the gastric neutral-red excretion test · the gastric neutral-red excretion test by s. sevltt...

J. clin. Path. (1948), 1, 217.

THE GASTRIC NEUTRAL-RED EXCRETION TESTBY

S. SEVlTTDepartment of Pathology, Birmingham Accident Hospital

AND

R. P. JEPSONSurgical Professorial Unit, Manchester Royal Infirmary

(R}EcIvED FOR PUBLICATION, MAY 18, 1948)

IntroductionThe stomach's acid secretion is commonly used

as a measure of gastric function, and variousstandardized tests have been devised. Owing,however, to the wide range of acid secretion innormal people, to the inconstant results obtainedby repeating a test on many individuals, to thefrequent occurrence of results well within thenormal range in patients suffering from gastritis,gastric carcinoma, or peptic ulcer, and to the factthat from 10 to 20 per cent of normal people mayhave spontaneous temporary or permanent achlor-hydria (Schiff, 1938; Palmer and others, 1940),estimation of the acid secretion of the stomachafter a popularity of many years is now fallinginto disfavour as a diagnostic test.In the search for a more reliable test, the excre-

tory function of the gastric mucosa to varioussubstances including dyes has been studied: oneof these dyes is neutral red.When a solution of neutral red is injected into

a vein of a normal individual, the dye is excretedwithin a few minutes into the gastric juice, stain-ing it pink: the concentration increases for aperiod and thereafter diminishes. During the nexttwenty-four to forty-eight hours the dye is excretedin the urine and colours the faeces.

In 1923 Glaessner and Wittgenstein introducedthe excretion of neutral red by the stomach as atest of gastric function, but the normal variationswere not worked out, nor was the relationship ofdye excretion to acid secretion fully understood bythese or subsequent workers (Davidson and others,1925; Zibalis, 1934; Winklestein, 1942; Lourja andMirkin, 1925). Twenty years later the excretion ofneutral red by the stomach was reintroduced byGillman (1943, 1944) as a test of gastric function.He used a standard technique whereby the rate of

dye excretion and the rate and intensity of con-centration of the dye in the gastric juice could bemeasured. He worked out the normal variationsin healthy young European adults in Johannesburg,and showed that normal results were repeatablewithin narrow limits. By studying the resultsobtained on three hundred cases, mostly Africannatives with disturbed gastric function, he claimedto have established criteria of abnormality whichcould be of diagnostic and prognostic aid.The intention of the present study was to check

the range of normality reported by Gillman; tostudy the anatomy and physiology of neutral-redexcretion; to compare the acid secretion of thestomach with its power to excrete dye, and to deter-mine if the test could be of use in this country inthe diagnosis of various gastric and duodenal dis-orders.

MethodsTechnique of the neutral-red excretion (N.R.E.)

test.-The Gillman method was adhered to with onlyminor modifications. A Ryle or Rehfuss tube waspassed through the mouth into the stomach of thepatient, who had fasted overnight. After the restingjuice had been drawn off, the stomach was washedout with 100 ml. or more of warm water by meansof a 20-ml. syringe until the aspirate became clear.When little or no mucus appeared in the washing,the stomach was emptied and was ready for the test.In a few cases, especially in carcinoma of the stomach,it was found difficult or impossible to obtain a clearand clean aspirate: these were cases with great excessof mucus in the juice, often together with alteredblood.Without delay, 5 ml. of a 1 per cent sterile neutral-

red aqueous solution was injected intravenously asrapidly as possible, and a stop-watch was started.Any remaining stomach contents were removed assoon as the injection was completed. Subsequentaspirations were carried out at 1-minute intervals

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S. SEVITT AND R. P. JEPSON

until the dye first appeared in the juice. After that,aspiration was repeated every 2 minutes for the next15 to 20 minutes, and later at 3- to 5-minute inter-vals. The period of the test was at least 30 minutes,and usually ten to fifteen aspirates were made. Theentire volume of gastric contents obtainable was re-moved at each aspirate and placed in a test-tube of5/8 in. diameter upon which was marked the numberof minutes from the time of the dye injection. Thecomplete removal of the gastric contents before andimmediately after injection and at subsequent aspira-tions was considered important; otherwise an un-known dilution factor would occur. Patients wereinstructed to expectorate and not to swallow salivaexcreted during the test.

Difficulty in aspiration sometimes occurred: if thetube was blocked by a plug of mucus, injection of20 ml. or more of air down the tube often cleared it:sometimes aspiration was facilitated by the patientbending forward.The concentration of the dye in each sample of

gastric juice was estimated by direct vision in a com-parator box using standard dilutions of the dye con-tained in identical test-tubes. The standard tube wasbacked with a tube containing uncoloured juice, andthe unknown with a tube of water. With a littlepractice the dilutions were easy to read.The standards were kept in well-stoppered test-

tubes ready for use. Deterioration in colour was notappreciable for three or four months. The dye wasmade up in decinormal hydrochloric acid and thefollowing dilutions used, viz., 1/300,000, 1/200,000,1/100,000, 1/75,000, 1/50,000, 1/30,000, and 1/20,000.The actual concentration of the dye was not usuallyrecorded but the arbitrary scale of Trace, 1, 2, 3, 4,5, and 6 used by Gillman was adopted (Trace =1/300,000, 1 = 1/200,000, etc.). Graphic representa-tion of each result was made by plotting dye concen-tration of the sample against the time in minutes (seeFigure).

Further studies of the fasting juice and subsequentunstained and stained gastric fractions were carriedout. The volumes were measured, the presence ofmucus, bile, and macroscopic blood were noted, andbenzidine tests for occult blood were performed."Free hydrochloric acid " and " total acid" estima-tions were performed on the fasting specimen andon four or five samples of juice removed at consecu-tive 5- to 10-minute intervals. The dye was firstremoved from the sample by filtration through twoor three thicknesses of filter paper, which absorbedthe neutral red. Sometimes refiltration was necessary.Titration was carried out on 5 ml. (or if available10 ml.) of the specimen with N/10 or N/20 sodiumhydroxide, using a drop or two of a mixture ofTopfer's reagent and phenolphthalein as indicators.If achlorhydria was present, qualitative tests for pep-sin were performed on two or three mixed samplesof juice using coagulated egg-white as a substrate.

In some of the cases (vide infra) in which excre-tion of neutral red did not occur or was very poor

within the first 30 minutes, aspirations were continuedevery 5 minutes for a further 30 to 60 minutes, some-times after an intramuscular injection of 0.5 mg. ofhistamine.

Fractional test meals.-These were not performedon the twenty " control " cases, but were carried outon patients exhibiting gastric symptoms, usually a fewdays before the neutral-red excretion test was per-formed.

Absence of toxicity.-In the dosage used, neutralred was found to be free from toxic or unpleasantlocal or general effects. Its excretion in the urineand its colouring of the faeces within the followingday or two was physiological. It was found advis-able, however, to warn the nursing staff and thepatient of this occurrence so as to prevent undueanxiety or alarm.

Observations

Results in N.R.E. tests in patients free fromgastro-intestinal symptoms.-N.R.E. tests wereperformed on twenty in-patients of a military hos-pital who were free of any gastro-intestinal symp-toms or signs. It was difficult to find perfectlyhealthy patients in hospital, but patient volunteerswith various non-gastric disorders were selected.They consisted of six convalescent minor surgical

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FIGURE.-Normal and abnormal neutral-red excretioncurves.

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THE GASTRIC NEUTRAL-RED EXCRETION TEST

w- I.I9:..PLATE VIII.-Photograph ofa colour drawing showing excretion of

neutral red by the mucosa of the upper half of the body of thestomach (black streaks = neutral red).

(Original drawing by Miss D. Davidson.)

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and fracture cases, three patients completely recov-

ered from old head injuries, two mildly neuroticpatients, and a case each of Bell's palsy, tineapedis, convalescent atypical pneumonia, endome-trosis, convalescent tonsillitis, progressive spasticpalsy, congenital cystic disease of the lungs, andconvalescent catarrhal jaundice. All these patientswere out of bed; they were fit and well and hadgood appetites.Only two of the patients were women; of the

men, four were Germans, one an Italian, anothera Pole, and the remainder were English, Scottish,or Irish. Two patients were 35 and 48 years ofage respectively, the remainder 18 to 26 years old.The details and shape of the N.R.E. curves were

studied (see figure). The general shape of eachcurve was similar; dye tinged the juice a faintpink within a few minutes, and the concentrationincreased- usually quite rapidly-to reach a maxi-mum which was maintained for a period and

which then declined. Particular attention was paidto :

(1) The excretion time (E.T.), or the period inminutes between injection of the dye and its firstappearance in the aspirate. In all cases this was

5 minutes or less. It is noteworthy that in seven

cases dye appeared within 1 minute, in thirteenwithin 2 minutes, and in seventeen cases within3 minutes of the injection.

(2) The concentration time (C.T.) or the periodin minutes between injection of the dye and itsearliest maximum concentration in the juice. Infifteen cases this was between 8 and 12 minutes;in the remainder it was 4, 5, 6, 14, and 19 minutesrespectively.

(3) The maximum degree of concentrationfound. (CM). Using the arbitrary scale of Trace,1, 2,- 6 (see above), the CM in 15 cases was be-tween 3 and 5; one was 6, three were 2, and one

was 1.

TABLE I

RELATIONSHIP OF NEUTRAL-RED EXCRETION TESTS TO THE ACID SECRETION OF THE STOMACH

No. of No. with No. with No. with No. withGroup of cases cases Classification of hyper- normal hypo- achlor- Total

in group N.R.E. tests acidity acidity acidity hydria

Non gastric controls 20 Normal N.R.E. 5 14 0 0 19

Hypo-excretory, Type a 0 1 0 0 1

Carcinoma ofstomach

10 Normal N.R.E.

Hypo-excretory, Type b

1 2

0 0

0 0 3

0

N.R. not excreted 0 0

Chronic dyspepsia 11 Normal N.R.E. 0 4and chronic gastri-tis Hypo-excretory, Type b 1 1 0

N.R. not excreted 0 1 0

Peptic ulcer 16

All Cases 73

Totals

2 2

4 5

I 0 5

0

3

2

4

Normal N.R.E. 4 6 0 0 10

Hypo-excretory, Types a and b 0 4 2 0 6

N R. not excreted 0 0 0 0 0

Normal N.R.E. 11 35 2 0 48

Hypo-excretory 1 6 2 3 12

N.R. not excreted 0 1 2 10 13

12 42 6 13 73

Statistical Ani!ysiv-.Chi-squared testing of all cases was done by grouping of classes owinglto the small numbers. In all arrangementsused there is a highly significant departure from random distribution (x2 = 35.06, 31.3 or 30.09: P<0-0001). The chief contribution to theChi-squared comes from the achlorhydric cases.

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TABLE I1ANALYSIS OF CASES WITH ABNORMAL N.R.E. TESTS

Case Dgs X-ray findings Laparotomy findings N.R.E. test Max. free HCI ofNo. Diagnosis barium meal, etc. and operation ET CT C gastric juice

9 Non-gastric con-trol 1 9 1 62%, Normal

(Bells palsy) acidity

21 Gastric carcinoma Filling defect near Never Never Nil Achylia gastricalesser curvature "coffee-ground"

fasting juice58 Gastric carcinoma Irregular filling de- Inoperable cancer of Never Never Nil Achlorhydria

fect on lesser curva- lesser curvatureture

62 Gastric carcinoma Large ulcer on lesser M align ant ulcer; Never Never Nil 8% hypo-aciditycurvature secondaries in liver

69 Gastric carcinoma Ulcer on lesser cur- High, inoperable car- Trace Never 1 at Achlorhydriavature cinoma 9 and 11 13 mins.

mins. Intermittentexcretion

71 Gastric carcinoma targe, pyloric filling Pyloric cancer: par- Never Never Nil Achlorhydriadefect tial gastectomy

72 Gastric carcinoma Filling defect near Inoperable carcinoma Never Never Nil Achlorhydriacardia

59 Carcinoma of Filling defect on Malignant infiltration 20 Never I Achlorhydriatransverse colon greater curvature of greater curvature Intermittent

excretion

2 Chronic dyspepsia Normal 24 Never Trace 50%, climbingcurve in F.T.M.Mucus + + +

4 Alcoholic gastritis Normal Never Never Nil Achylia gastrica(with hepatic

cirrhosis)10 Chronic gastritis Normal Never Never Nil Achylia gastrica28 Chronic gastritis Normal 10 Never Trace 70% climbtng

Intermittent curve in F.T.M.excretion hyperacidity

66 Chronic dyspepsia Mucosal puckering Stomach, duodenum Never Never Nil 42% normalonlesser,curvature, etc., normal acidity? gastric ulcer

69 Alcoholic gastritis Normal Never Never Nil Achlorhydria(with renal colic)

51 Chronic cholecys- Normal Never Never Nil Achlorhydriatitis

68 Chronic cholecys- Barium meal = Cholecystectomy and Never Never Nil Achlorhydriatitis normal. Gastros- appendicectomy

copy = atrophicgastritis

8 Gastric ulcer Ulcer on lesser cur- _ 9 Never Trace Trace of freevature HCI, Pepsin +.

"coffee-ground"fasting juice

25 Gastric ulcer Ditto - 3 7 1-2 12% low normalacidity42 Gastric ulcer Ditto 9 35 1 9% hypo-acidity20 Duodenal ulcer Shallow ulcer on 2 Never Trace 62% normal

medial border of aciditycap

50 Duodenal ulcer Ulcer on cap Partial gastrectomy 5 28 2 60% normal. ~~~~~~~~~~~~~~acidity56 Gastro-jejunal (Gastro-enterostomy Vagotomy Trace 25 2 1a% low normal

ulcer several years pre- 11-20 acidityviously) mins.

6 Intestinal colic Barium meal and 31 Never Trace Achlorhydriaenema = N.A.D. Pepsin +

61 Achalasia cardia Barium meal inde- - Never Never Nil 10% hypo-acidityfinite

14 Post-head-injury Normal Never Never Nil Achlorhydria,depression Pepsin

E.T. =Excretion Time. C.T. Concentration Time. Cx. -Maximum Concentration.Q

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The normal curve of N.R. excretion.-Exclud-ing one test, excretion of neutral red by thestomach began within 5 minutes of the injection;the dye was then concentrated to reach a maxi-mum within 19 minutes, and the maximum con-centration reached was between 2 and 6. In thecase of Bell's palsy showing a CM of 1 only, theE.T. was 1 minutes, C.T. 9 minutes (see Table II).Except for the maximum degree of concentra-

tion, these results are in general agreement withthose reported by Giliman (1944). He found thatnormal individuals excreted dye into the stomachwithin 10 minutes of the injection (usually 4minutes) and concentrated it to an intensity of 4 to6 within 20 minutes (average 14 minutes). If Gill-man's maximum concentration is to be accepted,then the ten N.R.E. curves with a maximurm of3, 2, or 1 would be considered abnormal. Noneof these patients had any symptoms or signs ofgastro-intestinal disorder, and they formed 50 percent of the present " control" series.

It was arbitrarily decided to consider a C.M of1 as abnormally low and to define a normal N.R.E.curve as one in which dye is excreted within 10minutes and reaches a maximum concentration of2 to 6 within 20 minutes of intravenous injectionof 5 ml. of 1 per cent neutral red.

Neutral-red excretion tests in patients withgastric or intesial disorders.-Tests were per-formed in a military hospital and in the Profes-sorial Surgical Unit, Manchester Royal Infirmary,on fifty-three patients with disturbed gastric orintestinal function. The cases fell into eightgroups, viz.: gastric carcinoma, peptic ulcer,"duodenitis," gastric neurosis, lienteric diarrhoea,chronic dyspepsia including chronic gastritis andalchoholism, cholecystitis, and a miscellaneousgroup.The ten patients with carcinoma of the stomach

were all in the 40 to 70 years age group; six weremen. 1n eight patients clinical and x-ray diagnosiswas confirmed by laparotomy. One patient hadprimary carcinoma of the transverse colon withdirect malignant infiltration of the greater curva-ture.Of the sixteen patients with peptic ulcer, four-

teen were men: six were under 30, seven between30 and 50, and three were more than 50 years ofage. Diagnosis of duodenal, gastric, or gastro-jejunal ulcer was made by correlating clinicalsymptoms and signs with x-ray evidence followinga barium meal. In ten cases laparotomy was per-formed and the diagnosis confirmed.The three patients with "duodenitis" were all

men under 30 years old. Diagnosis was made

when classical symptoms of duodenal ulcer ofrecent origin were present, and when no x-rayevidence of peptic ulcer was found.Of the four cases diagnosed as gastric neurosis,

three were men: all were under 25 years of age.In three an anxiety state was present: the otherwas a case of depression with vague gastric symp-toms following a head injury.The three cases with lienteric diarrhoea were all

young men: each gave a classical history of lien-teric and post-pandrial diarrhoea of several years'duration. Clinical and x-ray examinations werenegative.The chronic dyspepsia-gastritis group was a

heterogenous group of ten men and one woman.Four were under 30 years of age, three between30 and 50, and the remainder older men. Threeof the patients were chronic alcoholics and hadmorning anorexia with flatulent dyspepsia (onealso had mild polyneuritis and liver cirrhosis); theremainder gave a history of dyspepsia (flatulenceand/or discomfort after meals) with or withoutanorexia of some years' duration. In some casesthe history was typical of chronic gastritis, but inothers a satisfactory diagnosis could not be made.Cardiovascular disease was minimal or absent, andneither clinical nor x-ray examination was of posi-tive help. In three cases, laparotomy revealed noabnormality.The diagnosis of cholecystitis in one case (male,

age 59 years) was made on clinical grounds and anegative barium meal radiograph: in the other (awoman, age 62 years), the barium meal radiographshowed diminished motility of the stomach, andgastroscopy an atrophic gastritis. At laparotomy,cholelithiasis was found and cholecystectomy andappendicectomy were performed.The miscellaneous group were cases of subacute

nephritis, repeated attacks of acute gastritis,achalasia of the cardia, and severe intestinal colicof unknown origin.

Results of N.R.E. tests.-Tests on twenty-nineof the fifty-three patients were within the normalrange. The abnormal results fell into two groups,viz.: those in- which there was no excretion of dyeinto the stomach (anexcretory type) (13 cases), andthose in which the dye was poorly concentrated(11 cases). This group could be subdivided intotwo partially overlapping subgroups: (a) thosewith a normal E.T., CM less than 2 and a normalor delayed C.T., and (b) tests with normal ordelayed E.T. and CM 1 or less. The dye excretionin three was slight and intermittent. For descrip-tive purposes, these will be called hypo-excretorytypes a and b. The figure portrays examples of

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these curves in contrast with a normal result, andone in which the dye was not excreted.

Table I classified the tests according to diagnosis,the non-gastric controls being included for com-parison. Table II is a more detailed case and testanalysis of the patients with abnormal N.R.E. tests.Of ten patients with gastric carcinoma, five failed

to excrete neutral red; in two excretion was poorand intermittent. Four of the eleven patients withchronic dyspepsia or gastritis did not excrete thedye, in two excretion was poor and delayed orintermittent. Both cases of cholecystitis were ofthe anexcretory type, but one patient also hadatrophic gastritis. Of the sixteen peptic ulcerpatients, ten had normal N.R.E. tests, the re-mainder hypoexcretory curves. Each case of," duodenitis " and lienteric diarrhoea, and three ofthe patients with gastric neurosis excreted neutralred normally. One of the latter (post head-injurydepression) failed to excrete the dye. Of the fourmiscellaneous cases, the patient with achalasia ofthe cardia did not excrete neutral red and thepatient with undiagnosed intestinal colic had onlya very delayed and slight excretion of dye.From these results it is clear that abnormal

N.R.E. tests of the anexcretory and hypoexcretorytypes may occur in various organic disorders ofthe stomach and duodenum. Separate types ofN.R.E. curves were not detected for particulardiseases, and therefore the test cannot be of helpin discriminating between (say) gastric carcinomaand gastritis. It is doubtful whether the test candistinguish between organic and functional dis-order of the stomach, as cases of achalasia of thecardia (a local nervous disorder) and post-head-injury depression (with functional gastric symp-toms) had anexcretory results. Moreover, normalN.R.E. tests can be given by patients with well-established organic disease of the stomach such asgastric carcinoma or peptic ulcer.

In practice the N.R.E. test is of value when anabnormal result is obtained: the conclusion maythen be drawn that there is a disorder of the gastricmucosa of organic, functional, or possibly reflexorigin.

Gillman (1944) found that normal results wererepeatable after variable intervals of time. Toconfirm this, nine patients were subjected to asecond test two to five weeks after the first. Sevenhad given normal N.R.E. curves on the first occa-sion and when repeated these tests were still withinnormal limits; for example Case 3 (chronic gas-tritis) excreted neutral red at 3 minutes and con-centrated it to a maximum of 2-3 in 14 minutes:five weeks later, excretion of dye began in2 minutes and reached a maximum of 3 in 8

minutes. Two other patients failed to excrete thedye both at the first tests and when these wererepeated a few weeks later.Anatomy and physiology of neutral-red excre-

tion.-Attempts were made to compare the bloodlevel of the dye with its concentration in the gastricaspirate. In a patient with a blood volume of(say) 7 litres, the injection of 5 ml. of 1 per centsolution should produce a blood level of dye ofabout 1 in 140,000 or a plasma level of about 1 in270,000 if all the dye remains in the plasma andif none diffuses into the extracellular tissues or islost by excretion. In vitro experiments showedthat when neutral red is added to oxalated wholeblood, almost the entire quantity is present in theplasma, where its concentration could be estimatedcolorimetrically up to a concentration of 1 in250,000 and detected up to 1 part per million.Venous blood was removed from eight patients

(from the arm opposite to the injection) 5 to 20minutes after injection of the dye. In no case wasit possible to detect neutral red in the plasma,although at the same time dye was being excretedin the juice in concentrations of 1/50,000 to1/200,000. In two patients from whom blood wasremoved 5 and 6 minutes after injection, the dyewas just appearing in trace concentration in thegastric juice. The urine was free from dye and itwas considered impossible that excretion of dyecould have been responsible for a plasma levelof below 1 part per million. It was therefore con-cluded that neutral red rapidly diffuses into theextracellular tissues of the body, is present in aconcentration of less than 1 in a million in theplasma, and is concentrated by the gastric mucosaduring excretion at least 10 to 20 times.

In a patient with a duodenal ulcer (Case 49),the site of neutral-red excretion was establishedby a method suggested to one of us by ProfessorA. M. Boyd. This patient had an ulcer history oftwenty years' duration, verified by barium mealx-ray examination. The fractional test mealshowed hyperacidity and the N.R.E. test per-formed the day before the operation was normal(E.T. 2 minutes, C.T. 4 minutes, CM 4).

Premedication with omnopon, 1/3 gr., andscopolamine, 1/150 gr., was given an hour and ahalf before operation which was carried outunder ether anaesthesia. Partial gastrectomywas performed: during the course of the operation5 ml. of 1 per cent neutral red was injected intra-venously as soon as the greater curvature had beenfreed and the right gastric artery ligatured. Withina few minutes, the upper half of the gastric mucousmembrane was stained red by the dye (see Plate

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VIII) and there was a fairly sharp line of demar-cation halfway down the stomach below which nodye was excreted. The anexcretory area includedthe lower half of the body of the stomach, thepyloric antrum, and the pylorus. Excretion of dyestill persisted when the surface was washed withsaline.Was the lack of the excretion in the lower half

of the stomach due to tying the right gastric arteryand freeing of the greater curvature? To excludethis possibility the injection of dye was repeatedseveral times during the course of further gastrec-tomies for peptic ulceration and the same distribu-tion of dye excretion was obtained notwithstand-ing when the dye was injected or its relationshipto the tying of vessels.Was the lack of excretion in the lower half due

to antral gastritis ? The gastric mucosa lookednormal and subsequent histological examination ofvarious areas including the antrum showed noabnormality.

Evidence of the nervous control of dye excre-tion was obtained in other cases. In a patient witha gastro-jejunal ulcer (see Table II, Case 56) therewas a low normal gastric acidity befor,e operation,and excretion of neutral red occurred although thiswas delayed and rather poor. Four weeks aftera succcessful transpleural vagotomy the tests wererepeated. No excretion of dye was found, and thepatient had achlorhydria.

N.R.E. tests with normal results were performedon five patients, who for various reasons were latersubmitted to sympathectomy: three patients hadunilateral and two bilateral sympathectomy fromT 5 to L 3 performed; splanchnicectomy was alsocarried out. The tests were repeated 2 to 3 weekslater and no significant changes in the excretion orconcentration of dye by the stomach was found.

Several unsuccessful attempts were made both inrabbits and in man to discover the cellular originof dye excretion. Biopsies of the stomach wallfrom the fundus, body and pylorus were takenwithin a few minutes of an intravenous injectionof neutral red. The human material was collectedduring' the course of partial gastrectomies.Frozen and paraffin sections were made and ex-amined, stained and unstained, but no intracellulardye was seen even after treatment of the sectionwith acid (neutral red is yellow in alkalinesolution).These results indicate that the excretion of

neutral red is limited to the upper half of thegastric mucosa, that excretory activity is abolishedby vagotomy, and that the sympathetic plays nopart in the control of the excretion. The nervouscontrol of the dye excretion and acid secretion is

identical. Hydrochloric acid is secreted by themucosa of the body of the stomach, probably overa wider area than that responsible for neutral-redexcretion. The parietal (oxyntic) cells are notpresent in the pyloric region where an alkalinefluid is secreted. Stimulation of the vagus nervein dogs produces a flow of strongly acid juice,whilst stimulation of the sympathetic splanchnicnerves gives only a slow steady secretion of fluidrich in mucus, often alkaline in relation (Pavlov,1910; Carlson, 1923; Babkin, 1938). This lattersecretion comes mainly from the pyloric glandsand only to a minor extent from the body of thestomach.From similar anatomical sites of acid secretion

and neutral-red excretion, and from their iden-tical nervous control, it might be expected ontheoretical grounds that a relationship between acidsecretion and dye excretion exists, and that anabnormality in one property would be accom-panied by a parallel abnormality in the other, forexample achlorhydria with failure to excrete thedye.

Relationship of acid secretion to neutral-redexcretion by the gastric mucosa.-" Free " and"total" acid estimations were performed onspecimens of gastric juice from all patients,including the controls. Forty of the cases withdisturbed gastric or intestinal function -had frac-tional test meals performed: in fifty cases aciditywas estimated on several specimens of gastricjuice obtained during the course of the N.R.E.test, and in four of these acid estimations wereperformed on juice obtained after an injection ofhistamine.Acid secretion of the stomach has a wide range

of normality, and it is therefore difficult to decidewhen abnormality begins. Arbitrary figures haveto be adopted, and it was decided to accept a freeacid equivalent to 70 ml. of N/10 hydrochloricacid per cent (70 per cent) as the limit of the upperrange. Similarly the limit of the lower range wasaccepted as a free acid volume of 10 ml. of N/10hydrochloric acid per cent (10 per cent). If oneor more fractions of juice had a value of 70 percent or over, hyperacidity was said to be present:if all the specimens of juice analysed were lessthan 10 per cent, hypo-acidity was said to exist.From an examination of Tables I and II it is

clear that the ability of the stomach to excreteneutral red and to secrete hydrochloric acid are notnecessarily allied. Six patients (Cases 2, 9, 20,25, 50, and 56) with normal acid production hadhypo-excretory dye curves, one patient with hyper-acidity excreted the dye poorly (Case 28), andanother (Case 66) with normal acid production

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failed to excrete the dye at all. Gillman's (1944)statement that patients with very poor or noexcretion of dye are always achlorhydric is in-correct. However, there is a statistical correlationbetween normal acid production and normalneutral-red excretion and between achlorhydriaand lack of excretion of dye. This relationshipis probably the reflection of a common anatomicalmucosal site for secretion and excretion and of acommon nervous control of these activities.

Conclusions and SummaryThe excretion of neutral red by the normal

gastric mucous membrane follows the same pat-tern in different people within fairly narrow andeasily recognized limits. Normality and abnor-mality in dye excretion is readily established usingthe neutral-red excretion test standardized byGillman. When the test is repeated on the sameindividual, identical or closely similar results areobtained whether dye excretion is normal orabnormal. Rarely is an abnormal N.R.E. testgi,ven by a patient with a healthy gastric mucosa,and it can be said that abnormality in excretion ofthe dye is strongly suggestive of disordered gastricfunction.For these reasons the N.R.E. test is superior and

more reliable than the estimation of acid secretion.Other material advantages include safety, sim-plicity, and the short time it occupies. Further-more, the dye and the apparatus are easilyobtained, and if necessary the test can be per-formed by the clinician at the bedside.

All investigators agree that total failure toexcrete neutral red is diagnostic of gastric dys-function and many (Lourja and Mirkin, 1925;Morrison, 1938; Gillman, 1944) that -partial sup-pression of dye excretion is an abnormal resultpreceding the final stage of total suppression.Whilst the present investigation is in the main inagreement with these observations, there is nodoubt that patients with various disorders of thestomach and duodenum, including carcinoma andpeptic ulcer, may have normal dye excretion. Thismay occur notwithstanding the duration of the dis-order or the severity of the condition. A normalresult is then of no value in distinguishing a normalfrom a pathological stomach, and the test is notnearly as sensitive as Gillman suggests.

Furthermore, neither a normal nor an abnormalresult is of diagnostic use in differentiating betweencancer, gastritis, and other conditions. Althoughthe results of the peptic ulcer series suggest that

complete suppression of excretion does not occurin cases of simple ulcer, this may not be true andmay result from pure chance in the selection ofthe cases. Functional disorder of reflex or nervousorigin may or may not suppress excretory func-tion, so that the N.R.E. test is of no value in distin-guishing organic from non-organic disease.The problem of serial tests on the same patient

to determine the deterioration or recovery fromgastric disorder was not studied.Although a statistical relationship- exists between

excretion of neutral red and secretion of acid, thecorrelation has exceptions and in any individualcase disorder of one function may or may not beaccompanied by a parallel disorder of the other.

Finally, experiments and observation indicatethat, following the injection of neutral red, mostof the dye rapidly diffuses into the extracellulartissue fluid and is present in the blood in minuteundetectable traces (concentration < 1 part permillion) although at the same time it is beingexcreted by the gastric mucosa in concentrationsup to 1 in 50,000. The parasympathetic vagusnerves are probably the excitory nerves controllingdye excretion, and the sympathetic plays no partin the activity.The investigations of one of us (S.S.) were carried out

at the Military Hospital for Head Injuries, Wheatley,Oxford, and of the other (R.P.J.) in the ProfessorialSurgical Unit, Manchester Royal Infirmary. We areindebted to many clinical and laboratory colleagues andto members of the nursing staff of both hospitals for theirco-operation and assistance. In particular we wish tothank Dr. J. Bull for his statistical assistance in theanalysis of part of Table I1, Prof. A. M. Boyd for histimely suggestions and co-operation during the pursuanceof the work in Manchester, and Miss D. Davidson forher excellent drawing. We are indebted to Lieut.-Col.E. H. Hall, R.A.M.C., A.D.P. Southern Command, forhis interest. We acknowledge the permission of theArmy Medical Department, War Office, to publish thisinvestigation.

REFERENCESBabkin, B. P. (1938). Amer. J. digest. Dis., 5, 467.Carlson, A. J. (1923). Physiol. Rev., 3, 1.Davidson, P. B., Willcox, E., and Haagensen, C. D. (1925). J. Amer.

med. Ass., 85, 794.Gillman, T. (1943). S. Afr. J. med. Sci., 8, 50.Gitlman, T. (1944). Gastroenterology, 3, 188.Glaessner, K., and Wittgenstein, H. (1923). Klin. Wschr., 2, 1650.Lourja, R. A., and Mirkin, A. I. (1925). Arch. Verdauungskrankheiten,

34, 5.Morrison, S. (1938). Amer. J. digest. Dis., 5, 617.Palmer, W. L., Kirsner, J. B., and Nutter, P. B. (1940). Amer. J.

digest. Dis., 7, 427.Pavlov, I. P. (1910). "Work of Digestive Glands." Second ed.

Griffin: London.Schiff, L. (1938). Arch. intern. Med., 61, 774.Winklestein, A. (1942). Amer. J. med. Sci., 203, 419.Wright, Samson (1941). "Applied Physiology." Seventh edition,

Oxford University Press.Zibalis, S. (1934). Pr. Med., 42, 1260.

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