Gut 1993; 34: 1552-1558

Ileal and colonic epithelial metabolism in quiescentulcerative colitis: increased glutamine metabolism indistal colon but no defect in butyrate metabolism

I A Finnie, B A Taylor, J M Rhodes

AbstractPrevious studies have shown that butyrate is animportant energy source for the distal colon,and that its metabolism may be defective in

. ulcerative colitis (UC). A similar metabolicdefect in the ileum might account for theoccurrence of 'pouchitis' in UC patients aftercolectomy. A method has been developed thatallows the measurement of metabolism inileocolonoscopic biopsy specimens, and thishas been used to assess butyrate and glutaminemetabolism in quiescent UC and controls.Preliminary experiments showed optimalmetabolism ofbutyrate at 1 mmolll. In controlsglutamine metabolism was greater in theascending (mean (SD)) (4.9 (3.2) nmol/h[igprotein) than in the descending colon (1-4(0.7)) (p<005, Mann-Whitney U test), butbutyrate metabolism was similar in the tworegions (ascending 62-6 (44 2), descending51-5 (32.0)). Consequently ratios of butyrate/glutamine metabolism were higher in thedescending colon (20-6 (14-3)) than in theascending colon (14.3 (9.6)) (p<0 05). In UC,rates of butyrate metabolism were similar inthe ascending (92.5 (58.3) nmol/h/,tg protein)and descending (93.3 (115)) colon, and thesewere not significantly different from controls.In UC, glutamine metabolism was similar inthe ascending (6.2 (7.7) nmollh4tg protein)and descending colon (7-8 (7.9)); the meta-bolism in the descending colon was signific-antly greater than in controls (p<001).Butyrate (135 (56) nmollh4tg protein) andglutamine (24.1 (16.2)) metabolism in the ileumin UC, were not significantly different fromcontrol values (butyrate 111 (57), glutamine15-5 (15.6)). These results confirm that thereis regional variation of nutrient utilisationthroughout the colon, but they do not sup-port the hypothesis that UC is caused by adeficiency of butyrate metabolism.(Gut 1993; 34: 1552-1558)

of the colonic epithelium. Colitis, which ishistologically similar to ulcerative colitis, hasalso been described in association withpellagra.5How the various forms of nutritional colitis

relate to ulcerative colitis (UC) is uncertain, butstudies have suggested that metabolism of theshort chain fatty acid butyrate is impaired inUC.i In these studies, in common with manyothers, it has not been possible to discover if thechange was a primary defect or merely a second-ary effect of the disease process.The frequency of 'pouchitis' in ulcerative

colitis patients who have colectomy and pouchconstruction contrasts with the apparent rarity ofpouchitis in patients who have the same opera-tion for familial polyposis. '° This implies apotential abnormality of the ileal mucosa inulcerative colitis that is not evident when theileum and colon are in normal continuity, withthe possible exception of 'backwash ileitis'.In view of this it seems appropriate to studymucosal metabolism in ileal as well as colonicmucosa in UC patients and controls in theexpectation that any abnormalities that could bedetected in the histologically normal ileum ofulcerative colitis patients would be unlikely to besecondary phenomena.A further problem with the interpretation of

some previous studies of mucosal metabolism67has been concern that the procedure used forisolating a pure epithelial cell population fromresected bowel specimens may itself haveintroduced artefacts, and that these artefactualchanges might well have been different in theinflamed colitic tissue from those in the histo-logically normal colon.

This paper describes a technique that allowsmetabolism of nutrients to be assessed in wholeepithelial biopsy specimens, and this techniquehas been used to assess the metabolism ofbutyrate and glutamine by ileal and colonicbiopsy specimens from patients with idiopathiculcerative colitis and controls.

Departments of MedicineI A FinnieJ M Rhodes

and Surgery, Universityof LiverpoolB A TaylorCorrespondence to:Dr J M Rhodes, Departmentof Medicine, University ofLiverpool, PO Box 147,Liverpool L69 3BX.

Accepted for publication14 April 1993

There is strong evidence that colitis can resultfrom impaired mucosal metabolism. It has beenshown that the colonic epithelium obtains muchof its energy supply from fatty acids, particularlybutyrate, which are present in the colonic lumenas a result of fermentation of carbohydrates bybacteria. Colitis occurs in the defunctioned colonafter diversion of the faecal flow and subsideswhen the faecal flow is replaced.'2 The occur-rence of colitis in animals that are vitamindeficient3 or that are treated with inhibitors offatty acid metabolism4 supports the theory thatfatty acid metabolism is vital to the well being

Methods

MUCOSAL BIOPSY SPECIMENSAll specimens were taken using Olympus FB13U colonscopy forceps at routine colonoscopy,except in two patients in whom ileal specimenswere obtained through an ileostomy with thesame forceps. Bowel preparation in all colono-scopy patients was similar, consisting of Picolax,two sachets on the day before colonoscopyand clear fluids for 48 hours. All patients gavewritten informed consent, and the studies were

1552

Ileal and colonic epithelial metabolism in quiescent ulcerative colitis: increased glutamine metabolism in distal colon but no defect in butyrate metabolism

approved by the ethical committee of the RoyalLiverpool University Hospital.

SUBJECTS

Colonic studiesAscending and descending colonic biopsy speci-mens were taken from 12 patients with long-standing quiescent ulcerative colitis who werebeing examined as part of a programme to screenfor dysplasia/malignancy, and from 12 patientswho acted as controls, who did not have ulcera-tive colitis. The final diagnosis of the controlpatients was sporadic colonic polyps (n=4),diverticular disease (3), haemorrhoids (2),unexplained anaemia (1), carcinoma (1), andirritable bowel syndrome (1). All specimens inthese control patients were taken from a site atleast 5 cm from any macroscopic abnormality,and adjacent mucosal specimens, whichwere normal on histological examination wereobtained in all.The mean duration of colitis was 12 years

(range 6-18). The median age ofUC patients was43 (28-65), which was not significantly differentfrom the control median 48 (18-68) (p=021,Mann-Whitney U test). Sex ratios were similar,but none of the UC patients smoked whereas sixcontrols were current smokers. All the UCpatients were taking regular 5-amino salicylicacid preparations, but none was taking any formof corticosteroid.

Ileal studiesTerminal ileal biopsy specimens were taken atileocolonoscopy (four patients) for patients withlongstanding quiescent ulcerative colitis andby the ileostomy of two patients who had acolectomy for UC (histological reports state that2 and 4 cm of ileum had been resected with thecolectomy). Ileal specimens were taken fromeight patients who did not have UC and whoacted as controls. One of the UC patients whohad had an ileostomy had mild non-specificinflammatory changes histologically (but nosymptoms), but the ileal mucosa was histologic-ally normal in all other cases. The mean durationof ulcerative colitis was 14 years, and thesepatients were having colonoscopy to excludedysplasia/malignancy, except the patients withileostomies who gave informed consent forileoscopy specifically for this study. The medianages were similar (UC patients 48, controls 51).The final diagnosis in control patients wasdiverticular disease (n=3), colonic cancer (1),unexplained anaemia (1), and irritable bowelsyndrome (3). Three controls smoked, none ofthe UC patients did so, and all UC patientsexcept the two with ileostomies were takingregular 5-amino salicylic acid preparations.

MUCOSAL METABOLISMThe method that Veerkamp et al" devised tostudy fatty acid metabolism in skeletal musclewas adapted to study intestinal mucosalmetabolism. After removal, mucosal biopsyspecimens were placed in ice cold pregassed

Krebs-Henseleit buffer containing 11 mMglucose, and transported within 10 minutes tothe laboratory. Each specimen was divided byscalpel into approximately 10 pieces per speci-men (each piece approximately 1 mg), and thenplaced in a glass scintillation vial in 1 ml Krebs-Henseleit containing 11 mM glucose to whichhad been added either 1 iCi sodium [1-_4C]butyrate and 1 mM sodium butyrate, or 1 iCi L[14C(U)] glutamine and 1 mM glutamine. AnEppendorf reaction tube was suspended abovethe culture medium, the vial was gassed with95% 02/5% CO2 and closed with a rubber seal(see Fig 1). The vials were cultured for twohours at 37°C in a shaking water bath (GrantInstruments, Cambridge, UK) set at 120 oscilla-tions per minute. At the end ofthe culture period0-25 ml 10% perchloric acid was injected into theculture medium to stop the reaction, and 0-5 mlof a solution of 66% ethane 1,2 diol/33%ethanolamine was injected into the Eppendorftube to absorb '4CO2. The vials were kept at 4°Cfor 90 minutes to allow equilibration, after whichthe Eppendorf reaction tubes were removed andplaced in 10 ml scintillant, which was a mixtureof4 g/l Omnifluor in 2:1 toluene/methanol. Aftervigorous shaking, these were then counted in aBeckman scintillation counter. All counts werecorrected by subtraction of counts for 14Cliberated in control vials without specimens.

Total biopsy specimens protein content wasestimated at the end of the incubation periodusing a Lowry method12 after ultrasonica-tion (4x15 second bursts with an MSE (MSEInstruments, Crawley, UK) ultrasonicator atmaximum power setting).

Results were expressed as nmol substratemetabolised per hour, after correcting forspecific radioactivity of the substrate andnumber of 14C atoms per molecule. For eachpatient, each assay was performed in duplicate ateach site studied and the mean of the resultscalculated.

Comparisons of the metabolism betweendifferent sites in the same patient, and the same

Ethanolamine/ethanediol Perchlorate toinjected at end of stop reaction

incubation to collect14C02 T

Biopsy specimen and medium

Figure 1: Apparatus for measurement ofmetabolism bycolonic biopsy specimens. For detailed description see methodssection.

1553

Finnie, Taylor, Rhodes

Figure 2: Rate ofmetabolism ofbutyrate bycolonic biopsies. The rate ofmetabolism of '4C butyrateto 14C02 by colonoscopicbiopsy specimens taken froma resected colon wasfound tobe linearfor at least threehours. Metabolism ismeasured as number ofnmol14C02 producedfromsodium[1-_4C] butyrate perpg biopsy protein. Similartime course experiments forglutamine confirmed that rateofmetabolism was linearforthe duration ofthe cultureperiod (results in text).

Figure 3: Metabolism ofbutyrate at differentconcentrations. Conversionofbutyrate to C02 wasgreater at a concentration ofI mM than at 10mM(p<0 05), or at 0-067 mM(p<0 01), but did not differsignificantly from 5mM (p=0 33) (Kruskal-Wallis).

a)4-

0

,o

C

E

cu

E

0)

-c

CD

m

E _

._C.'5 *i.-mD+

C0E m0 o:

4-1

, _

m

400

300

0

200 _

100 _- I

1 2Time (h)

0

3

sites in UC patients and controls were analysedusing a non-parametric (Mann-Whitney U) test.(95% confidence intervals (95% CI) fordifference between means are quoted whereappropriate.)

VALIDATION EXPERIMENTSSmall pieces (about 3x2 cm) of resected colonwere obtained within 30 minutes of removalfrom patients having colectomy for cancer ordiverticular disease, and placed in ice coldKrebs-Henseleit buffer at pH 7-4 (SigmaChemical Co, Poole, UK) containing 11 1 mMglucose, gentamicin 100 rig/ml and nystatin 60pIml, which was pregassed with 95% 02/5%CO2. The tissue studied was taken at least 5 cmdistant from any macroscopic disease. Themucosal surface was then pinned out and biop-sied with colonoscopy biopsy forceps. The speci-mens were than analysed using the methoddescribed previously for mucosal metabolism.A series of experiments was performed to

detect the optimal concentration of butyrate inthe culture system. For these experiments speci-mens were cultured in the same concentration ofradiolabelled sodium butyrate but with differentconcentrations of unlabelled sodium butyrate.To assess the potential contribution of lamina

p < 0.01

p < 0.05

p = 033

1 5Total butyrate concentration (mM)

propria lymphocytes to measured metabolismwe obtained peripheral blood lymphocytes fromthe heparinised blood of a patient having colono-scopy for polyp surveillance with centrifugationin Lymphoprep (Flow Laboratories, Rickmans-worth, UK). Lymphocytes (12 8x 106) wereprepared from 10 ml heparinised blood.Viability was measured using trypan blue exclu-sion and was found to be >95% both before andafter two hours culture. Five 200 tl aliquotsrepresenting 2 56x 106 lymphocytes were eachplaced in Krebs-Henseleit with a colonoscopicbiopsy specimen taken from the same patientand metabolism measured as below for mucosalbiopsy specimens. The results were comparedwith those obtained when culturing colonicspecimens in the absence of the lymphocytepreparation.

Attempts were made to measure the relativecontribution to the overall metabolism ofmucosa, lamina propria, and muscularis layers.By gently scraping the mucosa and laminapropria from a small piece of resected colon witha scalpel blade and biopsying the tissue below, itwas possible to compare metabolism of mucosalbiopsy specimens with that of submucosal speci-mens (histologically shown to contain connectivetissue and muscularis).A further series ofexperiments was performed

to assess whether colonic bacteria could con-tribute to measured metabolism. This wasnecessary in case there were different adherentbacteria on the mucosal surface in disease andcontrols, and for these experiments faecal fluidwas aspirated at colonoscopy and added in 100,ul aliquots to a specimen in the culture system.Control experiments were performed adding 1001tl Krebs-Henseleit. In addition, experimentswere conducted with, and without 100 ig/mlgentamicin and 60 [tg/ml nystatin in the Krebs-Henseleit.

ResultsVALIDATION EXPERIMENTSThe rates of metabolism of butyrate (Fig 2) andof glutamine were found to be roughly linear forat least three hours. For butyrate oxidation therate in the first hour was (mean (SD)) 110 8(16-5) nmol/,tg protein, over two hours 85 8(11-1) nmol/h/,tg protein, and over three hours90 3 (8 8) (p<00001 for linearity test usinggrouped regression analysis (Arcus Pro-II). Forglutamine the rate ofmetabolism in the first hourwas 5 1 (1 1) nmol/tg protein, 4 6 (2 2) nmollh/pg protein over two hours, and 4*3 (1 0) overthree hours (p<00001 for linearity test).Metabolism of butyrate was optimal at 1 mM(Fig 3) and for this reason we decided to use1 mM butyrate for the remainder of the study.

Reproducibility was checked with six biopsyspecimens from adjacent sites in a single patientand coefficient of variation was found to be 12%for butyrate metabolism expressed per pgprotein per hour and 18% for glutamine meta-bolism.The mean overall coefficient of variation asses-

sed by comparison of paried biopsy specimenswas 20% for butyrate metabolism and 23% forglutamine metabolism.

nJ

1554

0 067 10

Ileal and colonic epithelial metabolism in quiescent ulcerative colitis: increased glutamine metabolism in distal colon but no defect in butyrate metabolism 1555

TABLE I Metabolism ofglutamine and butyrate by mucosalbiopsy specimens

UlcerativeControls colitis(n= 12) (n= 12)

Ascending colon butyrate 62-6 (44-2) 92-5 (58 3)Ascending colon glutamine 4 9 (3.2)* 6-2 (7 7)Ascending colon butyrate/glutamine 14-3 (9-6)t 14-6 (9 25)Descending colon butyrate 51-5 (32) 933 (115)Descending colon glutamine 1-4 (07)*t 7-8 (7 9)tDescending colon butyrate/glutamine 20-6 (14-3)t 15-9 (15-6)

Mean figures for metabolism (nmol/[tg protein/h) of each nutrientare given with standard deviation in brackets. Values with samesuperscript are significantly different at p<005 (*t) or p<0-01 ()using Mann-Whitney U test.

Ascending Descending Ascending DescendingControls Ulcerative colitis

Figure 4: Metabolism ofbutyrate (colon). Butyrate metabolism was similar in the ascendingcolon and descending colon ofcontrols. Metabolism in ulcerative colitis patients did not differsignificantly.

Submucosal specimens metabolised compara-tively little butyrate (5-6 (3 2) nmol/[tgprotein/h) (mean (SD), n= 5) and glutamine (0-2(0-2), n=5) compared with mucosal specimens(butyrate 66-2 (13-2), n=5, p<0 01, glutamine4-2 (0 6), n=5, p<0 01).The addition of peripheral blood lymphocytes

had no significant effect on butyrate metabolism(biopsy specimen with lymphocytes 49-2 (12-3)nmol/4ig protein/h, without lymphocytes 52 2(10-2)) (mean (SD) of five experiments) (95%CI=-22-4 to 24 6), or glutamine metabolism(biopsy specimen with lymphocytes 55 (2-2),without lymphocytes 4-5 (1-6)) (95% CI=-2-6to 3-2).The addition of 100 tl faecal fluid to a biopsy

specimen in culture did not significantly affectmetabolism of butyrate (biopsy specimen+faecal fluid 49-7 (10-1) nmol/tg protein/h,biopsy specimen+Krebs-Henseleit 52-2 (5 -8))(mean (SD), five experiments per group)(95% CI=-18-1 to 22-5) or glutamine (biopsyspecimen+faecal fluid 5-7 (1- 1), biopsy specimen+Krebs-Henseleit 6-8 (2-2)) (95% CI=-3-8 to2-2). The addition of gentamicin and nystatin tothe Krebs-Henseleit buffer had no significanteffect on metabolism of butyrate by colonicbiopsy specimens (with antibiotics 56 (10-3),without antibiotics 53-4 (7-9)) (95% CI=-16-2to 21-2).

Incubation with sodium [1-_4C] butyrate inthe absence of mucosal biopsy specimens led toan increase in scintillation in the Eppendorf vialof 512 (35) (mean (SD), n=6) disintegrations perminute (DPM) above background, which mayhave been because of the volatility of thebutyrate. This was small (<3%) in comparisonwith the radioactivity produced by substratemetabolism in the presence of mucosal biopsyspecimens (range 24 000-115 000 DPM).Incubation with 14C glutamine in the absence ofmucosal biopsy specimens gave results that wereless than 5% ofthe values obtained when a biopsyspecimen was present.

p < 0.01

COLONIC MUCOSAL METABOLISM (Table I)

- p<O-05

Ascending Descending Ascending DescendingControls Ulcerative colitis

Figure 5: Metabolism ofglutamine (colon). In controls, glutamine metabolism was significantlygreater in the ascending than in the descending colon (p<O-O5, Mann-Whitney U test). Incomparison, ulcerative colitis patients had significantly greater metabolism in the descendingcolon (p<O001), but values in the ascending colon were not significantly different.

ControlsNo significant difference was found in the rate ofmetabolism of butyrate by biopsy specimenstaken from the ascending and descending colon(Fig 4). Glutamine metabolism was significantlygreater in the ascending than in descending colon(p<005, Mann-Whitney U test) (Fig 5). Ratiosof butyrate/glutamine metabolism were signific-antly lower (p<0-05) in the ascending than in thedescending colon (Fig 6).

Ulcerative colitisButyrate metabolism was similar in ascendingand descending colon, and these values were not

significantly different from those found in con-

trols (Fig 4). In ulcerative colitis glutaminemetabolism was significantly greater in thedescending colon than in controls (p<0-01) andthere was no significant difference betweenglutamine metabolism in the proximal and distalcolon.

E

00.4_Q

oaL 400-

---

0Ec

E 200-co

.0Co

EDX 100-

m1

O E

40-

a)3 35-00.cm- 30-0E_uSE= 15-0

.0E

10-C

5Eco

2 5-(D1

u ,

500-i

Finnie, Taylor, Rhodes

-p < 0.05

TABLE II Metabolism ofglutamine and butyrate by ilealmucosa

Controls Ulcerative(n =8) colitis (n=6)

Butyrate 111 (57) 135 (56)Glutamine 15-5 (15-6) 24-1 (16-2)Butyrate/glutamine 13-6 (11-2) 11-5 (11 1)

Mean figures (SD) for metabolism (nmolV,ug protein/h) of eachnutrient are given.

250 r

Ascending Descending Ascending DescendingControls Ulcerative colitis

Figure 6: Butyratelglutamine ratio (colon). Incontrols, the ratio ofbutyratelglutaminemetabolism was significantlyhigher in the descending thanin the ascending colon(p<OO5). In ulcerativecolitis patients, this relationwas lost, and there was nosignificant differencebetween controls andulcerative colitis patients ineither site.

ECn -

.0uO

o L.E m

'o i

Cu0

4-'Em~

200k000150-

100Q-

50o

The mean ratio of butyrate/glutamine meta-bolism in the descending colon was greater incontrols than in UC, but this did not quite reachsignificance (p=009). Ratios for the ascendingcolon were similar.

ILEAL MUCOSAL METABOLISM (Table II)In controls the rate of butyrate metabolism in theileum was significantly higher than in the ascend-ing colon (p<005), and although the mean rateof glutamine metabolism was also higher inthe ileum this did not quite achieve statisticalsignificance (p=006). The ratios of butyrate/glutamine metabolism were similar in the tworegions. In UC the mean rate of butyrate meta-bolism was greater in the ileum than in theascending colon, although the differencebetween the rates was not significant (p=0-09);glutamine metabolism was significantly greaterin the ileum than in the ascending colon in UC(p<005). No significant difference was foundbetween the rates ofmetabolism of butyrate or ofglutamine by ileal biopsy specimens taken fromUC patients compared with controls, and theratios of butyrate/glutamine metabolism did notdiffer between UC patients and controls (Figs 7,8, and 9).Each data point in Figs 4 to 9 represent the

mean of two experiments.

DiscussionIn this study mucosal metabolism of butyrateand glutamine in the terminal ileum, ascendingcolon, and descending colon have been measuredin controls and patients with ulcerative colitis. Incontrols, we found similar rates of butyratemetabolism in the ascending and descendingcolon, but greater rates of glutamine metabolismin the proximal colon. Thus, although the ratioof butyrate:glutamine metabolism was greater inthe normal distal colon this resulted from

0

0

00

0

Controls Ulcerativecolitis

Figure 7: Butyrate metabolism (ileum). Butyrate metabolismin ileal biopsy specimens taken at colonoscopy (0) or throughan ileostomy (a) was similar in controls and ulcerative colitispatients.

50 r

E 40

.-Co0.X 30

L-

*5 20E C

10

30FE

,-T 254.4-5Cu.0" m+ X 203 Eo C 15o c

r , 10o0

5

00

i

Controls Ulcerativecolitis

0

S

0

0

0

Controls Ulcerativecolitis

Figure 9: Butyratelglutamine ratio (ileum). The ratio ofbutyratelglutamine metabolism in the ileum ofcontrols andulcerative colitis patients was similar.

75-

E0-0

a)ECDEco

-50)n

.CD0-0II-

cc

cc

60-

45-

30-

15-

0

.

U

.0

0

00

.U0

0

0

Figure 8: Glutamine metabolism (ileum). Ileal glutaminemetabolism did not differ significantly between controls andulcerative colitis patients.

35r

0

I00

u

U'

u

u-

1556

n-

Ileal and colonic epithelial metabolism in quiescent ulcerative colitis: increasedglutamine metabolism in distal colon but no defect in butyrate metabolism 1557

reduced glutamine metabolism rather thanincreased butyrate metabolism. Rates ofbutyrate metabolism in controls and ulcerativecolitis patients were similar for all three regionsstudied, but glutamine metabolism was signific-antly greater in the descending colon in ulcera-tive colitis. This resulted in a ratio of butyrate/glutamine metabolism that was slightly (but notsignificantly, p=0 09) lower in ulcerative colitiscompared with controls.

It has been established that glutamine is animportant food for many rapidly dividing cells,'3and the increased rate of glutamine utilisation inUC probably relates to the increased rate ofproliferation seen in this condition. The higherrate ofmetabolism of glutamine in the ascendingthan in the descending colon in controls is inagreement with the findings of Roediger'4 andindeed close examination of his work shows thatthe difference in the rates of butyrate metabolismin ascending and descending colon was in factsmall compared with the difference betweenrates of glucose metabolism and oxygen con-sumption. Fleming et all5 have reported nodifference in butyrate metabolism betweencaecal and colonic cells isolated from rats.

It is interesting that butyrate metabolism incontrols was greater in the terminal ileum than inthe ascending colon and this may suggest that themucosa here is exposed to the products ofbacterial fermentation.The similar metabolism of butyrate in controls

and in patients with ulcerative colitis, is incontrast with previous studies,67 where butyrateoxidation was impaired in mucosal cells isolatedfrom quiescent and active ulcerative colitis. Thisdiscrepancy probably results from the differenttechniques used to measure metabolism. Wehave used a technique, whereby mucosal meta-bolism can be measured in mucosal biopsyspecimens, and therefore patients in remissioncan easily be studied. In addition, our techniqueavoids potential damage to cells during the cellisolation procedure by EDTA and dithiothreitol.It also seems probable that cells from inflamedepithelium (colitis) will be more prone to toxicityfrom cytokines than will cells from control tissueand this may have introduced artefactualabnormalities in dispersed colitic cells. The factthat patients with active colitis were moreaffected than those with quiescent disease inRoediger's studies6 could reflect this. A furtheranxiety over the use of dispersed cells for meta-bolic studies comes from the finding that themetabolism of fatty acids entering intestinalmucosa from the lumen and plasma is different. 16Exposure to the surrounding solution of theparts of the cell membrane that are normally inapposition might well affect the metabolismwithin the cell. Two studies89 have reporteddefective butyrate metabolism in epithelialbiopsy specimens (as opposed to isolated entero-cytes) from patients with ulcerative colitis butthese have so far been published only in abstractform. Chapman et al8 used a similar technique tothe one described here, and found significantlylower rates of butyrate metabolism, and non-significant reductions in glutamine and glucosemetabolism in UC. Their results on butyratemetabolism therefore concur with the findings of

the dispersed cell experiments67 and are not inagreement with our findings. The lower rates ofmetabolism of glutamine and glucose, however,found in UC in that study are not in agreementwith either our or Roediger's6 findings, so theimplications of this report remain unclear. Onepossible reason for the difference betweenChapman's and our results is that their experi-ments were all performed in the presence of5 mM glutamine (M Chapman, personal com-munication) whereas our experiments werecarried out in the absence of glutamine. Thestudy by Williams et al9 reported no metabolismof either butyrate or glutamine by biopsy speci-mens taken from two UC patients, althoughthere was no report on tissue viability. Some ofthe reported difference in rates of metabolismcould relate to the severity of the underlyingcolitis.6One further possible explanation for the dis-

crepancy between some of our and Roediger'sfindings could be that the methods of assessingmetabolism were different. For example wherehe used production of ammonia, we have reliedon production of CO2 as the measurement ofglutamine metabolism. It is possible that theroute of metabolism of substrates, rather thanthe rate of their metabolism, is different in thediseased state.There are some potential disadvantages of

using biopsy specimens as opposed to epithelialcell preparations for metabolic studies. It couldbe argued that different specimens could havedifferent proportions of actively metabolising(mucosa) and inactive (lamina propria) tissue.For this reason we used a constant size of biopsyforceps throughout the study and reproduc-ibility of the assays was satisfactory. Any error asa result of variation in the proportion of activeand inactive tissue between patients (suchas might result if one patient's mucosa wasatrophic) should be alleviated by measuringglutamine metabolism in adjacent biopsy speci-mens and examining the ratio of butyrate/glutamine metabolised. The validation experi-ments suggested that lymphocytes probably con-tribute little to overall measured metabolism.Binder'7 found an excess of 1000 cells/mm2lamina propria in mildly active UC comparedwith controls, and assuming specific gravity= 1and that lamina propria accounts for about halfthe weight of a biopsy specimen, the excessnumber of cells in lamina propria of a 10 mgspecimen= 150 000. Selby et al'8 isolated anexcess of 44x 106 mononuclear cells/g UCmucosa, or 440 000 per 10 mg biopsy, so boththese estimates are considerably smaller than thenumber oflymphocytes studied with each biopsy(2 56x 106).One further effect worth considering is the

bowel preparation before harvesting of thetissue. Ardawi and Newsholme" have shownthat starvation reduces the rate of metabolism ofglutamine, but not that of ketone bodies bycolonocytes. By avoiding variations in bowelpreparation (apart from the two patients withileostomies) any such effect due to a change indiet should be equivalent in the UC and controlpatients.

It was notable that butyrate metabolism was

1558 Finnie, Taylor, Rhodes

greatest at a concentration of 1 mM with a fall offin metabolism at higher concentrations. Concen-trations of 10-15 mM are present in the coloniclumen,2t22 but separated cells and biopsy speci-mens might well react differently from an intactmucosal surface. Morita et al23 have reportedtoxic effects of sodium butyrate above 2 mM in acolon cancer cell line and it may be that previousstudies have used concentrations of butyrate thatare higher than optimal.

Studies have suggested that both diversioncolitis' and ulcerative colitis24 respond to rectallygiven short chain fatty acids, and recently acontrolled trial of butyrate enemas in ulcerativecolitis showed significant improvement inclinical and pathological parameters overplacebo.25 Vernia and colleagues26 have shownreduced concentrations of butyrate in the faecalwater of patients with ulcerative colitis, but notin Crohn's colitis. A reduced short chain fattyacid concentration has also been reported inpouch contents from patients with pouchitiscompared with those without.27 The response ofulcerative colitis to butyrate administration sug-gests that the mucosa is able to utilise butyrateadequately, and makes it more plausible that thecolitis results from a deficiency of, rather than aninability to metabolise, butyrate.

These studies show that mucosal metabolismcan be assessed effectively using biopsy speci-mens without the need for epithelial cell purifica-tion. The increased metabolism of glutamine bythe distal colon in ulcerative colitis seemsprobably to reflect a response to inflammationand consequent hyperplasia and is in keepingwith evidence that respiratory chain enzymes areincreased in ulcerative colitis.28 It seems unlikelyfrom this work that ulcerative colitis is a result ofa primary defect in butyrate metabolism.

Dr Finnie is supported by a grant from Mersey Regional HealthAuthority.

1 Harig JM, Soergel KH, Komorowski RA, Wood CM.Treatment of diversion colitis with short-chain-fatty acidirrigation. N EnglJ7 Med 1989; 320: 23-8.

2 Korelitz BI, Cheskin LJ, Sohn N, Sommers N. Proctitis afterfaecal diversion in Chron's disease and its elimination withreanastomosis: implications for surgical management.Gastroenterology 1984; 87: 710-3.

3 Wintrobe MM, Follis RH, Alcayaga R, Paulson M,Humphreys S. Pantothenic acid deficiency in swine. BullJ7ohns Hopkins Hosp 1943; 73: 313-33.

4 Roediger WE, Nance S. Metabolic induction of experimentalulcerative colitis by inhibition of fatty acid oxidation. BrJ7Exp Pathol 1986; 67: 773-82.

5 Segal I, Tim LO, Demetriou A, Paterson A, Hale M, LeriosM. Rectal manifestations of pellagra. IntJ' Colorect Dis 1986;1: 238-43.

6 Roediger WEW. The colonic epithelium in ulcerative colitis:an energy deficiency disease. Lancet 1980; ii: 712-5.

7 Ireland A, Jewell DP. 5-aminosalicylic acid (5-ASA) has noeffect on butyrate metabolism in human colonic epithelialcells. Gastroenterology 1989; 102: A176.

8 Chapman MAS, Grahn MF, Hutton M, Rogers J, WilliamsNS. Failure of colonic mucosa to oxidise butyrate inulcerative colitis [Abstract]. Gut 1992; 33: S40.

9 Williams NN, Brannigan A, Fitzpatrick JM, O'Connell PR.Glutamine and butyric acid metabolism measurement inbiopsy specimens (ex-vivo): a method of assessing treatmenton inflammatory bowel conditions. Gastroenterology 1992;102: A713.

10 Madden MV, Farthing MJG, Nicholls RJ. Inflammation inileal reservoirs: pouchitis. Gut 1990; 31: 247-9.

11 Veerkamp JH, Van Moerkerk HTB, Glatz JFC, VanHinsbergh VWM. Incomplete palmitate oxidation in cell-free systems ofrat and human muscles. Biochim BiophysActa1983; 753: 399-410.

12 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteindetermination with the Folin-phenol reagent. J Biol Chem1951; 193: 265-75.

13 Ardawi MSM, Newsholme EA. Metabolism in lymphocytesand its importance in the immune response. Essays Biochem1985; 21: 1-44.

14 Roediger WEW. Role of anaerobic bacteria in the metabolicwelfare of the colonic mucosa in man. Gut 1980; 21: 793-8.

15 Fleming SE, Fitch MD, DeVries S, Liu ML, Kight C.Nutrient utilisation by cells isolated from rat jejunum,cecum and colon. J Nutr 1991; 121: 869-78.

16 Gangl A, Ockner RK. Intestinal metabolism of plasma freefatty acids. Intracellular compartmentation and mechanismsof control. J Clin Invest 1975; 55: 803-13.

17 Binder V. Cell density in lamina propria of the colon. Aquantitative method applied to normal subjects and ulcera-tive colitis patients. ScandJ Gastroenterol 1970; 5: 485-90.

18 Selby WS, Janossy G, Bofill M, Jewell DP. Intestinal lympho-cyte subpopulations in inflammatory bowel disease: ananalysis by immunohistological and cell isolation tech-niques. Gut 1984; 23: 32-40.

19 Ardawi MSM, Newsholme EA. Fuel utilization in colonocytesof the rat. Biochem,n 1985; 231: 713-9.

20 Cummings JH, Hill MJ, Bone ES, Branch WJ, Jenkins DJA.The effect of meat protein and dietary fiber on colonicfunction and metabolism. Part II. Bacterial metabolites infeces and urine. AmJ3 Clin Nutr 1979; 32: 2094-101.

21 Rubinstein R, Howard AV, Wrong OM. In vivo dialysis offaeces as a method of stool analysis. IV. The organic anioncomponent. Clin Sci 1969; 37: 549-64.

22 Cummings JH, Pomare EW, Branch WJ, Naylor LP,Macfarlane GT. Short chain fatty acids in human largeintestine, portal, hepatic and venous blood. Gut 1987; 28:1221-7.

23 Morita A, Tsao D, Kim YS. Effect of sodium butyrate inHRT-18, a human rectal cancer cell line. Cancer Res 1982;42:4540-5.

24 Breuer RI, Buto SK, Christ ML, Bean J, Vernia P, Paoluzi P,et al. Rectal irrigation with short-chain fatty acids for distalulcerative colitis. Preliminary report. Dig Dis Sci 1991; 36:185-7.

25 Scheppach W, Sommer H, Kirchner T, Paganelli G-M,Bartram P, Christl S, et al. Effect of butyrate enemas on thecolonic mucosa in distal ulcerative colitis. Gastroenterology1992; 103: 51-6.

26 Vernia P, Gnaediger A, Hauck W, Breuer RI. Organic anionsand the diarrhea of inflammatory bowel disease. Dig Dis Sci1988; 33: 1353-8.

27 Clausen MR, Tvede M, Mortensen PB. Short-chain fatty acidsin pouch contents from patients with and without pouchitisafter ileal pouch-anal anastomosis. Gastroenterology 1992;103: 1144-53.

28 Mayall T, Macpherson A, Bjarnason I, Forgacs 1, Peters T.Mitochondrial function of colonic epithelial cells in inflam-matory bowel disease. Gut 1992; 33 (suppl 1): S24.