FAILURE OF ENDOGENOUS STIMULATION OF SECRETIN AND PANCREOZYMIN RELEASE TO INFLUENCE SERUM-INSULIN

448 be recognised at a glance by an experienced microscopist, and all such slides should be discarded. This error has been excluded in the present series. Secondly, the virus may not be present, at the time of collection of the specimen, in the nasopharynx. Deeper penetration into the respiratory tract, in the child with pneumonia, may have carried the agent beyond the reach of a throat swab. In 4 of the 5 cases with apparent false-negative F.A. results, the children had pneumonia. In the 5th (case 26) the clinical diagnosis was " measles ", and, although falling and significant antibody titres to R.s.v. were observed, the measles antibody rose significantly. It may be argued that, in children with pneumonia, sputum or nasopharyngeal secretions obtained after a coughing spell might offer a better prospect for diagnosis. This possibility has not been examined.

False positives by the F.A. technique were recorded in only 4 children (14%). Unfortunately, in 2 of these, specimens for serological testing were not obtained. The 3rd showed a rising antibody titre to measles virus, and the last showed no confirmatory evidence of R.S.V. or of any other virus infection. In view of the main virtue of the F.A. technique-that of speed-a small proportion of false-positive results may be acceptable to the clinician. This work was performed as part of an extended trial for the evaluation of immunofluorescent techniques in clinical virus diagnosis supported by a grant from the Western Regional Hospital Board, Glasgow; the Board’s generous support and encouragement is gratefully acknowledged. We are grateful to Dr. P. McKenzie and Dr. W. Campbell Love for allowing us to collect material from patients in their care; to Mr. A. S. Bryden, F.I.M.L.T., for assistance with the serological testing; and to Miss V. Jasnosz for assistance with the preparation of specimens for fluorescence microscopy. REFERENCES

Adams, J. M., Imagawa, D. T., Zike, K. (1961) J. Am. med. Ass. 176, 1037. Beem, M., Wright, F. H., Hamre, D., Egerer, R., Oehme, M. (1962) New Engl. J. Med. 263, 253. Chanock, R. M., Kim, H. W., Vargosko, A. J., Deleva, A., Johnson, K. M., Cumming, C., Parrott, R. H. (1961) J. Am. med. Ass. 176, 647. Gardner, P. S., Turk, D. C., Aherne, W. A., Bird, T., Holdaway, M. D., Court, S. D. M. (1967) Br. med. J. ii, 316. Sommerville, R. G. (1967) Arch. ges. Virusforsch. 20, 452.

FAILURE OF ENDOGENOUS STIMULATION OF SECRETIN AND PANCREOZYMIN RELEASE TO INFLUENCE SERUM-INSULIN RICHARD J. MAHLER College

M.D. New York Medical

ASSISTANT PROFESSOR

HERBERT WEISBERG M.D. Lausanne ASSISTANT PROFESSOR

From the Department of Medicine (Sections of Endocrinology and Gastroenterology), New York Medical College

The effect of endogenously stimulated and exogenously administered secretin and pancreozymin on blood-glucose and serum-immunoreactive-insulin was studied in healthy controls and in patients with histamine-fast anacidity. When the release of secretin was stimulated by duodenal acidification in three patients with histamine-fast anacidity, the serumSummary

immunoreactive-insulin response and the blood clearance of an intravenous glucose load were not enhanced. In the absence of a glucose load, in five anacid patients duodenal acidification did not lower fasting blood-glucose or raise serum-immunoreactive-insulin. Intravenous administration of either secretin or pancreozymin to four fasting healthy controls caused a small rise in serum-immuno-

reactive-insulin, which was associated with a slight fall in blood-sugar in the case of pancreozymin but not of secretin. These results indicate that the effect of intravenously administered secretin and pancreozymin on serum-insulin is not duplicated by the stimulation of their endogenous release. They suggest further that the endogenous release of these hormones does not normally influence the utilisation of ingested carbohydrate. Introduction

GLUCOSE when given by mouth is cleared more rapidly from the blood than when it is injected intravenously (Scow and Cornfield 1954, Conard 1955, Dupre 1964). This observation has led to the hypothesis (McIntyre et al. 1964) that ingested carbohydrate induces the release of an intestinal factor which stimulates insulin secretion more rapidly and efficiently than the rise in blood-sugar alone. The suggestion that the duodenum is the site of this factor is supported by recent work in which intravenous injections in dogs of the duodenal hormones, secretin and pancreozymin each produced a rise. in serum-immunoreactive-insulin (Meade et al. 1967, Unger et al. 1966, 1967). Similar results have also been obtained in man after injection of secretin (Conard 1955, Boyns et al. 1967) but not after injection of pancreozymin (Boyns

al. 1967). The increase in serum-insulin produced by intravenous injection of these hormones, however, does not necessarily mean that they regulate pancreatic insulin secretion under normal conditions. Other workers, for example (Boyns et al. 1966, 1967), were unable to enhance the seruminsulin response or the disappearance of blood-glucose of an oral or an intravenous glucose load in healthy persons by simultaneous duodenal acidification-a known endogenous stimulant for the release of secretin and et

pancreozymin. In health,

however, fasting duodenal pH may be spontaneously acid, and acidification of the duodenum may not induce further release of secretin and secondarily of pancreatic insulin. In view of this possibility, the present investigation was undertaken to determine whether duodenal acidification in anacid patients lowers bloodglucose, raises serum-immunoreactive-insulin, and enhances the clearance of an intravenous glucose load from the blood. In addition, to compare the effects of endogenously stimulated and exogenously administered secretin and pancreozymin, the serum-insulin and blood-glucose response to parenteral secretin and pancreozymin in

fasting

controls

was

investigated.

Subjects and Methods Duodenal Acidification in Anacid Patients in the Response Presence of a Glucose Load Three patients with pernicious anaemia and proven anacidity to maximal histamine stimulation were fasted overnight; on the morning of the test they were given 300 ml. of 0-1N hydrochloric acid by mouth, followed in 15 minutes by the rapid intravenous infusion of 0-5 g. per kg. glucose. The time taken to drink the hydrochloric-acid solution did not exceed 3 minutes, and the intravenous glucose infusion did not exceed 5 minutes. The completion of the intravenous glucose infusion was taken as " 0 " time. For control, an identical intravenous glucose load was given to the same patients on a different day after they had taken 300 ml. of 0-9% sodium-chlorine solution adjusted to pH 7 by mouth. During each test and control period, blood-samples were drawn at 0, 1, 3, 6, 10, 20, 30, 40, 50, and 60 minutes and assayed for blood-glucose and serum-immunoreactive-insulin. to

449

Response to Duodenal Acidification in Anacid Patients in the Absence of a Glucose Load After an overnight fast, four patients with pernicious anaemia and one patient with atrophic gastritis (all anacid to maximal histamine stimulation) drank 300 ml. of 0.1 N hydrochloric acid, in which a trace amount of barium sulphate dissolved. The time taken to drink the solution did not exceed 3 minutes. Each patient was then continuously screened radiographically, and the time of entry of the solution into the duodenum was taken as " 0 " time. This acidbarium mixture has been shown to acidify the duodenal contents in healthy subjects when the barium enters the duodenum (Bircher et al. 1965). Through an indwelling needle inserted into an antecubital vein, blood-samples were drawn before and after the solution was swallowed at -10, 0, 1, 3, 5, 10, 12, 15, 20, and 30 minutes. Each sample was assayed for blood-glucose and serum-immunoreactiveinsulin.

assayed for insulin by the double-antibody method of Morgan and Lazarow (1963). The rate of disappearance of glucose from the blood (Kg) was calculated according to the graphic method described by Conard (1955).

tion and

Results

was

to Parenteral Secretin and Pancreozymin in Healthy Volunteers Secretin (Boots Pure Drug Co. Ltd., Nottingham) was dissolved in 0-9% sodium chloride at a concentration of 11.5 units per ml. and injected intravenously into two healthy volunteers at a dose of 1.5 units per kg. and a rate of 4 ml. per minute.

Response

Cholecystokinin/pancreozymin (’ Cecekin ’, Vitrum, Stockholm) was dissolved in 0-9% sodium chloride at a concentration of 10 Ivy units per ml. and injected intravenously at a dose of 1.5 Ivy units per kg. into two other healthy volunteers at a of 4 ml. per minute. Both preparations were shown to be active in healthy subjects, and neither contained any immunoassayable insulin as determined by the method used in this investigation. All four volunteers had normal oral glucose tolerance and all tests were conducted after an overnight fast. On a different day, two of the volunteers received a comparable volume of 0-9% sodium chloride alone intravenously to serve as controls.

Response

to

Presence

of a

Duodenal Acidification in Anacid Patients in the Glucose Load

1 illustrates the change in serum-insulin and the of clearance of blood-glucose in three anacid patients after the simultaneous administration of an intravenous glucose load and either 0-1N hydrochloric acid or neutralised 0-9% saline solution by mouth. An increase in serum-insulin was seen in each case within 10 minutes of glucose administration. The early response after acid ingestion was practically identical with that following the ingestion of saline solution. In patients 1 and 2 (fig. 1) some divergence in the insulin response was noted later, with slightly higher insulin levels following the taking of saline solution in one patient and acid in the other. Similarly, the rate of blood-glucose disappearance was unchanged after the acid was taken as compared with saline ingestion (Kg=l-43, 1-76, 1-39 v. 1-35, 1-76, 1-30). Although the shapes of the individual curves of seruminsulin response varied from patient to patient, for each individual patient, the shapes of the curves either with acid or with saline solution were remarkably similar.

Fig.

rate

rate

Glucose and Insulin Determinations All blood-samples were collected from an antecubital vein into oxalated tubes for determination of blood-glucose on an reduction autoanalyser by a modification of the ferricyanide by centrifugawas separated method of Hoffman (1937). Serum

1’1g.

.’—°°°-ucose

acid

Response Absence

to

of

a

Duodenal Acidification in Anacid Patients in the Glucose Load

Oral administration of a 0-1 N hydrochloric acid/barium sulphate mixture to five fasting anacid patients in the absence of a glucose load failed to cause a rise in seruminsulin above baseline values (fig. 2). Despite early and repeated sampling of venous blood for 30 minutes after the radiographically determined entry of the acid-barium mixture into the duodenum, no serum-insulin concentration exceeded the normal range. Similarly, no consistent change in blood-glucose was noted during this period

(fig. 2).

disappearance and serum-insulin response after intravenous glucose load given together with O.lN or neutralised saline solution (solid line) in three patients with histamine-fast anacidity.

(interrupted line)

hydrochloric

450 rises above the baseline were 54% and 115%. Although the increase in serum-insulin following both secretin and pancreozymin injections were substantial when expressed as a percentage above baseline values, the absolute increase measured in , units per ml. in each case was small, and did not exceed the normal range for seruminsulin (see table). Within 40 minutes, all serum-insulin levels returned to pre-injection values. Throughout the entire post-injection period, there was no consistent change in blood-glucose after the injection of secretin (fig. 3). After the injection of pancreozymin, however, blood-sugar concentration fell 22% and 21% below the control values. Absolute values for serum-insulin and blood-glucose during the post-injection period are shown in the accompanying table. Discussion

endogenous stimulation of secretin and pancreonot enhance the clearance of an intravenous did zymin load from the blood nor did it increase serumglucose insulin concentrations. Furthermore, endogenously The

SERUM-INSULIN AND BLOOD-GLUCOSE CONCENTRATIONS AFTER INTRAVENOUS INJECTION OF SECRETIN, PANCREOZYMIN, OR SALINE SOLUTION IN 4 HEALTHY CONTROLS.

Fig. 2-Response to intraduodenal passage of 0·1N hydrochloric acid in five fasting patients with histamine-fast anacidity. Mean, range, and individual readings are shown.

Response

to

Parenteral Secretin and

Pancreozymin in Healthy

Volunteers

The range of baseline values for serum-insulin in four controls was 13-66 jjt. units per ml. Intravenous administration of either secretin or pancreozymin produced a prompt rise in serum-insulin in all of them (fig. 3). Within 3 minutes of injection, the concentration of serum-insulin in the two volunteers who were injected with secretin rose 162% and 226% above baseline levels, and in the two who were injected with pancreozymin the corresponding

*Serum-insulin ([iU per ml.).

fBlood-glucose (mg.

per 100

ml.).

stimulated secretin and pancreozymin did not lower the fasting blood-sugar. These observations, together with those of Boyns et al. (1967), make it unlikely that either - secretin or pancreozymin alone is the intestinal hormone responsible for the more rapid clearance of an oral glucose load over that of an intravenous load. Intravenous injection of secretin or pancreozymin caused a small and transient rise in serum-insulin, and pancreozymin caused a corresponding fall in the fasting blood-sugar. This discrepancy between the effect of exogenously administered and endogenously stimulated secretin and pancreozymin may be due to the fact that the injected hormones were administered in pharmacological amounts not usually attained under physiological conditions in vivo. This hypothesis may be properly tested only when methods become available for measuring the levels of these hormones in the blood, determining their rate of secretion by the duodenum, and assaying their effect in physiological amounts on insulin secretion and glucose utilisation. For our purpose we have assumed that the secretin mechanism is intact in patients with histamine-fast Fig. 3-Response to intravenous administration of secretin, pancreozymin, or saline solution in four fasting healthy controls.

anacidity secondary to atrophic gastritis or gastric atrophy and is responsive to stimulation by the entry of acid into the duodenum.

Although

this has

not

been

specifically

451

proven, it may be reasonably assumed from the knowledge that (1) our anacid patients had no clinically evident impairment of pancreatic function, as one would expect if their secretin mechanism were not intact; (2) patients with pernicious anaemia display a normal exocrine pancreatic response to secretin stimulation (Dreiling 1968); and (3) the secretin mechanism has been shown to respond to food as well as to acid (Wang and Grossman 1951) and is probably maintained by this stimulus in anacid patients. Nevertheless, our results must be interpreted within the limitation of our present inability to test the responsiveness of the secretin mechanism

directly. The increase in serum-insulin after the exogenous administration of pancreozymin is in accord with previous findings in the dog (Unger et al. 1967) but not in man (Boyns et al. 1967). This difference may be explained by the fact that we took samples of venous blood within a minute of injection and at closely timed intervals thereafter, whereas Boyns et al. (1967) took their samples at longer intervals, with the earliest sample taken at 10 minutes. Since the peak increase in serum-insulin after stimulation by either secretin or pancreozymin appears within 1 to 3 minutes, and subsides rapidly (Conard 1955, Unger et al. 1966, 1967), this transient response may not have been detected by previous workers. In conclusion, therefore, endogenously stimulated secretin and pancreozymin release did not promote insulin secretion, enhance the blood clearance of an intravenous glucose load, or lower fasting blood-glucose in anacid patients. These results suggest that secretin and pancreozymin alone are not responsible for the more rapid clearance of an oral glucose load. Since recent work has shown that orally administered protein (Floyd et al. 1966a), intravenously administered aminoacids (Floyd et al. 1966b), gastrin (Unger et al. 1967), and intestinal glucagon (Samols et al. 1966, Hanson et al. 1967) all produce increases in plasma-insulin, the intestinal regulation of plasma-insulin is apparently more complex than was hitherto believed. We are grateful to Dr. Rachmiel Levine and Dr. George B. Jerzy Glass for advice and encouragement; to Dr. R. A. Camerini-Davalos for assays; and to Miss Linda L. Salkin, R.N. for technical assistance. This investigation was supported by grant no. U-1836 of the Health Research Council of the City of New York and graduate training grant TI-AM-5237-02 from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. Requests for reprints should be addressed to R. J. M., New York Medical College, Department of Medicine, 1249 Fifth Avenue, New York, N.Y. 10029. REFERENCES

Bircher, J., Mann, C. V., Carlson, H. C., Code, C. F., Rovelstad, R. A. (1965) Gastroenterology, 48, 472. Boyns, D. R., Jarrett, R. J., Keen, H. (1966) Lancet, i, 409. (1967) Br. med. J. ii, 676. Conard, V. (1955) Acta gastro-enter. belg. 18, 655. Dreiling, D. (1968) Personal communication. Dupre, J. (1964) J. Physiol., Lond. 175, 58. Floyd, J. C., Jr., Fajans, S. S., Conn, J. W., Knopf, R. F., Rull, J. (1966a) J. clin. Invest. 45, 1479. (1966b) ibid. p. 1487. Hanson, J., Ohneda, A., Eisentraut, A., Unger, R. H. (1967) Clin. Res. 14, 43. Hoffman, W. S. (1937) J. biol. Chem. 120, 51. McIntyre, N.. Holdsworth, C. D., Turner, D. S. (1964) Lancet, ii, 20. Meade, R. C., Kneubuhler, H. A., Schulte, W. J., Barboriak, J. (1967) Diabetes, 16, 141. Morgan, C. R., Lazarow, A. (1963) ibid. 12, 115. Samols, E., Tyler, J., Megyesi, C., Marks, V. (1966) Lancet, ii, 727. Scow, R., Cornfield, J. (1954) Am. J. Physiol. 179, 435. Unger, R. H., Ketterer, H., Dupré, J., Eisentraut, A. (1966) Lancet, ii, 24. (1967) J. clin. Invest. 46, 630. Wang, C. C., Grossman, M. I. (1951) Am. J. Physiol. 164, 527. — — —

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ENCEPHALOPATHY AND VISCERAL FATTY INFILTRATION OF PROBABLE TOXIC ÆTIOLOGY

JOHN F. T. GLASGOW M.B., B.Sc. Belf., M.R.C.P., M.R.C.P.I., D.C.H. PÆDIATRIC REGISTRAR

J. A. J. FERRIS ASSISTANT STATE

M.B. Belf. PATHOLOGIST, NORTHERN IRELAND

From the Royal Belfast Hospital for Sick Children, the Nuffield Department of Child Health and the Department of Forensic Medicine of the Queen’s University of Belfast Summary

A child who died after

a

3-day

illness

was

found to have acute encephalopathy and visceral fatty infiltration. Evidence obtained suggested that the patient’s illness and death were caused by poisoning with a commercial paint-thinner. Introduction

Two reports published in 1963 (Anderson 1963, Reye al. 1963) described what appeared to be a distinct clinicopathological entity of childhood, characterised by acute encephalopathy and fatty infiltration of certain viscera. These articles referred to a total of forty-one patients aged between 5 months and 81/2 years. Subsequently, more than thirty similar cases have been described (Corlett 1963, Elliott et al. 1963, Maloney 1963, Utian and Wagner 1963, Steiskal and Kluska 1964). Two reports of what was probably the same condition had been published previously (Brain and Turnbull 1929, Curry et al. 1962). The clinical features of the syndrome are somewhat bizarre. It often seems to start with an upper-respiratorytract infection and vomiting, which becomes persistent. A profound disturbance of consciousness follows, with convulsions, irregular breathing, and changes in muscle tone and the deep reflexes. A low level of glucose in the blood and cerebrospinal fluid (c.s.F.), metabolic acidosis, and some abnormality of liver-function tests are commonly present. At necropsy, brain oedema and gross fatty infiltration of the liver and kidneys are constant findings. The mortality-rate is high, and survivors may be mentally retarded. The cause of the disorder remains obscure (Utian et al. 1964, Randolph et al. 1965, Becroft 1966). The following case-report is therefore of considerable interest, since evidence of a toxic xtiology was found. et

Case-report The patient, a 4-year-old girl previously in good health, had been unwell for 3 days with an apparently mild illness associated with occasional vomiting. On the 2nd day of the illness, vomiting became worse, and on the day of admission the level of consciousness began to deteriorate and occasional convulsive movements of the arms and legs occurred. There was no history to support a diagnosis of poisoning, though the family of twelve lived in an industrial area of the city where various chemical poisons were in commercial use. On examination, the child was comatose and made convulsive of a decorticate nature, particularly when she was moved. There was no retinopathy. The pupils were equal and widely dilated, and reacted slowly to light. Bodytemperature was 99-6°F. Deep reflexes were present, and plantar responses extensor. There was no neck stiffness. Blood-pressure was 140/80 mm. Hg. There was no jaundice

movements