monitoring as a diagnostictechnique in invasive candidiasis

Vol. 12, No. 4JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1980, p. 594-6020095-1 137/80/10-0594/09$02.00/0

Quantification of Arabinitol in Serum by Selected IonMonitoring as a Diagnostic Technique in Invasive Candidiasis

JOHN ROBOZ,* ROBERT SUZUKI, AND JAMES F. HOLLANDDepartment of Neoplastie Diseases, The Mount Sinai School of Medicine, City Unitersity of Neu, York, New

York, Neu, York 10029

D-Arabinitol was identified by mass spectrometry as a metabolite of Candidaalbicans and Candida tropicalis. For quantification, serum was deproteinizedwith acetone, the supernatant was evaporated to dryness, the silyl derivative wasformed, and a portion was injected into a combined gas chromatograph-massspectrometer system. Erythritol or 2-deoxy-galactitol was the internal standard.The protonated molecular ions, obtained in chemical ionization with isobutane,were monitored. For 39 normal subjects the mean endogenous arabinitol concen-tration was 0.52 ig/ml (standard deviation, +0.34). An increment of 0.2 ig ofarabinitol per ml could be quantified. Of 11 cases with diagnosed invasivecandidiasis, 9 had arabinitol levels >1.2 ,ig/ml (8 tM) in the range of 1.2 to 25.0kg/ml; the remaining 2 cases had levels in the normal range. Six cases of diagnosedcolonized candidiasis showed normal arabinitol levels.

Infection as a contributory cause of death incancer patients may reach as high as 50% de-pending on the type of underlying disease (5).Fungal infection occurs at some time in 10 to25% of all cancer patients. It has been reportedthat fungal septicemia in patients with acuteleukemia has increased from 2 to 6%c during thelast 5 years (3). In a recent study of 42 autopsiesperformed on leukemia and lymphoma patients,severe fungal superinfections, predominantlycandidiasis and aspergillosis, were found in 52%;most of the patients were not diagnosed andhence were not treated antemortem (11). Hos-pital-acquired fungemia also appears to be onthe increase in patients with a variety of diseasesand who have undergone surgical procedures,due to the use of antibiotics, indwelling cathe-ters, and parenteral hyperalimentation (6).

Episodes of fungemia often resolve sponta-neously without the need of potentially toxicantifungal therapy (2). In immunosuppressedpatients, however, fungemia is usually not tran-sient and is often invasive in nature. By invasivewe mean the infiltration and colonization of themicroorganisms into normally sterile deep-seated tissues and organs. Probably as many as90%k of all invasive Candida infections are notdiagnosed early enough to permit effective treat-ment. Reasons for the lack of recognition includethe protean nature of symptoms as well as thelack of rapid and reliable diagnostic tests. In arecent review of the available diagnostic tech-niques (10) the authors concluded that "tradi-tional cultural and serological determinations ofantibody response to Candida have not been

satisfactory for the diagnosis of invasive Can-dida infection, particularly in the host with com-promised resistance." Conventional blood cul-tures were reported positive in 50 to 70% of caseswith disseminated Candida infection (7). How-ever, positive blood cultures are not always di-agnostic of invasive disease because they mayresult from transient candidemia or from con-tamination of needles. Also, often the Candidaorganisms cannot be recovered (cultured) fromthe bloodstream of patients with deep-seatedinvasion.

Miller et al. (8) reported a characteristic peakin the gas chromatographic profiles of extractsfrom patients with candidiasis. The peak wastentatively identified as mannose, presumablyoriginating from the mannan content of the cellwall of Candida. In another approach, Weinerand Yount (13) used hemagglutination inhibi-tion to detect mannan in human serum; in seracollected serially, mannan antigenemia was de-tected early in the course of systemic infectionin only 4 out of 14 patients.The objective of this paper is to present meth-

odology for the quantification of arabinitol inthe serum of patients suspected of having inva-sive Candida infection. Arabinitol has beenshown to be a metabolite of Candida albicansand Candida tropicalis, and elevated levels ap-pear to be associated with invasive candidiasis.The technique described is based on mass-spec-trometric monitoring of selected ions of arabin-itol and an internal standard. This approach hasthe advantages of inherent specificity for arabin-itol as well as a capability to reliably quantify

594

ARABINITOL IN CANDIDIASIS 595

small increments in concentration. Concurrentlywith and independently from the present work,Kiehn et al. have developed a gas-chromato-graphic technique for the quantification of ara-binitol in serum as a potential diagnostic tool forsystemic candidiasis (4). The conclusions of ourwork are similar to those in their paper. Theadvantages of the mass-spectrometric approachare herein discussed.

MATERIALS AND METHODSChemicals and reagents. D-Arabinitol (arabitol),

i-erythritol (meso-erythritol), D-adonitol, and D-xylitolwere purchased from Sigma Chemical Co. (St. Louis,Mo.). The silylating reagent N,O-(bis)trimethyl-silyltrifluoroacetamide + 1% trimethylchlorosilane(BSTFA + 1% TMCS), silylation grade pyridine, andacetic anhydride were purchased from Pierce Chemi-cal Co. (Rockford, Ill.). Gas chromatographic columnmaterials were from Supelco, Inc. (Bellefonte, Pa.). Ailsolvents were of "distilled-in-glass" quality (Burdick& Jackson Laboratories, Muskegon, Mich.). Gas chro-matographic carrier and chemical ionization reagentgases were of "high-purity" grade (Matheson Scien-tific, Inc., Rutherford, N.J.). Culture media and relatedmaterials were purchased from BBL MicrobiologySystems (Cockeysville, Md.). Membrane filters (0.45um) were from Millipore Corp. (Bedford, Mass.).

2-Deoxy-galactitol, one of the internal standardsused, was synthesized from 2-deoxy-galactose (SigmaChemical Co.) by NaBH4 reduction, as follows. Thesugar was reacted with a 4 M excess of NaBH4 inaqueous solution for 1 h at room temperature. Thereaction was stopped by the dropwise addition ofacetic acid (50%) until no more hydrogen gas wasevolved. The solution was evaporated with nitrogen tonear dryness, and 1 volume of 20% acetic acid inmethanol was added. The solution was reevaporated,and the procedure was repeated three more times toensure the removal of excess borate. Finally, the so-lution was evaporated to dryness, and a stock solution(in 10% methanol-90% distilled water) was prepared.Purity was checked by forming the O-methyl-oxime-trimethylsilyl derivative to prove that no unreacted 2-deoxy-galactose remained and also by forming thepentaacetate derivative to prove that the total amountof sugar impurity present was <5%.Candida cultures. C. albicans and C. tropicalis

were isolated from clinical specimens submitted to themicrobiology laboratory of Mount Sinai Hospital.Identification was based on sugar fermentation andassimilation studies and chlamydospore formation oncorn-meal Tween-80 agar. Stock cultures were subcul-tured weekly on beef heart infusion agar and main-tained at room temperature. Cultures were grown inyeast nitrogen base broth supplemented with 1% dex-trose. The broth was prepared according to the man-ufacturer's instruction by dissolving 6.7 g of yeastnitrogen base and 10 g of dextrose in 10 ml of steriledistilled water, followed by gentle stirring and heatinguntil dissolved. The solution was filter sterilized andstored at 4°C. Before use a portion was diluted 1:10 insterile distilled water. A 5-ml portion of this medium

was inoculated with single-colony isolates. The cul-tures were grown unshaken at 37°C for 18 h. Forgrowing Candida species in human serum, pooledserum was first heat inactivated at 56°C for 30 minand then inoculated and incubated as described above.After incubation, the bulk of the culture was removedby centrifuging at 1,500 x g for 10 min. For removingany residual organisms, the supernatant was filteredthrough a 0.45-gm membrane filter.

For determining the number of organisms per mil-liliter of medium, samples from the cultures werediluted 1:10' with normal saline, and a 0.1-ml portionwas plated on 5% sheep blood agar. After incubatingat 37°C for 24 h, the colonies were counted, and thenumber of yeast cells per milliliter of medium wascalculated.

Cell hydrolysis. A 1-volume amount each (typi-cally 0.2 ml) of fungal cells, cell-containing serum, orfiltered (cell-free) serum was diluted with 1 volume of8 N methanolic hydrochloric acid and hydrolyzed in asealed container at 80°C for 60 min. After neutralizingwith solid K2CO:i to pH 6 to 7, the liquid was trans-ferred into a Corex tube with the aid of 2 volumes ofmethanol and centrifuged at 27,000 x g at 0°C for 15min. The supernatant was decanted and evaporatedto dryness with dry nitrogen in a water bath kept at50°C. The dry residue was silylated as described in thesection on sample preparation. The pellet remainingafter centrifugation was discarded.Computer searching for characteristic metab-

olites. A simple computer program was developed forthe point-by-point comparison of the elution profilesobtained by collecting complete mass spectra of allchromatographically separated components from nor-mal serum and a serum in which Candida was grown.The program compared each mass in the two profileson a segment-by-segment (peak-width) basis by sum-ming the respective ion intensities and taking theirratios. When the ratio exceeded a user selected value(threshold), the location (scan number range) and thenumerical value of the ratio were printed out.

Standards and calibration samples. Both eryth-ritol and 2-deoxy-galactitol were used as internalstandards. The pure compounds were dissolved inwater-methanol (9:1) to provide solutions of knownconcentrations. An adequate amount of internal stand-ard was added to yield a final concentration of 1 to 2gg of either sugar or both sugars per ml of serum.

For establishing optimal analytical conditions bothfor sample preparation and also for chromatographicseparation, pooled normal serum samples were spikedwith known quantities of arabinitol and the internalstandards. For obtaining calibration curves for quan-tification, samples containing increasing quantities ofarabinitol and a fixed amount of internal standardwere prepared. The concentration range of the cali-bration samples covered the entire range of concentra-tions expected in the samples from the patients. Theamount of internal standard added was the same forboth calibration samples and samples from the pa-tients. A full set of calibration samples was analyzedwith every set of samples from the patients.

Preparation of serum samples. Blood sampleswere drawn into a tube containing no heparin or

VOL. 12, 1980

596 ROBOZ, SUZUKI, AND HOLLAND

preservative. Serum was obtained by letting wholeblood clot at room temperature for 20 to 25 min,followed by centrifugation at ca. 500 x g at roomtemperature for 10 min. Serum samples were stored at-20°C until used.TMS technique. The trimethylsilyl (TMS) tech-

nique was performed as follows. To a 0.2-ml sample ina glass test tube (10 by 75 mm) the internal standardswere added and mixed by fast blending in a Vortexmixer for 15 s. For precipitating proteins, 1 ml ofacetone was added, and the tube was centrifuged at1,200 x g for 15 min at room temperature. The super-natant was removed from the tightly packed precipi-tate and evaporated to dryness in a small screw-capvial with the aid of dry nitrogen, in a water bath keptat 500C.The solid residue was silylated by adding 200 il of

BSTFA + 1f7 TMCS-pyridine mixture (3:1) and heat-ing at 100°C in a metal heating block for 5 min. Thesilylation vial was sealed tightly with a Teflon-faceddisk. A 4-jl portion was injected into the gas chro-matograph-mass spectrometer system.

Pentaacetate technique. To a serum sample of0.2 to 2.0 ml, 4 volumes of acetone were added, andthe precipitated proteins were removed by centrifug-ing at 1,200 x g for 15 min at room temperature. Thesupernatant was evaporated (dry nitrogen) to approx-imately 0.2 ml and diluted with 0.8 ml of distilledwater. After acidifying with 2 drops of 6 N HCl, lipidswere extracted twice with 3 ml of ethyl acetate, andthe aqueous layer was evaporated under vacuum. Theresidue was acetylated with acetic anhydride-pyridine(2:1 by volume) for 2 h at 600C in a metal heatingblock, the reaction was stopped by adding 1 ml ofdistilled water, and the acetates were extracted threetimes with 2 ml of hexane-chloroform (3:1 by volume).The organic extract was backwashed twice with 2 mlof distilled water, dried with solid Na2SO4, and evap-orated to dryness with nitrogen gas. The residue wasdissolved in 100 1d of hexane, and a 4-ml portion wasinjected into. the gas chromatograph-mass spectrom-eter system.Gas chromatography-mass spectrometry. Ail

quantification work was carried out with a combinedgas chromatograph-mass spectrometer-computer sys-tem (model 3300 quadrupole-type mass analyzer andmodel 6000 computer; Finnigan Corp., Sunnyvale,Calif.) equipped with a chemical ionization source andcomputer capability for selected ion monitoring. High-precision mass measurements were made with a com-bined gas chromatograph-mass spectrometer-com-puter system (model MM 70-70 magnetic double-fo-cusing mass analyzer and model DS 2250 data system;VG-Organic, Ltd., Altrincham, Cheshire, England).

In the TMS technique, a glass column (inside di-ameter, 1 m by 2 mm) filled with 3% OV-17 (80%methyl-20% phenyl silicone gum; Ohio Valley Spe-cialty Chemical, Inc., Marietta, Ohio) on ChromosorbW HP (80/100 mesh) was employed for chromato-graphic separation. Depending on which internalstandard was used, the column was operated isother-mally at 200°C (2-deoxy-galactose) or 175°C (erythri-tol). The injector temperature was kept at 2500C.Trapped endogenous constituents from serum sampleswere removed periodically by heating the column to

J. ClIN. MICROBIOL.

250°C and keeping it at that temperature until nomore effluent could be detected. The sample port wascleaned after every 20 to 25 analyses by replacing 1 to2 cm of column material from the top of the column(dark deposits).

In the acetate technique, a glass column (insidediameter, 1.5 m by 2 mm) filled with 3% SP 2340 (75%cyanopropyl-25'/ methyl silicone gum; Supelco, Inc.)(100/120 mesh) was employed isothermally at 220°C;the injector temperature was 250°C.

Isobutane was used both as the gas-chromato-graphic carrier gas and as the reagent gas in thechemical ionization source. There was no separatorbetween the chromatograph and the ion source; theconnecting tube was kept at 240'C. The pressure ofisobutane in the ionization source was adjusted toyield the highest intensity for the m/e = 414 peak ofperfluorotributylamine.

Operational parameters of the mass spectrometerwere adjusted daily for maximum sensitivity at a res-olution of about 600 with the TMS derivative of purearabinitol. The emission current was 0.4 to 0.8 mA,and the ionization energy was 100 eV. The electronmultiplier was operated with 3 kV on the conversiondynode and 1.2 kV on the amplification stage; thepreamplifier was set at 10-' A/V. Mass spectra foridentification were obtained in the full-scanning mode.For quantification the selected ion-monitoring modewas employed. The effluent was vented for 30 s toavoid contamination of the ion source by the excesssilylation reagent and pyridine.The following masses were monitored in the TMS

technique: m/e = 513 (arabinitol), m/e = 411 (eryth-ritol), and m/e = 527 (2-deoxy-galactitol). Pentaace-tates were analyzed by monitoring the base peak (M- 59) at m/e = 303.The high-resolution mass-spectrometer system uti-

lized gas-chromatographic columns and analyticalconditions similar to those described above. Heliumserved as the chromatographic carrier gas; it was re-moved with a heated glass jet separator. For chemicalionization, the mass spectrometer ion source was pres-surized directly with isobutane. The scan cycle was 2.4s/decade. The ion acceleration voltage was 4 kV, andthe resolution was set to ca. 10,000. Mass measure-ments were made both bv manual peak matching andwith the aid of the computer.

RESULTS AND DISCUSSIONIdentification of arabinitol. Because inva-

sive Candida infections are often present whenthe organisms cannot be recovered from bloodcultures, a metabolite which could be detectedin blood serum was sought as a sensitive markerthat might indicate Candida infection. Accord-ingly, both C. albicans and C. tropicalis weregrown in human serum for 18 h, cells wereremoved from the sample, and the remainingserum was filtered (0.45-pm membrane) andtreated as described above. Figure lA shows thetotal ion current profile (reconstructed gas-chro-matographic profile) of a serum sample in whichC. albicans was grown. These profiles are nor-


-jZw

Fh.z

-J

w

A

ARABINITOL

b.... âh, .... oia' .... 5 20b 2SScon No.

Ion B

-ARABINITOL

A-'l t.50 I 15b 201bZ 250

Scon No.

FIG. 1. (A) Reconstructed chromatogram profile(total ion monitoring) of human serum in which C.albicans was grown. (B) Mass chromatogram ofm/e= 513 ofA. Rel. int., Relative intensity.

malized plots of the sum of the ion abundancesin each mass spectral scan as a function of thescan number; i.e., every scan represents a com-

plete mass spectrum which is stored in memory.The profile shown in Fig. 1A could not be vis-ually distinguished from that of a normal serum

sample. However, computer comparison re-

vealed a significant difference for mass 513 inthe area of scan 30. The basis of the computercomparison was the matching of individualmasses recalled from the full mass spectra(known as mass chromatograms). The peakaround scan 30 in the mass chromatogram formass 513 (Fig. 1B) of the infected serum samplewas several hundred times larger than that ofthe control serum. The magnitude of this com-

ponent appeared dependent upon the number ofCandida organisms present.When the complete mass spectrum corre-

sponding to scan 30 was recalled (Fig. 2A), theabundance of mass 513 was the highest in thespectrum (base peak). This mass spectrum was

identical to that of pure (silylated) arabinitol(Fig. 2B); here mass (m/e = mass/charge ratio)is plotted against normalized abundance. Thebase peak corresponds to the protonated molec-ular ion of arabinitol, with the uptake of fiveTMS groups by the molecule. The small peak at

m/e = 423 corresponds to the loss of an 0-trimethylsilyl group.

Figure 3 shows a total ion current profile ofhydrolyzed and silylated C. albicans cells. Ap-proximately 107 ceils were grown, the cells wereremoved by centrifugation, and the cell pelletwas hydrolyzed and subsequently silylated.When scan 63 was recalled from the computermemory and corrected for background, the massspectrum was identical to that of pure arabinitol(Fig. 2). The other peaks in Fig. 3 were notidentified because they either did not appear inthe serum after the organisms were removed orcould not be separated from interfering endoge-nous serum constituents.Because the m/e = 513 peak was also detected

in all control samples, identification by high-precision mass measurement was warranted.The peak assumed to be endogenous arabinitolgave a precise mass of 513.272, corresponding tothe molecular composition of C20H5305Si5, whichis the formula of the protonated silyl derivativeof arabinitol. The mass measurement (averageof seven scans) was within 1.9 millimass units ofthe mass calculated from the formula. The samemass and formula were obtained for the com-pound originating from Candida grown in serumas well as for the one obtained directly from themicroorganism upon hydrolysis. Arabinitol wasfound to be a metabolite of both C. albicans andC. tropicalis.

Figure 4 shows the monitoring of arabinitollevels in two samples from a patient with acutemyelocytic leukemia. Here only the protonatedmolecular ions of arabinitol and erythritol weremonitored to achieve maximum sensitivity(while retaining specificity). The sample shownin Fig. 4A exhibits a normal endogenous arabin-itol level, corresponding to 0.7 ,tg/ml. Figure 4Bshows an increase in the level to 2.5 ,ug/ml,coinciding with the diagnosis of invasive candi-diasis based upon positive culture from a liverbiopsy specimen.

Because the TMS technique does not fullyresolve arabinitol and the two other pentitols,adonitol (ribitol) and xylitol, a technique basedon the pentaacetate derivatives was also devel-oped. The chemical ionization (isobutane) massspectrum of the pentaacetate of arabinitol (Fig.5) yielded a detectable but weak (M + 1)+ ion.The base peak was the (M - 59)+ ion at m/e =303, corresponding to the loss of a CH3C00group. Figure 6A shows chromatographic sepa-ration for equal amounts of the three pentitolsby monitoring the m/e = 303 ions. Figure 6Bshows the monitoring of pentitols in the serumof a patient with disseminated C. albicans infec-tion. The levels of adonitol and xylitol were<10% that of arabinitol in the majority of normal

VOL. 12, 1980


z

il

a u

A

40hm/c

4b

(M+l)+513

TMSOC-4-1TMSOC-H

TMSO-C-HF+4-OTMS

H~-y--OTIMSH

- -

1. . -b .

m/eEXACT MA1SS - 513.27212 C20'%3% 9S

FIG. 2. (A) Mass spectrum (isobutane) of scan 30. (B) Mass spectrum (chemical ionization with isobutane)of the TMS derivative of authentic arabinitol. Rel. int., Relative intensity.

,On

ARABINITOLz

w ,,.,.,.,,,.,,,., ..........,...,,,...,...,..............11, ,è5 t~00 t8 0 250I 300 35

Scon No.

FIG. 3. Reconstructed chromatogram profile of hydrolyzed C. albicans. Rel. int., Relative intensity.

samples as well as in those from infected pa-tients. On a few occasions the level of adonitolwas as high as 50% that of arabinitol. We havenot found any evidence that adonitol or xylitolwas a metabolite of Candida species grown inhuman serum.

The TMS technique is considerably faster (12patient samples together with 6 calibration sam-ples can be analyzed by a technician in 7 h),

simpler, and more sensitive than the one basedon the separation of pentaacetates. All levelsquoted for arabinitol by the TMS techniquemight include contributions from endogenousadonitol or xylitol or both. In case of doubt or

when additional confirmation is warranted, therelative quantities of all three pentitols may bedetermined with the pentaacetate technique.The possibility that arabinitol might also be

a byCLI B

(M+I)t513

z

-J.w

Cr1

.b11 1

J. CLIN. MICROBIOL.

1


A B10o

m/e 513

ARABINITOL ARABINITOLw

zw'00-w ~~~~~~~~~~~~m/e411

INT. STD. INT. STD.

4

0 1l6 200 300 0 106 200 301SCAN NUMBER

FIG. 4. (A) Single ion monitoring of m/e = 513 (arabinitol) and m/e = 411 (erythritol, internal standard[int. std.] in a leukemic patient without infection (normal range). (B) Data for the same patient as in A at thetime ofdiagnosed (liver biopsy) invasive candidiasis.

100

(M-59) 303ACO-C-H

z AcO-141

W 363oe ^(Mi.H)+

t 5b 20à 25' 30à 350m/6

FIG. 5. Mass spectrum (chemical ionization with isobutane) of the pentaacetate derivative of authenticarabinitol. Rel. int., Relative intensity.

AAdonitol m/e 303

Arobinitol

e /Ét B m/e503

240 300 400 500 600 700 80Scon No.

FIG. 6. (A) Single-ion monitoring of m/e = 303 (M -59 peak) of the pentaacetates of adonitol, arabinitol,and xylitol in a calibration mixture. (B) Single-ion monitoring of the pentaacetates in the serum of a patientwith invasive candidiasis.

VOL. 12, 1980


a metabolite of other microorganisms which fre-quently infect immunosuppressed patients wasalso investigated with the computer program tocompare profiles of normal serum and of serumsamples in which Aspergillus fumigatus, Asper-gillus niger, Pseudomonas aeruginosa, Klebsi-ella pneumoniae, and Escherichia coli weregrown. No detectable differences were foundbetween normal and infected serum sampleswith any of these organisms. Other relevantorganisms (e.g., Cryptococcus, Histoplasma) arebeing investigated.

Speculations about the biochemistry of theformation of arabinitol as a metabolite of Can-dida are beyond the scope of the present work.Based upon studies on the utilization of sugarsby yeasts (1), a possible route of the catabolismof L-arabinose to L-arabinitol by the Candidaorganism may take place with the aid of nicotin-amide adenine dinucleotide phosphate and adehydrogenase (12). Arabinose was not presentin the serum in which Candida species weregrown. It is presumed that the organism makesand excretes arabinitol from unknown precur-sors. Another, though less likely, explanation forthe presence of arabinitol may be that arabinitolfrom dead and decomposing cells of Candida iscontinuously washed out into the bloodstreamfrom the site of the primary infection.

Quantification. Erythritol and 2-deoxy-ga-lactitol were selected as internal standards be-cause their extraction, chromatographic, andmass spectral properties closely parallel those ofarabinitol. The mass spectra of the silylatedinternal standards exhibited base peaks at m/e= 411 and m/e = 527, respectively, correspond-ing to the protonated molecular ions. Eitherinternal standard was adequate; in more recentwork 2-deoxy-galactitol was preferentially em-ployed because of more convenient chromato-graphic behavior. In agreement with an earlierreport (9), relatively large amounts of erythritolhave been found in patients with renal failure.2-Deoxy-galactitol levels were not elevated inpatients with renal failure; however, other inter-ference peaks of high abundance did appear.When interference with the internal standardwere severe, quantification of arabinitol wasmade with the technique of external calibration(i.e., determining arabinitol levels in the samplesfrom patients by comparing measured areas tothose obtained upon separately injecting knownquantities of pure derivatized arabinitol).A calibration curve is shown in Fig. 7. This

was obtained by adding increasing amounts ofarabinitol to a serum sample while keeping theconcentration of the internal standard fixed. Ev-ery point on this curve corresponds to the aver-

J. CLIN. MICROBIOL.

age of four independent samples. The coeffi-cients of variation for all points were in the rangeof 3.9 to 9.4%. Other reproducibility sets yieldedcomparable ranges. Regression analysis gave acorrelation coefficient of 0.998.

Extrapolation of the calibration curve pro-vides a value for the endogenous arabinitol inthe sample when normal serum is used to estab-lish the calibration curve. When an adequateamount of a serum sample from a patient isavailable, it is advantageous to use that sampleas the medium to which known amounts ofarabinitol are added (standard addition tech-nique). This approach, although obviously en-tailing more work, provides more reliable datafor patients with serum having interfering sub-stances due to renal failure or some other cause.When large increases occur in the arabinitol

level above the level of endogenous arabinitol,gas chromatography is adequate. However, largechanges are likely to be associated only withvery advanced disease. For providing as early anindication of infection as possible, small incre-mental changes must be determined. In conven-tional gas chromatography identification isbased only on retention time measurements. Itis also difficult to quantify increments of lessthan a factor of two among the ever present"chemical-noise" peaks. In a series of runs withserum spiked with arabinitol in increments of0.2 tg/ml in the range of 0 to 1.0 ,ug/ml (withfour replicates at each point) we determinedthat an increment of 0.2 ,ug/ml (1.35 ,M) can bedetected with selected ion monitoring. Becauseonly preselected peaks are monitored, specificityfor arabinitol is retained, and, at the same time,the presence of chemical-noise peaks is irrele-vant. As shown in Fig. 4, the peak correspondingto the protonated molecular ion of arabinitol (atm/e = 513) is easily seen, and its area can bemeasured by the computer without interferenceor contribution from chemical noise that mayoccur at the same chromatographic retentiontime as arabinitol.

Clinical applications. Normal endogenousarabinitol levels were determined for 39 subjects(ages 16 to 59). The mean arabinitol level was0.52 ,ug/ml (standard deviation, ±0.34). Whenplotted, the curve approximated normal distri-bution. On this basis it was estimated that thereis only a 2.6% probability that an arabinitol levelof 1.2,ug/ml (8,M) is normal.Of 11 cases with diagnosed (autopsy or posi-

tive blood culture) invasive candidiasis (C. al-bicans), 9 had arabinitol levels -1.2 ig/ml(range 1.2 to 25.0 ,ug/ml); the remaining 2 caseshad levels in the normal range. Six cases oforopharyngeal or vaginal candidiasis without


ArabinitolInt. Std. 2.0

1.0 Regression

r=0.998

Endog. Arabinitol0.5811g/ml

1.0 Q5 0 0.5 1.0 I.5 20UÀg/ml added Arabinitol

FIG. 7. Calibration curve for the quantification of arabinitol; normal pooled serum or the patient's ownserum is spiked. Extrapolation provides endogenous (Endog.) arabinitol. For quantification of other samplesendogenous arabinitol was subtracted so that the curvepassed through the origin. Int. std., internal standard.

clinical findings indicative of invasion revealedarabinitol levels within the normal range.The arabinitol levels of one patient with acute

myelocytic leukemia were monitored for a pe-riod of 1 year. Arabinitol levels increased fromnormal to 2.5 ,g/ml. Maximum levels coincidedwith the diagnosis of candidiasis based on liverbiopsy cultures. After the initiation of antifungaltherapy, arabinitol levels remained relativelyhigh for several weeks, followed by a slow de-crease to normal.We have found that severe (requiring dialysis)

kidney dysfunction can result in elevated serumarabinitol levels, leading to a false diagnosis ofCandida infection. This was observed with pa-tients having cancer, as well as with patientshaving other underlying diseases. Such casescould often be detected by strong interferencein the monitoring of the internal standard (2-deoxy-galactitol). We are currently investigatingthe relationship between deteriorating kidneyfunction and serum arabinitol levels to deter-mine how serum creatinine levels might be uti-lized to differentially diagnose Candida infec-tion in patients with kidney dysfunction.

Early diagnosis of invasive candidiasis is es-sential for effective treatment. The selected ionmonitoring technique described here permits themonitoring of blood levels for precise measure-ment of small incremental changes in arabinitolcontent which might provide a biochemical di-agnosis of otherwise undiagnosable invasive can-didal disease. The technique is sufficiently spe-cific and sensitive to justify serial sampling inpatients at high risk in an attempt to diagnoseinvasive candidiasis before clinical signs appear.Our data do not yet allow more than a gross

correlation between arabinitol concentrationand the extent, location, or intensity of Candidainfection. Similarly, prognostic connotations ofarabinitol concentration with respect to sponta-neous cure, therapeutic response to antifungalagents, and survival have not yet been estab-lished. Studies of this nature are currently inprogress.

ACKNOWLEDGMENTSThis work was partially supported by Public Health Service

grant CA-15936 from the National Cancer Institute and by theT. J. Martell Memorial Foundation for Leukemia Research.We gratefully acknowledge the contributions of Kuldip

Sandhu and E. Bottone, who prepared microorganisms andconsulted on the microbiological aspects of the work; GenieBeer, who prepared samples and operated the mass spectrom-eter; and D. Millington (VG-Organic, Ltd.), who assisted inobtaining precise mass measurements. Special thanks are dueto D. Arrnstrong, E. Bernard (Memorial-Sloan Kettering Can-cer Center), J. Giron, and J. Cuttner (Mount Sinai School ofMedicine), who provided samples from patients.

LITERATURE CITED1. Barnett, J. 1976. The utilization of sugars by yeasts. Adv.

Carbohydr. Chem. Biochem. 32:125-170.2. Editorial. 1971. Candidiasis: colonization vs infection. J.

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