standard band (included) to the front edge of guanosine; and (3) the internal standard band onl y plus the are a from guanos ine (incl uded) 10 the stan (included) . Each silica gel fraction sc raped from the plate was quantitatively tran sferred 10 the Ultrafree unit , 300 ~L of water was added. vortexcd, and centrifuged, and 150 ~L of ultr afi ltrate (corresponding to I0 ~ L of orig inal urine ) was inject ed onto the HPLC co lumn. Reference Ran ge Study Specimens.-Random morning urine speci mens submitted to the laboratory for screening of inborn errors of metabolism were tested for amino acids. monosaccharides, di saccharide s, and oligosaccharides, and succi nylnucleosides ." Or ganic acid s and glycosaminoglycans were used when no abnormality was found. Quality Control.-ehemlrak normal or pathologic general c hem istry control serum (Med ical Analysis Systems, Inc .) was used to assess quantification accuracy of uric acid determ inatio ns, The material was processed as plasma (50 ~L ) and analyzed once per ne w batch of the buffer stock substrate. Periodically, a Supclco nucl eoside test mixture (catalog no. 4-73 10) was analyzed to verify qoantificatio n

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722

LABORATORY DlAG NOStS OF PURINE AND PYR IMIDINE DISORD ERS

accuracy of several nucleoside determinations (inosine. uridine, and g uanos ine) .

RESULTS Ch romatography.- The co lumn effi ciency expressed in theoretical plates was n = 29.000 and 68.000 for guanosine and deoxyadenosine. respectivel y. The intern al standard retention time coefficient of variation (CV) was 0.5 1%. The within-run precision (CV) ranged from 3.6% for inosine to 5.1% for adenine; between -run precision ranged from 5.7% for guanosine to 6.25% for uracil. The reco very of ultrafiltrate ranged from 96.8% for hypoxanthine to 100.5% for guanosine; the recovery after TLC fractionation was 42% for dcoxyadcnosine and 83% for deoxyguanosine or better for bases and other analytes . Separations of reference mixtu re (50 ut, of Chemtrak + 50 ul, of calibration solution) and disease-specific metabo lites are shown in Figure I. Identification of Disease-Sp ecific Pattems.s-Dlhydropyrimidine dehydrog enase deficiency is biochemically characterized by thymine and uracil in the urine. The concentration nf thymine in our specime n reached on ly 129 mmo l/l., Uracil was preseot at a concentration of abou t 250 mmol/L, Microbial degradation of pseudouridi ne (a ubiq uitous nucleoside originating from degrada tion of Iransfer RNA" ) during shipment also contributed to this increase. This speci me n did not contain S-hydroxymel hylur.lcil, which would appear between uracil and pseudouridine peaks. Both metabolites indicative of adenylosuccinate lyase defi ciency were chromatographically resolved and ident ified; suecinyladenosine yielded a clear and quantifiable signal at 285 nrn, where absorption of hippuric acid appearing before succinyladenosine is minima l. The pattern of LeschNyhan syndrome treated with allopurinol showed high peaks for uric acid. hypoxanthine , allopurinol, and oxypurinol. A characteristic excretion profile o f inosine. guanosi ne. deo xyinosi ne, and deo xyguanosine in conjunction with minimal ex cretion of uric acid is indicati ve o f purine nucleoside

phosphorylase deficiency. Xanthine oxidase deficiency and molybdenum cofactor deficiency showed increased levels of xanthine and min imal excre tion o f uric acid. Adenine phosphoribosyltransferase deficiency biochemically manifests with excretion of norma lly undetectable 2,8-dihydroxyadenine. characteristically with an abso rption maximum at

306 nm; both analytic modes identified the condition, and mode II was better for metabolite quantification. Urea cy cle diso rders are characterized by pyrimidin e overproducti on. The pattern of acu te relapse of ornithine carbamoyltransferase deficiency showed increased excre-

~1a)'u

Clio "rue. AuguSI 194n , \'0172

ac id. uracil. and uridine excretion in a newborn with argin inos uccina te lyase deficien cy during an acute attack; this patient subsequently died. A male patient with mental retardation. behavioral problems. and psych iatric sy mptoms. in whom arginin osuccin atc lyase defi ciency was diagno sed at age 14 years. excreted nonnal amo unts of orotic acid and uracil but no undine; his nitroge n balance was maintained by massive excreti on of argininosucci nate. The pattern s show n were identified in nonfractionarcd

urine (reconstituted from filter papers used for mailing except for dihydropyrimidine dehydrogenase sent frozen and urea cycle disorder s dia gnosed in the laboratory) on the basis of retention limes and 254/270/2 85 nm ratios and in-line UV spectrosco pic evaluation. The reference value s for uric acid. hypox anthine, xanthine. and uracil are shown in Figure 2. DISC USSION C" rum atograp"y.--Our HPLC of purines and pyrimidines proved to be a stable method with minima l sample preparation and fully suitable for screening up to 17 specimens a day. Co lumn temperature control is reco mmended beca use retention times of se ve ral compounds (for examp le, succi-

nylnucleoside s) vary up to I minute/I ' C. T his chrornato graphic system remains stable for about 2,000 analyses if preco lumns are change d after about 200 injection s. as evidenced by reproducible retent ion times (CV for internal standard retention time, less than 0.5%) and separation of xanthine and uridine. Although tctrahydrofuran impro ved symmetry of the adeni ne peak, it introduced variabilit y with retention times because of its volatility and was found unnece ssary for separation.":" Quantification at two 10 three wavelengths is needed to obtain valid results. Purity of the peak is ensured if calculat ed conce ntrations at diffe rent

wavelengths match. If interferences occur, the sample is fractionat ed with use of norm al-phase TLC; the sarne approach is taken as the confirm atory technique when the presence o f a metabolit e of interest is indicated. Once calibrated, the system is stable for severa l months. Use of strong hydro xides (for example, NaOH and KOH) to "facilitate" dissolving of uric acid and 2,8-dihydroxyadenine results in their decompo sition; therefore. saturated Li,CO, is the solvent of choice. Specific identification of dihydropyrimidines was done by mode I for dihydrothym ine and mode // for dihyd rourac il, but ammonium acetate buffer was replaced by otherwise identical ammonium phosphate buffer to eliminate absorbance of acetate at 200 to 240 nm. Mix-

tion of orot ic acid and highly increased excretion of uracil

lures of urine spiked w ith dihydropyrimidine s were evaluated becau se urine speci mens from a patient with dihydro pyrimidin ase deficiency" were unavailable .

and und ine; uridine ex cretion disappea rs in remissio n. and uracil. orotic acid. or both remain increased in asy mptomatic patients. We observed almost an ide ntica l patt ern of orotic

Sample Preparation.c-Scvcsei hundred compounds absorbi ng in the UV range 230 to 320 nm can be present in urinc. Nonnal-phase TLC offers different fractionation se-

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Mayo C1 in Prcc, August 19117, Vol 72

LABORATOR Y DIAGNOSIS OF PURINE AND PYRIMIDI NE DISORDERS

723

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Fig. I. Anal yses scanned from 235 10 320 nm for two-mode high-perform ance liquid chro matography (y-axes in abso rbance unit s. full sca le: x-axes in mi nutes). S·254. 5·270, and S-285 nm. Stand ard three- wavelength, mode I chro ma togram of a mixture of standar d + Chcmt rak abnormal serum (50 ml + 50 ml) + 25 mL of internal standar d. shown at se lected wave lengths. Numbering of pcaks is identica l throughout all chro matograms: / = intern al standard: 2 =orondirc: 3 = oro tic acid; 4 = uraci l; 5 =uric acid; 6 =tyrosine ; 7 =hypoxanthine; 8 = xanthine; 9 = uridin e; 10 = thymine; / / adenine; / 2 inosine : 13 = guanosi ne; 14 = dcoxyinosi ne; IS = deoxyguanosine; 16 = thymidine; 17 = succ iny ladenosine; / 8 = trypto phan; / 9 = adenos ine; 20 = deoxyadcnos mc: 21 = 2.8 -dihydroxy adenine; 22 = pseudouridine; 23 = succi noam ino imidazo le-earboxamide ribosi de; 24 = oxy pu rinol; 25 allopu rino l. OCT( h)·2 70 nm. Mode II. female heterozygous for ornithine ca rbamoy hransferase deficiency, 6 hours after allopurino l load, c harac terized by incre ased excretion of oro tidine, orotic ac id, and high ly increased uracil ; undine no t detectab le. APRT·310 nm. Mode II. adenine phosphori bos yltransferase de ficiency. DPD ·270 nm . Mode I, d ihyd ropyrim idine dehydrogen ase defici ency. ASL·270 nm . Mode I, adenyJosuccinate lyase defi ciency. treated with am ino imidazole-earboxamide riboside. adenine. and allopurinol. LN·254 nm. Mode I, Lesch-Nyhan syndrome , tre ated with allopurin ol. PNP-254 um , Mode I, purine nucl eoside phosphory lase defi ciency. XOD -254 nrn, Mode I, xanthine oxidase defi cien cy . OCf-270 nm, Mode I, ma le hemizygous for o rnithine carbamoy ltransferase deficiency during an acute attack, charac terize d by increased excre tion of orotic acid, uraci l, and urid ine.

=

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lectivity in comparison with anion-exc hange chromatography and yields no fraction ove rlap; thus, compo unds di fficult to sepa rate (urac il + 5-hydroxym ethy luraci l + pse udouridine; succinyladenosi ne + hippuric acid ) se gregate fully

in d iffere nt frac tions . The use of intern al standard throughout the procedu re allows quan titative data handl ing. The recovery is also satisfactory fo r deoxynucleo sides except for dcoxyadenosine, for which deg radation occurs even if a lka-

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124

LABORATORY DIAGNOS IS OF PURINE AND PYRIMIDI NE DISORDERS

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Mayo Cl io Proc, August 1997. Vol 72

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Fig. 2. Distribu tions of concentrations versus age of subjects (y-axes in mmol/mmol crea tinine; x-axes in years) and reference ranges for uracil, uric acid. hypoxanth ine. and xanthine . Age-gro ups: younger than 2 years . 3 to 5 years, 6 to 11 years, and 12 years or older; " number of measurement s within a group.

=

line so lvents are used (probably because of the acidic character of silica gel). We prefer ultr afiltration" over trichloroacetic acid" with back-extraction or organic solvent deprotein ation. Hypoxanthine in plasm a ca n be eva luated only if fonned ele ments are se parated in a refrigerated ce ntrifuge within 15 minut es afte r venipuncture. lnterf erenceso-«The tec hnique reveals abo ut 40 major peaks that correspo nd to endoge nous and exoge nous compoun ds in urine. Interferences due to new drugs and their metabo lites remain an analytic challenge beca use the spec imens analy zed are frequen tly from pati e nts rece iving therapy. One metabolite of ibuprofe n, co mmonly used by pediatricians , appears between hypo xanthin e and xanthine. and a second metabolite coe lutcs with inosine, both having an " inos ine-like" UV spectrum (the identit y of both metabolites is co nfinned by gas chromatog raphy-mass spectrometry) . Acetam inoph en (confirmed by gas chromatographymass spectrometry) appea rs as a major pea k after the 3methylurid ine pea k a nd before hip pu ric ac id and Nacetyllyros ine. Acyclo guanosine elutes at abo ut 22 minut es, separated from measured co mpo unds ; howe ver, one o f its metabolites coe lutes with hypoxanth ine. Spec tral com pari-

sons by the diode -array detector of peaks and known co mpoun ds have proved valuable in eva luation of this chro matographic procedure and in detect ing interferences. Reference Values.-elinically insig nificant ge nder and year-to-year di fferences in se rum uric aci d co ncen tratio n were established in a gro up rangin g from 0 to 11 years of age ; the steady downwa rd trend we observed for all four co mpounds is ca used by the increase in creatinine excre tion with adv anci ng age . Oth er factors such as diet may influence exc retio n of uric aci d. We do not think that this is a critica l co nfounding factor. however , beca use findin g of true hyperuricosuria, proved also by a 24~hour urine co llection, sho uld elicit differentia l d iagnostic testing (for exa mp le, for purine disorde rs. glycoge neses. t3·oxidation diseases. and lactic acidosi s). Th e pathobiochemistry and relation ships of genotypes to bioche mica l and clinical pheno types of purine and pyrim idine disorde rs rem ain poorly unde rstood despite the prog ress in ident ifying new e ntities dur ing past decades. Nothing is known abo ut the mechanism of induction of severe central nervous system symptoms. We sugges t that all patients who have psyc homotor retardation. seiz ures. or muscle wasting

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Mayo C lio Proc , Augu st 1997, Vol 72

LARORATORY DIAGNOSIS OF PURINE AND PYRIMIDINE DISORDERS

and hypoton ia sho uld be scree ned by a pane l of tests, includ ing stud ies of purines and pyrimid ines. Reasonable approaches are either ( I) to sc ree n for purine and pyrimid ine disorders afte r e limina ting other metabolic di sord ers as d iagnostic possibilities (van Gennip AH. Personal co mm unication) (bea ring in mind ex istence of variants that can shift the age at mani festation 10 later decades of life) or (2) to sc ree n specimens se nt to the metabol ic labo ratories for " screening for metabolic disorders." Indeed , both gro ups represent a high ly skewed sample of patients with substantial pro babil ity of ncc urrcnce of metabolic path ologic co nditions . In fact , we recenll y identified the first US patient with adenyin. succ inate lyase.de ficiency by ana lys is of a urine spec imen submitted for sc ree ning for inborn errors of metabolism ."

ACKNOWLEDGMt:NT

12.

13.

14.

IS.

16.

Urine speci me ns from patient s with purine and pyrim idine disorders were kindly made avai lable tn us by Dr. A. H. van Ge nnip (di hydro pyrimidine deh ydrogenase), Dr. G. Van den Berghe (adeny losucc inate lyase), and Dr. J. Krijt (hypoxanthine o r gua n ine ph osp ho ribo syltran sfer ase , ade n ine phosph oribosyltransfcrase, purine nucl eosid e phosphorylase, oro iate phosphori bo syltransferase, and xanthine o xidase de ficiency).

19.

R EFE R EN C F.~

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I.

2. 3. 4. 5.

6. 7.

8.

9. 10.

I I.

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