Life Sciences, Vol. 32, pp. 787-794 Pergamon Press Printed in the U.S.A.

URINARY PROFILES OF VOLATILE AND ACID METABOLITES IN GERMFREE AND CONVENTIONAL RATS

Mary Holland, Gerald Rhodes, Mark DalleAve, Donald Wiesler and Milos Novotny

Dept. of Chemistry, Indiana University Bloomington, IN 47403

(Received in final form November 4, 1982)

Summary

Qualitative and quantitive differences in the urinary excre- tion of volatile and acidic metabolites in germfree and con- ventional rats were examined by capillary gas chromatography and gas chromatography/mass spectrometry. A number of carbonyl compounds, including several short-chain aliphatic ketones and acetophenone , were higher in the conventional urines, while many heterocyclic compounds (furan derivatives, benzothiazole and others) were lower. Both qualitative and quantitative differences were observed in the urinary ex- cretion of acidic metabolites. Three meta-hydroxy phenolic acids appeared only in the conventional rat urines, while levels of many other aromatic and aliphatic acids were also higher.

I t i s w ide ly known t h a t t he g a s t r o i n t e s t i n a l m i c r o f l o r a can d r a m a t i c a l l y a l t e r t he metabol ism o f numerous compounds, y i e l d i n g unique m e t a b o l i t e s which may then be observed in the u r i n e or f e c e s o f expe r imen ta l animals (1) . In a d d i t i o n , c e r t a i n changes in the m e t a b o l i c s t a t e o f an organism, such as s t a r v a t i o n , cause a l t e r a t i o n s in the m i c r o f l o r a which a re in t u r n r e f l e c t e d by changes in the normal c o n s t i t u e n t s o f p h y s i o l o g i c a l f l u i d s (2) . Consequent ly , in m e t a b o l i c p r o f i l i n g s t u d i e s i t i s d e s i r a b l e to l e a r n which m e t a b o l i t e s may or may not be a s s o c i a t e d wi th the a c t i o n o f i n t e s t i n a l b a c t e r i a .

A c o n s i d e r a b l e amount o f r e s e a r c h has been c a r r i e d out conce rn ing the metabol ism of s p e c i f i c f o r e i g n compounds and c l a s s e s o f compounds by i n - t e s t i n a l microorganisms . Ea r ly s t u d i e s by S j o v a l l e t a l . i n d i c a t e d the impor tance o f d e h y d r o x y l a t i o n r e a c t i o n s o f the i n t e s t i n a l b a c t e r i a in the metabol ism o f b i l e a c i d s (3 ,4 ) . Examples o f o t h e r i n t e r a c t i o n s a re the h y d r o l y s i s o f g l y c o s i d e s ( i n c l u d i n g g l u c u r o n i d e s ) , s u l f a t e e s t e r s , amide e s t e r s , s u l f a m a t e s , and n i t r a t e s ( 1 , 5 , 6 ) . Compounds may a l s o be modi f i ed through d e h y d r o x y l a t i o n , deamina t ion , h e t e r o c y c l i c r i n g f i s s i o n , and numerous r e d u c t i o n r e a c t i o n s t h a t mammalian enzymes may not perform (1 ,5 ) . Also , c e r t a i n p roduc t s o f m i c r o b i a l a c t i o n may subsequen t ly be modi f i ed by mammalian enzymes, g i v i n g a d d i t i o n a l m e t a b o l i t e s . These v a r i o u s r e a c t i o n p roduc t s can then be absorbed or r eabsorbed from the i n t e s t i n e , e n t e r t he gene ra l c i r c u l t a i o n , and be c l e a r e d i n t o the u r i n e . Prominent examples o f t h e s e i n t e r a c t i o n s a re e v i d e n t in the p u b l i s h e d i n v e s t i g a t i o n s on the appearance o f c h a r a c t e r i s t i c p h e n o l i c a c id s in the u r i n e through the m o d i f i c a t i o n o f c a f f e i c a c i d , c innamic a c i d , and s i m i l a r s u b s t r a t e s by m i c r o b i a l enzymes ( 1 , 7 , 8 ) .

0024-3205/83/070787-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

788 Urine Metabolites of Germfree Rats Vol. 32, No. 7, 1983

The present study was designed to examine differences in the urinary excretion of a wide range of volatile and acidic metabolites in germfree and conventional rats. Numerous metabolites can now be followed simultaneously and quantitatively by the so-called metabolic profiling methods (9,10), which are essentially multicomponent analytical determinations performed through high-resolution chromatographic methods. Mass spectrometry is typi- cally used to identify or verify the individual profile components. This approach to studies of normal metabolites and those altered by disease states was pioneered by Evan Homing (ii), who coined the term "metabolic profile".

An increasing amount of research in metabolic profiling has recently been focussed on low-molecular-weight neutral (volatile) and acidic urinary constituents. Metabolic alterations in such compounds were found to be as- sociated with human diabetes (12,13) and other metabolic disorders (9,10). In addition, certain metabolic parallels have been observed in experimentally diabetic rats (14,15). As these compounds appear to be by-products and terminal products of a wide variety of metabolic processes, interpretation of profiling data is currently complicated by a lack of knowledge concerning their metabolic origin.

In order to clarify possible contribution of an intestinal flora to the urinary metabolic patterns of volatiles and acids, chromatographic pro- files of the urines from germfree and conventional animals were qualitatively and quantitiatively compared in this study. The urinary metabolites which appear to be primarily microbial in origin could then be identified.

Materials and Methods

Sample Collection: Germfree and conventional Sprague-Dawley male rats (8 animals in each group) were obtained at six weeks of age from Harlan In- dustries, Indianapolis, IN. The germfree animals were maintained in sterile isolators at the Harlan Industries facility throughout the experiment. All animals were supplied with the same diet (Lab-blox, Wayne Feeds, Bloomington, IN) and water ad libitum.

24-hour urine samples were collected from each germfree animal using polycarbonate metabolism units (Maryland Plastics, Federalsburg, MD) which had been autoclaved prior to sterile introduction into the isolator. Interim samples were kept frozen during collection by passing liquid nitrogen from an exterior tank through coils of copper tubing wound around the base of each urine collection cup. The nitrogen flowed out of the isolator through ad- ditional tubing, insuring no contact with the inner environment of the iso-

lator. Following urine collection, the germfree animals were removed from the isolator and immediately sacrificed and tested to verify their germfree status. 24-hour urine samples were collected from each of the conventional animals using the same polycarbonate metabolism units with the interim samples frozen over dry ice.

Prior to analysis, the urine samples were brought to room temperature, filtered and diluted to a standard volume with distilled, deionized water. The urines were then divided into suitable aliquots and quickly refrozen at -20°C until analyzed. No preservatives were used.

Analysis of Urinary Volatiles: Analysis of the volatile constituents of the urine samples was carried out using a headspace concentration method followed by capillary gas chromatography [14). In this method, volatile

Vol. 32, No. 7, 1983 Urine Metabolites of Germfree Rats 789

compounds are purged from the heated urine headspace with high-purity helium and adsorbed onto Tenax GC (2,5-diphenyl-p-phenylene oxide porous polymer from Applied Science Laboratories, State College, PA). The volatiles are then desorbed from the Tenax GC in the injection port of the gas chromato- graph and trapped at low temperature onto a short section of a capillary column prior to chromatographic separation. The general procedures and in- strumentation utilized for the acquisition of metabolic profiles have been previously described (14,16).

A glass capillary column (60 m x 0.25 mm i.d.) coated with UCON 20-HB- 2000 (Applied Science Laboratories) and containing 0.015% bis-triphenyl- phosphonium chloride as surfactant was employed for the analysis of volatile metabolites. The separations were carried out using a Perkin-Elmer 3920 gas chromatograph equipped with an automatic injector, flame ionization de- tector, mass-flow controller and a Perkin-Elmer PEP-2 data acquisition system, as previously described (14,16). All samples were run at a flow rate of 2 ml/min, and temperature programmed from 28-160°C as indicated in Figure i.

Identification of the volatile constituents was accomplished using the same capillary column in a Hewlett-Packard Model 5980A. combined gas chroma- tograph/dodecapole mass spectrometer computer system. Low resolution electron impact ionization spectra were obtained at 70 eV and a scan rate of 100 amu/sec. The chromatographic conditions were the same as above, with the capillary column directly interfaced with the mass spectrometer ion source (maintained at 220 ° C). Structural assignments were verified by a comparison of retention times and mass spectra from the authentic compounds whenever possible.

Analysis of Organic Acids: Urinary profiles of the organic acids were obtained using the following procedure. A 2 ml aliquot or urine was acid- ified (pH i) with 6N HCI and saturated with NaCI. I0 ~I of a 2 mg/ml solution of p-chlorobenzoic acid was added as an internal standard. Next, the urine was extracted twice with 4 ml of ethyl acetate and once with 4 ml of diethyl ether. The organic extracts were combined and dried for 30 minutes over an- hydrous Na2SO~, followed by evaporation to dryness with a stream of nitrogen gas.

Trimethylsilyl esters of the acids were formed by adding I00 ~I Regisil RC-3 (bis-trimethyltrifluoracetamide plus 10% trimethyl chlorosilane; Regis Chemicals, Rockford, IL), and heating in sealed vials for 60 minutes at 60°C. Following derivatization, 0.2 ~i sample aliquot was injected into the gas chromatograph (splitless injection).

The derivatized acids were chromatographed on a Varian Model 1400 gas chromatograph equipped with a flame ionization detector. Peak area and re- tention time data were recorded through the Perkin-Elmer PEP-2 data acquis- ition system. Separation of the acid derivatives was accomplished with a glass capillary column (30 m x 0.25 mm, i.d.), coated with 0.4% SE-50 methyl- silicone stationary phase (Applied Science Laboratories). Samples were run at a flow rate of 2 ml/min and temperature programmed from 50-260°C at 4°/min ~after an initial two minute isothermal period at 50°C.

Identification of the derivatized acids was carried out with the same SE-30 glass capillary column in the combined gas chromatograph/mass spectro- meter/computer system described previously. Chromatographic conditions were identical to those described above. Structural assignments were confirmed

790 Urine Metaholites of Germfree Rats Vol. 32, No. 7, 1983

TABLE I

VOLATILE METABOLITES OF GERMFREE AND CONVENTIONAL RAT URINE Concentrations Reported as Percent of the Germfree Value ± Standard Error

Peak

2 5 6 8 9

12 15 17 19 23 25

No. Compound Name Germfree Conventional/Germfree x 100

2-hexanone I00 ± 24 248 ± 39 4-heptanone i00 ± 13 1300 ± 38 2-heptanone i00 ± 37 3000 ± 37 5-hepten-2-one I00 ± 16 309 ± 36 2-methylthiofuran i00 ± 27 47 ± 32 5-methyl-2-hexenal i00 ± 44 1250 ± 56 4-nonanone i00 ± 16 260 ± 41 2-acetylfuran i00 ± 35 52 ± 41 N-acetylpyrrole I00 ± 26 39 ± 19 acetophenone i00 ± 27 1350 ± 35 benzothiazole i00 ± 42 40 ± 28

Peak

1 3 4 7

I0

Ii

*Tentative Identification

Compounds not significantly different in germfree vs. normal rat urine

No. Compound Name Peak No. Compound Name

dimethyldisulfide 13 4-methyl-3-penten-2-one 14

*a methylvinylfuran 16 methylpyrazine 18 3-hepten-2-one + 20 2,6-dimethylpyrazine 21 4,6-dimethylpyrimidine 22

24

2-heptenal 5-methyl-S-hexen-2-one furfural benzaldehyde 2-methylpyrrole 5-methylfurfural 2-methyl-N-acetylpyrrole 4-methylthio-2-butenal

~g

22

E

g

5 19 22 25

TEMP I°C) 3/. 90 g6 13L, 160-- : ~ i ' , ' ' I "I

FIG. I Urinary volatile chromatograms from conventional (A) and geznnfree(B) rats.

Vol. 32, No. 7, 1983 Urine Metabolites of Germfree Rats 791

through comparison of retention times and mass spectra of the authentic compounds as much as possible.

Results and Discussion

Volatile Compounds: Typical volatile metabolite profiles from germfree (bottom chromatogram) and conventional (top chromatogram) rats are compared in Figure i. Table 1 lists the identified metabolites, along with the peak numbers to which they correspond, and the average quantitative differences between the germfree and conventional urinary volatile metabolites. The values given indicate the average peak area value from the conventional pro- files as the percent of the average peak area for that metabolite in the germ- free profiles. Quantitative data are given only for those compounds which were judged significantly altered from germfree excretion levels (Students t-test, p < .05). Although headspace analysis methods do not allow direct absolute quantitation, the concentration of the individual urinary volatile metabolites lies in the range of 100-700 mg/24 hr. period.

No consistent qualitative differences were observed in the volatile fraction of germfree and conventional urine, however, several interesting quantitative differences were noted.

A number of heterocyclic compounds including benzothiazole, N-acetyl pyrrole, 2-methylthiofuran, 2-acetylfuran, furfural, and methylvinylfuran were found to be lower in the conventional rat urine profiles. It is in- teresting to note that heterocyclic ring fission by intestinal bacteria has been demonstrated in chemically diverse ring systems containing either nitrogen or oxygen (1,2). The decreased levels observed in the conventional urine indicate that the intestinal microflora may influence the degradation of these compounds.

Of additional interest is the considerably higher level of acetophenone in the conventional profiles. Acetophenone may arise from phenyl-B-oxopropion- ic acid, which is in turn formed from phenylpropionic acid and possibly cin- namic acid (17). The well documented role of the intestinal bacteria in the metabolism of cinnamic acid (i) indicates the involvement of the microflora in the production of acetophenone.

Several carbonyl compounds, containing from 6 to 9 carbon atoms per molecule, were significantly higher in the conventional rat urines. Ad- ditional carbonyl compounds, including 5-methyl-S-hexen-2-one and 4-methyl- thio-2-butenal showed small, though not statistically significant elevation. The data presented here indicate a general trend of higher concentration of these carbonyl compounds, implying that the intestinal microflora play some role in the production of such compounds. It is reasonable to assume that these carbonyls arise partially from the interaction of the various lactic acid bacteria with dietary fat and carbohydrates, since compounds such as 2-heptanone, 2-hexanone, and pentanal are known to be produced in vitro by numerous strains of bacteria through fat metabolism (19,20).

Organic Acids: Typical organic acid profiles from germfree (right chroma- togram) and conventional (left chromatogram) animals are shown in Figure 2. The identified metabolites are listed in Table II along with the peak numbers to which they correspond. The average quantitative differences are also given for the organic acid constituents which demonstrated significant (Student's t- test, p < .05) differences in the conventional profiles relative to the germ- free profiles. The data are presented in the same manner as those of Table I;

792 Urine Metabolites of Oermfree Rats Vol. 32, No. 7, 1983

TABLE II

ACIDIC METABOLITES OF GERMFREE AND CONVENTIONAL RAT URINE Values Reported as Percent of Germfree Value ± Standard Error

Peak No. Compound Name

2 lactate 8 3-hydroxy-3-methylbutyrate 9 benzoate

Ii succinate 12 2-methylsuccinate 13 fumarate 17 adipate + malate 20 phenyllactate 22 pimelate 27 p-hydroxyphenylpropionate 29 hippurate 30 isocitrate

Compounds not significantly different in

Peak No. Compound Name

3 2-hydroxyisobutyrate 4 glycolate

- 5 3-hydroxybutyrate 6 2-hydroxybutyrate 7 3-hydroxy-2-methylbutyrate

10 phosphate 15 glutarate

G e r m f r e e

I00 ± 18 I00 ± 26 i00 ± 20 i00 ± 21 1 0 0 ± 17 IO0 ± 21 100 ± 27 i00 ± 31 100 ± 12 1 0 0 ± 1S 100 ± 37 I00 ± 13

germfree

Peak No.

16 18 21 * 23 24 28 32

Conventional/germfree x i00

150 ± 20 358 ± 67 185 ± 22 194 ± 25 266 ± 26 191 ± 22 143 ± 33 169 ± 23 151 £ 13 814 ± 21 651 ± 41 74 ± 14

vs. conventional rat urines

Compound Name

3-methylglutaconate muconate

2-hydroxy-5-methylsuccinate p-hydroxybenzoate

p-hydroxyphenylacetate aconitate

p-hydroxycinnamate

Compounds identified only in urine of germfree animals: Peak #i phenol Peak #25*p-hydroxy-e-methylphenylacetate Peak #19 m-hydroxybenzoate Peak #26 m-hydroxyphenylacetate

Peak #31 m-hydroxycinnamate

*Tentative Identification

A

17

1." 15 " ~ ,,

: ] 32

I i ,

12

B

11 13 . 1011 I 1/.I/

12

3 6

28

~9

3o

FIG. 2 Urinary organic acid chromatograms from germfree (A) and conventional (B) rats.

Vol. 32, No 7, 1983 Urine Metabolites of Germfree Rats 793

the c o n v e n t i o n a l peak a rea ave rages a r e g iven as t he p e r c e n t a g e o f the co r r e spond ing germfree peak a rea ave rage .

This study was primarily directed towards a qualitative and semi- quantitative assessment of differences in the excretion of acidic metabolites in urine. Absolute quantitation would require recovery and detector response studies for each measured metabolite. Although this is only a rough estimate, the peak area corresponding to the internal standard is representative of an excretion rate of 310 mg of organic acid metabolite per 24 hour period.

Both qualitative and quantitative differences were observed in the urinary excretion of organic acids in the conventional rats relative to the germfree animals. Three meta-hydroxyphenolic acids (m-hydroxy benzoic, m-hydroxyphenylpropionic, and m-hydroxycinnamic acid) were absent in the germ- free samples, as verified by the mass spectral information. Such compounds were either not excreted by the germfree animals or were excreted at the levels below the detection limit of the mass spectrometer. The absence of m-hydroxyphenolic acids in germfree rat urine has been previously reported in studies which examined the interaction of intestinal bacteria with dietary constituents such as caffeic and ferulic acid (8), various flavonoids (I), and other compounds, such a L-DOPA (3,4-dihydroxyphenylalanine)(6). Such studies have demonstrated that the intestinal bacteria are solely responsible for some of the reactions leading to m-hydroxyphenolic acid metabolites.

Several additional aromatic metabolites that were significantly higher in the conventional rat urines include phenol, benzoic acid, phenyllactic acid, p-hydroxy-e-methylphenylacetic acid and p-hydroxyphenylpropionic acid. All of the compounds, with the exception of p-hydroxy-e-methylphenylacetic acid, are known to be produced through the action of intestinal bacteria on the previously mentioned dietary constituents as well as tyrosine. However, these compounds must also have an entirely endogenous source as well; except for phenol, they were identified in the germfree rat samples through mass spectrometry. It is interesting that p-hydroxy-~-methylphenylacetic acid was not found in the germfree mass spectra, nor has it been previously reported as a metabolite attributable to the intestinal microflora. Goodwin (17) in- dicates that o-hydroxy-~-methylphenylacetic acid is probably formed from ~-hydroxyphenyllactic acid. Presumably, p-hydroxy-e-methylphenylacetic acid originates from p-bydroxyphenyllactic acid. The observed increase in phenyl lactic acid in the conventional rat urine and the absence of p-hydroxy-~-methyl phenyl acetic acid in the germfree rat urine indicate microbial involvement in one or more of the possible reactions leading to these compounds. L- tyrosine is one known metabolic source of p-hydroxyphenyllactic acid; per- largonin, a flavonoid, is another (1,18). In both cases, the intestinal micro- flora is known to be involved (1,18).

The h i g h e r l e v e l o f h i p p u r i c a c i d in t he c o n v e n t i o n a l u r i n e s i s a t l e a s t p a r t i a l l y accounted f o r by the known a r o m a t i z a t i o n o f d i e t a r y q u i n i c ficid to y i e l d benzo ic ac id (1 ,2 ) . The benzo ic ac id i s then con juga t ed wi th g l y c i n e and e x c r e t e d as h i p p u r i c ac id .

The remaining acids which are elevated in the conventional rat urines are either dicarboxylic, hydroxy acids or, in the case of malic acid, a hydroxy- dicarboxylic acid. The involvement of intestinal bacteria in production of these compounds in vivo is suggested and several bacterial reactions are pro- bably involved. Increased levels of lactic and succinic acids may be at- tributed in part to the fermentation of glucose and other dietary constituents by numerous types of intestinal bacteria (19). Lactic, fumaric, succinic and

794 Urine Metabolites of Germfree Rats Vol. 32, No. 7, 1983

and mal ic a c i d s may a l s o a r i s e th rough the b a c t e r i a l deamina t ion o f a s p a r t a t e or a spa r ag ine through r e a c t i o n s known to occur in v i t r o in many ane rob i c b a c t e r - i a (19). S i m i l a r b a c t e r i a l t r a n s f o r m a t i o n o f d i e t a r y amino ac id s or ca rbo- hyd ra t e s i s p robab ly r e s p o n s i b l e f o r the i n c r e a s e d l e v e l s o f some o f the shor t cha in hydroxy- or d i c a r b o x y l i c a c i d s observed in t h i s s tudy. I t i s impor tan t to no te t h a t some d i f f e r e n c e s in m e t a b o l i t e l e v e l s may a l s o a r i s e from d i f - f e r e n c e s in i n t e s t i n a l a b s o r p t i o n due to the p r e sence o f the m i c r o f l o r a .

Conc lus ions : The s u b s t a n t i a l number o f q u l i t a t i v e and /o r q u a n t i t a t i v e d i f f e r e n c e s observed in the u r i n a r y m e t a b o l i c p r o f i l e s demons t ra tes the impor- t ance o f c o n s i d e r i n g p o s s i b l e a l t e r a t i o n s in the i n t e s t i n a l m i c r o f l o r a in the i n t e r p r e t a t i o n o f p r o f i l i n g da ta from s u b j e c t s wi th an abnormal m e t a b o l i c condi - t i o n . A number o f m e t a b o l i t e s , both v o l a t i l e and a c i d i c , were i d e n t i f i e d as p a r t i a l l y or t o t a l l y a t t r i b u t a b l e to the a c t i o n o f t he m i c r o f l o r a ; t h i s may a id in t he i n t e r p r e t a t i o n o f p r o f i l e da ta from o t h e r i n v e s t i g a t i o n s .

Acknowledgments: The au thors would l i k e t o thank Dr. Char les Lat tauda and Mr. Dwight Owens o f Harlan I n d u s t r i e s f o r t h e i r l a r g e r o l e in making t h i s s tudy p o s s i b l e . This r e s e a r c h was suppor ted by g ran t no. 24349 from the Na- t i o n a l I n s t i t u t e o f General Medical S c i e n c e s , U.S. Pub l i c Hea l th S e r v i c e .

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