GREEN SPUTUM

12 Alstad 1951). By one or other means hypertensive headaches can be relieved in most cases. Dizzy attacks, breathlessness on exertion, and cardiac asthma are usually relieved, often without the use of other therapeutic measures. Papilloedema, retinal exudates, and

haemorrhages usually disappear. Electrocardiographic improvement is the rule. In our experience, however, such results are obtained only when the regime used has led to a considerable decrease in the average blood-pressure over the twenty-four-hour day. We conclude that about 1 in 4 or 5 hypertensives react sufficiently well to oral H.M.B. to permit an adequate measure of control of the blood-pressure without important side-effects. In contrast, by careful adjustments of the regime, an adequate, though seldom an ideal, control of the bloodpressure can be secured in almost all hypertensives with subcutaneous

H.M.B.

CONCLUSIONS AND SUMMARY

in blood-pressure, much doses of hexamethonium bromide are needed by larger the oral than by the subcutaneous route. The effects of oral administration continue longer but in most patients they are less predictable from one occasion to another. Symptoms of intolerance develop in a large proportion of patients when effective doses are given by mouth. Among the symptoms encountered were malaise, diarrhoea, abdominal distension, nausea, vomiting, and occasionally collapse. Constipation has not been To

produce therapeutic falls

prominent. Oral therapy is

more likely to be successful when the subcutaneous dose needed to control blood-pressure is small, either from natural sensitivity, after sympathectomy, or where excretion is delayed, as in some patients with chronic nephritis. Sometimes a salt-poor diet makes oral therapy possible with hexamethonium bitartrate. Patients whose blood-pressure reduction is inadequate or who have important side-effects from oral H.M.B. should be maintained on the insulin-like regime of subcutaneous H.M.B. The best results can be obtained only by adjusting the dose, times of administration, and mode or modes of administration in terms of whole-day observations of the patient’s response. In practice such tests must be made by trained technicians. Failure to ameliorate hypertensive manifestations with hexamethonium salts is usually due to a defect in technique, such as neglect to make use of the more satisfactory falls of blood-pressure in the standing and sitting postures, neglect of the changing sensitivity to R.M.B., incorrect usage, or unwillingness to use the subcutaneous route when the oral route is unsatisfactory.

We are indebted to Miss M. Poppelwell for secretarial work, and to Miss A. N. Hoggan and Miss J. Rivers for technical assistance. Messrs. May & Baker Ltd. supplied the hexamethonium salts. The expenses of the work were defrayed in part by the Medical Research Council of New Zealand.

GREEN

SPUTUM *

A. JOHN ROBERTSON M.D. MEDICAL

REGISTRAR,

Lpool,

M.R.C.P.

ROYAL

INFIRMARY, LIVERPOOL

"Certum est, quod in omnibus pectoris morbis, sputa attentam mereantur considerationem."—VAN SWIETEN (1764).

IT has been noticed for many years that patients with bronchiectasis may bring up green sputum. I had been impressed by the frequency with which the converse appeared to be true, and patients with a history of green sputum very often were found to have bronchiectasis. It was felt that the relation between these two was worthy of further study ; so I tried to find out why sputum became green, and its connection with bronchiectasis. Green sputum has been described for centuries, and Hippocrates used it as a prognostic sign ; he declared that patients with pains in the lungs whose expectoration was very green and frothy did no good (Adams 1886). This gloomy outlook was maintained throughout the 18th century, and both Boerhaave (Van Swieten 1764) and Stoll (1787) wrote that, if the sputum became bilious after the sixth day of a pneumonia, the patient would die on the seventh or ninth day. The first serious attempt to explain why the sputum was green was made by Andral (1821), who attributed the varied colours of sputum to the quantity of blood He found experimentally that he that it contained. could produce yellow, green, and other colours by mixing water with various quantities of blood. Laennec (1834) also thought that the shades of green which he saw in the sputum pot depended on blood existing in a greater or less proportion." Grisolle (1841), who was interested in green sputum, disagreed with the previous eminent writers. He thought that the opinion of Hippocrates in singling out green sputum as being a bad sign in pneumonia was probably false, though he was not sure of its exact significance himself. He repeated Andral’s experiments many times and never succeeded in producing the green colour. Professor Traube’s views on the aetiology of the green colour were published by his pupil Nothnagel (1864), and are summarised as follows :

(1) Jaundice plus respiratory infections : (a) " biliary pneumonia " ; (b) bronchial catarrh with mucus, in the presence of

jaundice. (2) Following (a) (b) (c)

chronic

pneumonia :

croupus pneumonia, as yet unresolved ; croupus pneumonia, going on to lung abscess ; subacute caseous pneumonia, whether tuberculous

or

not.

REFERENCES

Arnold, P., Goetz, R. H., Rosenheim, M. L. (1949) Lancet, ii, 408. Rosenheim, M. L. (1949) Ibid, p. 321. Barlow, R. B., Ing, H. R. (1948a) Nature, Lond. 161, 718. (1948b) Brit. J. Pharmacol. 3, 298. Campbell, A., Robertson, E. (1950) Brit. med. J. ii, 804. T. C., De Elio, F. J. (1947) Brit. J. Pharmacol. 2, 268. Chou, Frankel, E. (1951) Lancet, i, 408. Freis, E. D. (1951) Ibid, p. 909. Locket, S., Swann, P. G., Grieve, W. S. M. (1951) Brit. med. J. i, 778. Paton, W. D. M., Zaimis, E. J. (1948a) Nature, Lond. 161, 718. — — (1948b) Ibid, 162, 810. (1949) Brit. J. Pharmacol. 4, 381. Restall, P. A., Smirk, F. H. (1950) N.Z. med. J. 49, 206. Saville, S. (1950) Lancet, ii, 358. Smirk, F. H. (1949) Brit. med. J. i, 791. (1950a) Lancet, ii, 477. (1950b) N.Z. med. J. 49, 637. (1951) Amer. Heart J. 42, 530. Alstad, K. S. (1951) Brit. med. J. i, 1217. Turner, R. (1950) Lancet, ii, 353. —

—

—

—

—

—

—

Fig. I-Optical pathway in Beckman spectrophotometer.

It was thought that in the cases with jaundice the- pigment was bile, and that in the postpneumonic group the pigment was a hsematin compound, which was changed by oxidation in an analogous fashion to the colour changes in a bruise.

—

—

* A paper delivered to the Thoracic Feb. 23, 1951.

Society

in London

on

13 Rosenb a c h

to be excluded.

The first is by conversion to bile pigbiliverdin-as ments-e.g., suggested by Traube (Xothnagel 1864). The second is from bacteria-e.g. Strep. zvridans and certain pneumococci. Hart and Anderson (Hart and Anderson 1933, Anderson and Hart 1934) have described the mechanism whereby these organisms produce a green colour on blood-agar. They showed that this green is not an intrinsic bacterial pigment but a conversion product of haemoglobin, which Lemberg and Legge (1949) later found to be choleglobin. It was thought best to deal with this problem by finding some solvent for the green colour, analysing the solution spectroscopically, and searching for haemoglobin derivatives-e.g., choleglobin and biliverdinand for the two pigments pyocyanin and fluorescin, which are formed by Ps. pyocyanea.

(1875) noticed that occasionally sputa left standing i n jars would turn grassgreen, and he gave experimental1 evidence in of favour

bacterial

con-

tamination,

by inoculating milk some

with of this

METHOD

and producing the

sputum, same

Some

Frick (1889) further investigated the

5CC

samples oj sputum

green.

green Fig. 2-Absorption

curves

of green sputum.

collecfrom patients with bronchiectasis or lung were

ted

and described the three groups of conditions in which he had found green sputum : jaundice ; after pneumonia, as described by Traube ; and bacterial contamination in the sputum jar by pyocyaneus organísm&bgr;. This list of Frick’s has been copied from textbook to textbook, usually with omission of his emphasis that pyocyaneus organisms are contaminants. Apart from a description of an epidemic of green sputum (Combemale and Francois 1890) there does not seem to have been anything further published on this

bacteriological side,

subject.

abscesses, and attempts made to make a solution suitable for spectroscopy. It was found to be

extremely THEORETICAL

It will be obvious that

difficult to get rid of protein in the Fig. 4-Absorption curves of five samples of green form off sputum on reduction, showing peaks at 637 mc.

the green colour might

and tne mucus

POSSIBILITIES

arise from at least five sources- bac-

teria, erythrocytes, leuco-

cytes, bronchial

alveolar and blood-serum or its breakdown products. or

cells,

"albumin,’" wnicn

was

curves of two samples of green well-marked change of pigment vivid green on reduction.

also

A

Beckman o meter was

pigment-

used, and

forming organisms,

optical path-

such as f6M dorno1las does pyocyaitea, are commonly incriminated, not fit in with the clinical facts. Not only are these organisms rarely found in sputum cultures, but also many patients give a history of coughing up green phlegm in the morning, and say that later on in the day their sputum has changed to yellow or even white. It seems unlikely that any bacterial infection would behavein this wav. It was also unlikely that the colour arose from the erythrocytes ; firstly because green sputum would then really mean occult bleeding, and secondly because the sputum of patients with haemoptysis alone does not become green. There are, however, two ways in which any hæmoglobin present could give the green colour, and these had to more

always present

spectroph 0t

Although Fig. 3-Absorption sputum, showing

almost

greatly with spectroscopy. In addition, any bacterial pigments present had to be extracted. After much trial and error the method adopted was simply to centrifuge the sputum, preferably after allowing it to stand for some time, and then to emulsify the greenish upper layer with chloroform, and again centrifuge for a long time. In this way it was possible to get a green solution which was clear enough for spectrophotometry, and it could be concentrated either by boiling under low pressure or by dialysis. interfered

its

way is shown ill fig. 1. In this instrument light is

this

passed through a quartz prism and spread into such

a

broad

spectrum that it to

thought

is possible send very

narrow

bands

the The of amount

through solution.

transmi8sion at any verdoperoxidase

light

Fig.

S-Absorption

curve

of

(Agner 1941).

particular

14 can be measured with the photometer, and be plotted showing the absorption peaks may which take the place of the less accurate " bands" of the direct-vision spectroscope.

wave-length curves

SPECTROSCOPIC FINDINGS

from the green solution are shown in will be seen that there are absorption peaks at 690, 625, 570, and 425 mu, which changed on reduction with sodium hydrosulphite to 637, 590, and 475 m. Fig. 3 shows a portion of the curve between 550 and 700 mu. from two samples, emphasising the well-marked change which the pigment undergoes on reduction (when it becomes an even more vivid green). The remarkably constant absorption peak at 637 mfl. in five separate specimens of reduced pigment is shown in fig. 4. At this stage great difficulty was experienced in identifying the compound because there are no satisfactory tables of absorption bands, and it was through almost fortuitous reading of some rather small print in the work on haematin compounds by Lemberg and Legge (1949) that Agner’s work was discovered. It will be seen from fig. 5, adapted from Agner (1941), that the curve of verdoperoxidase looks very similar to that of green sputum. Three criteria are necessary before establishing spectroscopically that any two substances are identical :

Typical fig. 2. -It

curves

all fulfilled, and the colouring as identified verdoperoxidase. By thereby some of adopting Agner’s procedures the green solution could be further purified, and fig. 6 shows the final method of dealing with the sputum. It will be seen that pyocyanin when present was extracted with chloroform in the same way that its discoverer Fordos (1860, 1863) had done nearly a hundred years ago. The pigment fluorescin, whose well-marked fluorescence in ultraviolet light was at first puzzling, was separated out (when present) by its failure to be precipitated with ammonium sulphate, and it could be identified by its spectroscopic properties (Turfitt 1936, 1937). After this, increasing amounts of unwanted protein were removed from the solution by precipitation with alcohol and ammonium sulphate, until fairly pure verdoperoxidase remained. The final green solution gave an intense blue reaction with tincture of guaiacum and hydrogen

(1) The two compounds must have absorption peaks at the wave-length. (2) The amount of light transmission (optical density) at these various peaks must be relatively the same in both

same

cases.

treatment with such reagents as sodium hydrocarbon monoxide, and sodium cyanide the absorption must again be identical.

(3) On

sulphite, peaks

These criteria

were

matter

peroxide. VERDOPEROXIDASE

It had long been known that tincture of guaiacum was stained blue by pus (Klebs 1868), and Meyer (1903) showed that this seemed to be due to the leucocytes. He found that blood from patients with myeloid leukaemia gave a strong blue reaction to guaiacum, whereas blood from lymphatic leukaemia did not, and he thought that the reaction was due to an oxidase present in the myeloid cells. However, Linossier (1898) distinguished between oxidase and peroxidase reactions by showing that the guaiacum reaction with pus would only take place in the presence of hydrogen peroxide. One problem in deciding the enzyme responsible for such a change is that hmmoglobin and other hsemin compounds can also catalyse peroxidase reactions. Agner (1941) isolated the peroxidase found in the granulocytes and studied its properties in detail. He first used empyema fluid, later demonstrated the same enzyme in the white cells in myeloid leukaemia, and finally identified verdoperoxidase as a cause (at least in part) of the green infiltration of the sternum in a museum specimen of chloroma some 27 years old. He showed that this enzyme was bound with the structure of the granulocyteand was set free when the granulocyte disintegrated. It was distinguished from most other peroxidases by being green when prepared in its final pure state by cataphoresis-hence the name verdoperoxidase, which Agner has since changed to myeloperoxidase in view of the origin of the enzyme. SIGNIFICANCE OF GREEN SPUTUM

It has been shown that in green sputum various substances can be identified. If there should be an infection with Ps. pyocyanea (this is rare), pyocyanin and fluorescin may be present, which The remaining green seems to can be extracted. be almost entirely due to verdoperoxidase, which can be readily identified spectroscopically. An analogy may now be drawn with the excretion of bile. When red cells disintegrate bile pigments are formed, and if there is interference with their excretion the serum becomes visibly jaundiced. Similarly, when the granulocytes disintegrate verdoperoxidase is set free, and if there is failure to excrete this enzyme the purulent material becomes visibly The significance of these findings is that green sputum is purulent sputum in which there is failure to excrete verdoperoxidase, which accumulates and colours the phlegm green. This happens whenever there is stagnation, which is especially found in disease of the bronchial wall. Two clinical examples may be given : (1) during

green.

Fig. 6-Procedure

for

obtaining

a

suitable solution

spectrophotometric analysis.

of green

sputum

for

15 the bronchiectatic may not cough much, and be green, but once this has may revert to its normal colour unless there is continued difficulty with expectoration ; and (2) in a lung abscess there is destruction of leucocytes with failure to excrete any enzyme ; hence a well-marked green colour may be found when the abscess is coughed up. the

night

by morning his sputum may gone the purulent material

CONCLUSION

the classifications of Traube (Nothnagel 1864) and of Frick (1889) should be discarded, and it will be noticed that the previously described postpneumonic group (where Traube thought that the colour was due to a haematin compound) can well fit into the verdoperoxidase group. The following classification is proposed in their place : (1) Stagnation of purulent sputum with poor excretion of verdo-

I

suggest that

peroxidase set free by the breakdown of leucocytes-e.g., bronchiectasis, lung abscess, inhalation pneumonia. (2) Bacterial contamination of sputum with Ps. pyocyanea; usually outside the body, but occasionally inside. (3) Severe jaundice, especially with hepatobronchial nstulse.

It is hoped that these observations may be of value in routine history-taking in chest cases. If a patient tells us that his sputum is, or has been, green, we must try to decide the cause for this ; and, if it should fall into the verdoperoxidase group, as it nearly always will, we must investigate this patient carefully to be sure that early bronchiectasis, or even a lung abscess, is not being missed. It should be of further use in the assessment of progress after any chest infection involving the bronchial tree. If the sputum remains green its drainage is inadequate, and the most strenuous efforts should be made by postural drainage to overcome this stagnation and to ensure a return to the yellow colour. SUMMARY

Previous theories about the nature of the colouring sputum are discussed. A method for obtaining a green solution from the

matter in green

sputum, suitable for spectrophotometric analysis, is described. The absorption curves in the spectrum are shown to be those of verdoperoxidase. It is suggested that a hitherto undescribed cause for the green colour is failure to excrete verdoperoxidase, and that this happens in the stagnation of purulent

sputum. The

aetiology

of green

sputum is given. carried out, through

The investigation was the courtesy of Dr. 0. T. Mallery, in the biochemistry department of the University of Michigan Hospital, Ann Arbor, Michigan, U.S.A., where the most generous facilities in advice and equipment were given to me. I had the advantage of constant discussion with, and encouragement from, Dr. Joseph P. Chandler, with whom all the spectrophotometric readings were made, and to whom I am especially indebted. I was working at the time in Dr. John Alexander’s department of thoracic surgery as a Dorothy Temple Cross travelling research fellow, and I am grateful to the Medical Research Council for the funds which

HYSTEROSALPINGOGRAPHY INFERTILITY

IN

FEMALE

A COMPARISON OF LIPIODOL AND VISKIOSOL SIX

DEREK FREETH M.D. Lond., M.R.C.O.G. CHIEF ASSISTANT, DEPARTMENT OF OBSTETRICS AND GYNÆCOLOGY, CENTRAL MIDDLESEX HOSPITAL, LONDON

THE

of hysterosalpingography is to diagocclusion of the fallopian tubes and to establish the incidence of sterile marriages due to tubal dysfunction. The elimination of all other factors barring conception, whether anatomical, cervical, hormonal, or metabolic, will obviously be worthless until tubal obstruction has been overcome, and an accurate knowledge of the condition of the tube is essential in nose

primary

patency

use

or

formulating prognosis

and

therapy.

HISTORICAL

In 1910 Rindfleisch was the first to attempt radiography of the uterus by injecting a bismuth emulsion through the cervical os. His historic paper makes interesting reading, for it is clear that he understood the importance of observing the state of the fallopian tubes besides demonstrating abnormalities of the uterus. During the evacuation of an incomplete abortion the dared uterine cavity was found to be abnormal, and he to try " injecting bismuth nitrate into the uterus. He was unable to interpret his results because there were no previous pictures for comparison, but he seems to have been dealing with a bicornuate uterus with a "

pregnancy in the left horn. In 1914 W. H. Cary injected 10 ml. of ’Collargol’ solution through the cervix while the patient was under anaesthesia for laparotomy. Radiography showed the left tube to be blocked, and this was confirmed at operation. Cary gave the first warning of the danger of making this investigation in the presence of pelvic inflammation. Rubin (1914) also described the use of collargol for testing tubal patency, but his report appeared two months after Cary’s. In 1916 Dartigues and Dimier described their method of intra-uterine radiography with collargol to demonstrate uterine and ovarian tumours. They suggested that pelvic tumours could be outlined by comparing the shadows of intra-uterine collargol with bismuth enemas given at the same time. Douay (1927) claimed that Dimier deserved credit for first injecting an opaque fluid into the uterus, but Dimier’s attempts were made in 1913 with 8 ml. of 10% collargol, and one of the patients afterwards died of peritonitis, so the research was abandoned. In 1920 Rubin described the non-operative determination of patency of the fallopian tubes by intra-uterine inflation with oxygen, and the demonstration of gas under the diaphragm by X rays. Rubin briefly referred to his use of the halogen salts, and in the discussion following this paper Polak (1920) stated he had usedArgyrol ’ and thorium to display the uterus and tubes. In 1921, in another important paper, Rubin criticised the use of collargol on the grounds of non-absorption, and thorium

-

they provided.

REFERENCES Adams, F. (1886) The Genuine Works of Hippocrates. New York vol. I, p. 204; vol. II, p. 271. Agner, K. (1941) Acta physiol. scand. 2, suppl. 8. Anderson, A. B., Hart, P. D’A. (1934) J. Path. Bact. 39, 465. Andral, G. (1821) Paris Thesis, no. 89, vol. 165, pp. 54, 64. Combemale, F., Francois, A. (1890) C.R. Soc. Biol. Paris, 9, 266. Fordos, M. (1860) C.R. Acad. Sci., Paris, 51, 215. (1863) Ibid, 56, 1128. Frick, A. (1889) Virchows Arch. 116, 266. Grisolle, A. (1841) Traité pratique de la pneumonic. Paris ; pp. 215, 540. Hart, P. D’A., Anderson, A. B. (1933) J. Path. Bact. 37, 91. Klebs, E. (1868) Zbl. Med. Wiss. 6, 417. -

Laennec, R. T. H. (1834) A Treatise on the Diseases of the Chest. 4th ed., London ; p. 201. Lemberg, R., Legge, J. W. (1949) Hematin Compounds and Bile Pigments. New York. Linossier, G. (1898) C.R. Soc. Biol. Paris, 50, 373. Meyer, E. (1903) Münch. med. Wschr. 50, 1489. Nothnagel (1864) Berl. klin. Wschr. 1, 273. Rosenbach, O. (1875) Ibid, 12, 645. Stoll, M. (1787) Aphorismi de cognoscendis et curandis febribus. Vindobonae; p. 30. Turfitt, G. E. (1936) Biochem. J. 30, 1323. (1937) Ibid, 31, 212. Van Swieten (1764) Commentaria in Hermanni Boerhaave aphorismos de cognoscendis et eurandis morbis. Leyden; vol. II, p. 772; vol. IV. p. 83. —