REFERENCES.
[ [1] Tanquerel: Traité des Maladies de Plomb ou Saturnines. Paris, 1839.
[ [2] Lancereaux: Gaz. Méd., 1862; Tribune Méd., 1896.
[ [3] Meillère, G.: Le Saturnisme, chap. iv.
[ [4] Teleky: Deutsch Zeitschrift für Nerven Heilk., vol. xxxvii., 1909.
[ [5] Déjerine-Klumpke: Thesis on Des Polynephrites en Général et des Paralysies et Atrophiques Saturnines en Particulier. Paris, 1889.
[ [6] Teleky: Ibid.
[ [7] Teleky: Ibid.
[ [8] Gombault: Arch. Phys., 1873.
[ [9] Moebius: Ueber einige Ungewöhnliche Fälle von Bleilähmung. Cent. für Nervenheilk. 1886.
[10] Tanquerel: Ibid.
[11] Sajous: Archiv für Laryng., iii., 1882.
[12] Morell Mackenzie: Brit. Med. Journ., epitome, p. 1202, 1893.
[13] Seifert: Berl. Klin. Woch., 1884.
[14] Lockhart Gibson: Brit. Med. Journ., vol. ii., p. 1488, 1908.
[15] Galezowski: Jahr. f. Aug., p. 382, 1877.
[16] Folker: Brit. Med. Journ., ii., p. 1556, 1898.
[17] Robert Jones: Brit. Med. Journ., September 22, 1900.
[18] Rayner: Journ. of Mental Science, 1880.
[19] Savage: Clifford Allbutt’s Medicine, vol. vii., p. 657.
[20] Goodall: Ibid., p. 693.
[21] Elschnig: Wien. Med. Woch., Nos. xxvii, xxix., 1898.
[22] Mannaberg: Ber. Klin. Woch., 1896.
[23] Bikler: Arch. für Augenheilk., b. xl., 1900.
[24] Weber: Thèse de Paris, 1884.
[25] Folker: Ibid.
[26] Lockhart Gibson: Ibid.
CHAPTER X
CHEMICAL INVESTIGATIONS
Very great assistance is afforded by chemical and histological diagnosis in the determination of cases of lead poisoning, especially when the case is likely to involve proceedings under the Workmen’s Compensation Act. In addition, a large amount of information is afforded to the certifying or appointed surgeon and the medical practitioner by adoption of certain easily carried out methods of diagnosis. It will be our purpose in the present chapter to describe as far as possible methods by which chemical or other investigation of a case of lead poisoning can be pursued, and the clinical methods of diagnosis which are applied in ordinary routine.
The majority of the methods described, especially the chemical investigation of material obtained from alleged fatal lead poisoning for the purpose of determining the presence or absence of lead, the histological examination of such tissues, and the examination quantitatively of excretions for lead, are processes which can only be carried out in a fully equipped laboratory, and certainly do not belong to the ordinary routine of medical work. The medical practitioner cannot be expected in the ordinary course of his routine work to examine blood-films for basophile staining or to make differential blood-counts. Especially is this the case in the routine examination of large numbers of factory hands. Further, many of the processes involved in either the chemical or histological examination of the tissues require so much special apparatus, without which such work cannot be undertaken, that the mere cost of the necessary instruments precludes the investigation being carried on except in special laboratories. At the same time, our purpose is to point out how additional methods of research may be made use of in obscure cases, and how recourse should be had to a well-equipped laboratory in doubtful cases. Further, the coroner, when ordering a post-mortem examination, may ask for a histological and chemical examination.
Methods of Chemical Diagnosis.
—The presence of lead may require to be determined qualitatively and quantitatively, and the procedure may differ slightly as to which process it is necessary to adopt. The quantitative determination of the amount of lead present in organs or excretions of the body is of far more importance than the estimation or determination of the fact of its presence. We have already referred to the work of Gautier[1], who has found lead present in the tissue of normal persons with such constancy that French observers, at any rate, now speak of “normal lead,” to distinguish it from lead which may be found in pathological conditions. There is, however, little doubt that the quantity of lead existing in the human body is exceedingly small. It is possible, with certain refined methods of chemical examination, that qualitative traces of this substance might be found. On the other hand, the methods of determination of lead are some of the most difficult in toxicological analyses, on account of the presence of other metals, particularly iron, which are exceedingly difficult to get rid of, and may easily lead to errors.
1. Qualitative Tests.
—The group reagent for lead is sulphuretted hydrogen in acid or alkaline solution. Lead is precipitated by this reagent as a black precipitate. Where no other metals are present, and where no organic matter is present at the same time, there is very little difficulty, and the usual method of the determination of lead in water by means of sulphuretted hydrogen is exceedingly easy.
Potassium iodide gives a yellow precipitate soluble on warming, and forms large crystals on the tube. Hydrochloric acid and chlorides give needle-shaped crystals, soluble in heat, and crystallizing out when cold. But the double chloride of potassium and lead is more soluble in heat, and still more soluble in cold, than is the pure chloride. This fact is made use of in the process to be described presently in separating lead from organic mixtures.
The direct examination is rarely possible or satisfactory, but the potassium cupric acetate method is one that may be applied to the qualitative estimation of lead in the tissues.
Method for detecting Small Quantities of Lead qualitatively.
—Dry and incinerate the material to be tested. Extract with hot dilute nitric acid, after repeated incinerations, finally extracting with ammonium acetate. Filter, incinerate, and take up the residue in dilute nitric acid. Evaporate to dryness and add a few drops of dilute acetic acid, and transfer drop to microscope slide.
To the drop on the microscope slide add one drop of dilute copper acetate solution, and two to three drops of saturated solution of potassium nitrate. Stir up the drops and well mix with a platinum wire, allow to stand for a few minutes, and then examine with a two-thirds objective. If lead is present, violet-black cubes of potassium copper lead nitrate appear (K2CuPb[NO3]).
This test is said to give a reaction in the presence of 0·00003 gramme.
Determination of Lead in the Urine.
—The quantity of lead passed by the kidneys into the urine is always small, even in acute lead poisoning. Further, the lead is excreted in organic combination, and is therefore difficult to detect. For absolute quantitative examination it is essential to evaporate the whole bulk, using at least ¹⁄₂ gallon of the fluid, and proceeding with the concentrate in the manner indicated in the estimation of lead.
For the qualitative examination many methods have been suggested, but all of them are more or less fallacious.
In certain instances in acute lead poisoning, or where a relatively large quantity of lead is excreted by the kidney, acidulation of the fluid with strong sulphuric acid direct will at times produce a precipitate of lead sulphate, which may be filtered off and the filtrate examined by the usual tests—namely:
A white precipitate with dilute sulphuric acid.
A yellow precipitate with potassium chromate, hardly soluble in nitric acid, but soluble in alkalies.
A blue flame on heating on a platinum wire, and finally, if sufficient substance is present, the reduction to metallic form with the blowpipe flame.
A method has been recommended of suspending a small bag of calcium sulphide in the sample to be examined, the supposition being that, if the calcium sulphide in the bag showed blackening, it would be due necessarily to lead. This is highly questionable, and in the hands of one of us (K. W. G.) has not given satisfactory results.
A further method, which is quite simple in application, and occasionally gives confirmatory results, may be applied in the following manner: The urine to be examined is inoculated with the Bacillus coli communis. For this purpose a small quantity of fæces may be used. The B. coli in its growth makes use of the organic substances in the urine, and at the same time sets free sulphuretted hydrogen. The urine left is filtered, the filtrate dissolved in 10 per cent. nitric acid (minimal quantity), and the filtrate examined by the usual tests. This method has occasionally given quite good results in the hands of one of us (K. W. G.), and is, moreover, an exceedingly easy one to carry out.
Passing sulphuretted hydrogen direct through the fluid is of no value, as it is necessary first of all to split up the organic compound before it will react to sulphuretted hydrogen.
Electro-Chemical Methods.
—Of all the methods at present in use for the estimation of the presence of lead in the urine, the electro-chemical gives by far the most satisfactory results. Several methods are described:
The first method consists in using magnesium, which is left for some hours in the urine, which has been previously strongly acidulated. In this manner Marsden and Abram[2] say that they have been able to detect 1 part in 50,000 in the urine without difficulty. The method adopted is as follows:
“A strip of pure magnesium is placed in the fluid to be examined. Ammonium oxalate in the proportion of about 1 gramme to 150 c.c. is added. If lead is present, it is deposited on the magnesium. A deposit is seen within half an hour, but we have usually left it twenty-four hours. The slip is then washed with distilled water and dried. Confirmatory tests: (1) Warm the slip with a crystal of iodine (yellow iodide proves lead, cadmium may be ignored); (2) dissolve deposit in HNO3, and apply usual tests. The magnesium can be used again after careful washing with acid and distilled water. The surface of the magnesium, when used, must be bright and free from oxide. The delicacy of the method has been tested with aqueous solutions containing known quantities of lead, also with normal urine to which known quantities of lead have been added. In all cases a control experiment was performed to insure the freedom of the materials from lead. Lead has been detected when present in the proportion of 1 part to 50,000, whether in simple aqueous solution or in urine.”
Shufflebotham and Mellor[3] describe the following method as one by which lead may be detected in organic tissue, and in each case this necessitated a large amount of evaporation. The method has value, but the difficulty of dealing with large quantities of fuming nitric acid, and the addition of this acid from time to time during the operations, render it difficult unless a good fume chamber is at hand. Shufflebotham and Mellor state that they obtained no reaction with the potassium chloride-hydrochloric acid method suggested by Dixon Mann.
“On the Detection of Lead in Urine and Post-Mortem Specimens.—A piece of kidney of 20 c.c. capacity was cut up into about a dozen pieces. These were placed in an evaporating basin, and about 50 c.c. of fuming nitric acid were poured into the dish. Dense brown fumes of nitrogen oxides were evolved. When the action had subsided (in from two to three minutes), the dish was placed upon a sheet of asbestos, and allowed to simmer over the Bunsen flame for about an hour. If the frothing appears in danger of running over the sides of the dish, stirring with a glass rod or removal of the flame for a short time may be necessary. Twenty-five c.c. of the fuming acid were added at intervals of a quarter of an hour, and this process was repeated three times. The destruction of the organic matter was so complete that the whole of the piece of kidney passed into complete solution. The solution was then evaporated down to a few c.c., neutralized with caustic soda, filtered, and treated with hydrogen sulphide. A dark-brown precipitate of lead sulphide was obtained. With potassium chromate a yellow precipitate of lead chromate was obtained with the same specimen of kidney which gave a negative result with the KClO3-HCl method of destroying the organic matter. Our reagents, dishes, etc., were then examined with a blank test, but we found no lead.
“Urine.—We then sought the presence of lead in the urine of Cases 2, 3, and 4. Half a gallon of urine was evaporated down to dryness in each of two basins. In one basin the residue was heated until it was charred. Both residues were then treated separately with fuming nitric acid, as just described. The uncharred residue passed into solution, and on cooling deposited a white sediment. The mother-liquor was neutralized and tested in the usual way. A brown precipitate of lead sulphide was obtained in Case 2, while in Case 3 a well-marked black precipitate was obtained. The urine of Case 4 gave a negative result. The charred residue did not pass completely into solution, and the tests for lead were not so well defined as when the residue was uncharred. This shows that care must be taken to prevent charring of the residue during evaporation.”
A method has recently been described by Hebert[4]—a modification of Trillet’s. This method is based upon the fact that peroxide of lead, when mixed with tetramethyl of diphenyl-methylen, gives in acetic acid solution a fine blue coloration. Unfortunately, a number of other peroxides give the same blue coloration, amongst them manganese, potash, copper, magnesium. In addition, the sodium peroxide used to convert the lead present into the peroxide also gives a bright blue coloration with the reagent, even if present in minute quantities.
The test is made in the following manner: The substance is incinerated, sulphuric acid added in the usual way, and the substance evaporated to dryness. It is treated with a cold solution of sodium hypochlorite. The hypochlorite is then removed partly by washing and partly by heating, and the reagent is then added directly to the substance in the capsule, and if a peroxide is present the blue colour results.
Unfortunately, this dissociation-point of the hypochlorite and the temperature at which peroxide of lead is changed back again to the oxide are very close together, being only about 25° C. In addition, it is very difficult to remove the last traces of the substances which give a blue coloration in addition to lead. One of us (K. W. G.) has made extensive trials with this method, as, if it had been a reliable process, it would have been one which would have considerably facilitated the estimation of lead in small quantities. The method has been adopted by certain French observers, who by drawing 2 c.c. of blood from the median basilic vein, and estimating the lead present in this small amount by means of the blue coloration, have sought to show that at least 25 milligrammes of lead were circulating in the blood of the body. In addition to other grave considerations, the fact that the reagent itself is colourable by certain other peroxides which exist in the blood-ash renders these figures entirely untrustworthy.
2. Quantitative Methods of Estimation.
—Two methods may be used in the detection of lead in organic substances, either in organic fluids or in solids, and are generally termed the “wet” and “dry” methods, from the original treatment of the substance.
In the dry method the substance is incinerated with or without the addition of sulphuric and nitric acid; in the wet the material is treated with hydrochloric acid and potassium chlorate. Subsequent treatment in both cases is on the same lines.
Method of Fresenius and Von Babo[5]
—Moist Method.—The substance which is suspected to contain the poison, if solid, is reduced to a pulp, and is mixed with sufficient water until of the consistency of thin gruel. The urine should be evaporated to one-fourth or one-sixth of its volume. Fæces should be well stirred up with distilled water. The substance is then placed in a large flask together with crystals of potassium chlorate; each 100 grammes of the substance require 3 to 4 grammes of potassium chlorate. Pure HCl of the same weight as the original substance is then added, the flask is placed on a water-bath and gently heated. Care must be taken that the heating is not too brisk, as otherwise the evolution of the chlorine peroxide takes place too rapidly. If necessary, additional crystals of potassium chlorate are added from time to time until the fluid becomes limpid and of a slight yellow colour, or, if there is much organic matter, until it assumes the appearance and colour of thin oatmeal gruel. On account of more gradual evolution of chlorine, the chlorate that is present before the flask is heated acts much more energetically, weight for weight, than fragments added after the liquid is heated, as a great deal of the gas then escapes without rendering any service. If the substance contains sugar, starch, or alcohol, extra care must be taken to avoid frothing over. When the fluid contents are clear or reduced to a thin consistency, the liquid is transferred to an evaporating basin, and allowed to remain on a water-bath until the smell of chlorine has disappeared; it is then filtered while hot. The whole of the organic matter is not destroyed by this process, fatty substances especially being resistant; but if the organic matter is reduced to small fragments, any mineral poison present will be liberated.
The objections raised against this process are that some important poisons—such, for instance, as arsenic and antimony, specially the former—are liable to escape partially in the form of vapour, and that others, such as lead and silver, may remain as insoluble precipitates on the filter. As regards the first objection, it is to be observed that, when the hydrochloric acid is diluted with water (as in a moist method of destroying organic matter), any arsenic which may be present in the hot solution is not given off with its acid aqueous vapour, arsenious chloride dissolved in hydrochloric acid being volatile only when the solvent is concentrated. Any possibility of loss may be avoided, however, by furnishing the flask, in which the organic matter is being destroyed, with a condenser and receiver. The second objection, as far as lead is concerned, is met by taking care to filter the solution whilst hot; if only a limited amount of lead is present, it then remains in solution as chloride so long as the liquid is hot, and will consequently pass through the filter. A considerable quantity is kept in solution in the cold, as it forms a combination with potassium chloride, which is more soluble than lead chloride alone. If a large amount is present, it will not all be found in the filtrate; the substance left on the filter, therefore, must always be tested for lead. In toxicological work, however, the amount of lead present is not as a rule more than will remain dissolved in the cold. Silver chloride, being insoluble either in hot or cold water, will not pass through the filter; consequently the salts of silver require dealing with in a special manner.
Dry Method.—This is effected by heating the finely divided substance to redness, so that it is either carbonized or completely incinerated. When cold, the residue is drenched with nitric acid, and sufficient heat is afterwards applied to drive off the free acid. The nitrate of the metal is then dissolved in water, filtered, and dealt with according to the kind of metal present.
The dry method is unsuitable in the case of the more volatile metals, as arsenic, antimony, and, in a lesser degree, lead, tin, and zinc. Further, it is extremely difficult and troublesome to carry out with large masses of organic matter. It is convenient with small amounts, and in the absence of the more volatile metals yields good results.
The following two methods are given by Glaister[6] on the one hand, and Dixon Mann[7] on the other. Both methods are good. It will be seen that Glaister recommends the estimation of the lead as sulphide.
When minute quantities of lead are present in combination with large amounts of organic matter, the dry process is tedious, difficult to carry out, and uncertain in its results. The plan adopted in the elimination of lead by Dixon Mann is as follows:
The urine is evaporated down to the consistency of gruel, and the fæces mixed in distilled water to a like consistency. They are then treated by the wet method, as given above. The filtrate after cooling is placed in a glass cell, the bottom of which consists of a sheet of vegetable parchment; the cell is immersed to such a depth in a deeper cell, containing distilled water acidulated with a few drops of sulphuric acid, that the liquids in the inner and outer cells stand at the same level. A piece of platinum-foil enclosing a surface of about 50 square centimetres, constituting the kathode, was submerged in the liquid contained in the inner cell, a similar piece of platinum-foil, constituting the anode, being immersed in the outer cell. The pieces of foil are so placed as to be opposite each other, separated by the parchment diaphragm. A current, 3 or 4 volts, is then passed through for from six to eight hours, after which the foil is removed from the inner cell and gently washed and dried. The metallic lead is dissolved off the foil with dilute nitric acid aided by heat, and after driving off most of the free acid the solution is decomposed with dilute sulphuric acid with an equal volume of alcohol added. It is then set aside for twenty-four hours. The precipitate of lead sulphate is washed with water containing 12 per cent. of alcohol, until all the free acid is removed; it is then separated by decantation, ignited, and weighed. The amount of lead is calculated from the weight of the sulphate; 100 parts of sulphate are equal to 68·319 parts of metallic lead.
Whether the moist or the dry process is used, the residue after the primary filtration should be tested for lead, which may be present as sulphate and remain undissolved. If the original substance contains lead as sulphate, the salt should be dissolved with heat in an aqueous solution of ammonium tartrate to which a little free ammonia has been added, then precipitated with sulphuretted hydrogen (100 parts of lead sulphide equal 86·61 parts of metallic lead). It is better, however, to convert the sulphide into sulphate by treating it with nitric and subsequently sulphuric acid, after which it is ignited, weighed, and the amount of the metal calculated by the lead sulphate factor.
Having obtained the substance by decomposing the organic matter, two methods of estimation may be made use of:
(1) Colorimetric; (2) gravimetric.
Where the estimation is to be made gravimetrically, the substance is always obtained as a sulphate, and the lead estimated by weighing as a sulphate. This process is an exceedingly tedious one when a large number of small samples have to be estimated, as in the determination of the amount of lead dust present in the air. On the other hand, it is probable that the use of the balance in the estimation of lead as sulphate is more accurate where larger quantities up to 10 milligrammes are present; but where only 2 or 3 milligrammes are present in the amount of substance examined, the experimental error in washing is too large to warrant the expenditure of time required in this form of estimation, and the colorimetric method is used.
Detection of Lead in Organic Mixtures.
—Acidulate the organic substances, reduced to fine proportions, with nitric acid, heat for some time, then permit to cool; filter, wash residue, and mix washings with filtrate; concentrate filtrate; pass H2S; place mixture in warm place to allow precipitate to settle; after which decant supernatant fluid, collect precipitate on tared filter, thoroughly wash, dry on water-bath, and weigh. One part of sulphide is equivalent to 0·9331 part of lead oxide and 1·5837 parts of acetate of lead.
The electrolytic method is better adapted for the detection of minute quantities of lead, as, for example, in the urine or fæces or in vomited matter. The urine may be evaporated to a viscous state; the others, finely broken up, are treated in the same way, after HCl is added, as recommended above, the mixture heated, and pinches of powdered chlorate of potash added, as necessary, to break down organic matter. The heating is continued until the odour of chlorine disappears, after which it is filtered and the filtrate allowed to cool. The filtrate is then placed in the outer cell of a two-celled arrangement, not unlike a dialyser, the bottom of which is formed of vegetable parchment, the outer cell containing distilled water acidulated with H2SO4. Into the inner cell is placed a piece of platinum-foil measuring about 50 square centimetres of exposed surface, which is connected with the kathode or negative pole of four Grove cells, and into the outer cell is placed a like-sized piece of platinum-foil connected with the anode or positive pole. These pieces of foil are so placed in relation to one another that they are only separated by the parchment. The galvanic circuit being now closed for some hours, any lead in the filtrate will be deposited on the platinum-foil connected with the kathode in the inner cell. The foil is then removed, carefully washed, and the metallic lead dissolved by dilute nitric acid aided by heat, after which the solution is concentrated until most of the free acid is driven off; dilute sulphuric acid is added to throw down the sulphate, alcohol being also added to expedite precipitation. The precipitate is allowed to settle for twenty-four to thirty-six hours, filtered on a tared filter, washed with water containing 12 per cent. of alcohol, dried, ignited, and weighed. One part of sulphate is equivalent to 0·68319 part of metallic lead and to 1·25 parts of acetate of lead.
The estimation of the lead, especially if the amount be small, may be more accurately made by the volumetric colorimetric method. The metallic lead deposited on the platinum-foil is dissolved in nitric acid, and distilled water added, and an aliquot portion placed in a Nessler glass. A few drops of freshly-prepared H2S water, or H2S gas itself, may be added to or bubbled through the contents of the glass so as to form lead sulphide. The colour formed is now matched in a similar glass, using a standard solution of lead nitrate, and forming the lead sulphide as before.
Some have advocated the magnesium wire deposition test, originally devised by von Jaksch, and modified by Hill Abram, for the detection of lead in the urine of persons who are suspected to be suffering from chronic lead poisoning (see [ante]).
The colorimetric method of estimating lead has been made use of with very great success by Duckering in the estimation of lead in the air of potteries. The method is that devised by Mr. Vernon Harcourt[8], with a few modifications. The whole of the method is given, as observation on the quantity of lead present in the air of lead factories helps greatly in suggesting rational methods of precaution.
“After drying and weighing the filters, the following is the method. Solutions required:
“Nitric Acid.—One part of pure concentrated nitric acid to three parts of water.
“Caustic Soda.—One hundred grammes of pure caustic soda dissolved in 250 c.c. of water.
“Sugar.—A saturated solution of sugar in water.
“Sulphuretted Hydrogen.—A saturated solution of sulphuretted hydrogen in water.
“Coloured Solution.—Cotton-wool dissolved in concentrated nitric acid and evaporated to dryness, and the residue dissolved in a little water and filtered; the solution is deep yellow in colour.
“Standard Lead.—A solution of lead acetate or lead nitrate made up to contain exactly 0·0001 gramme of lead per c.c. of solution.
“The bulk of the dust in the filter was removed to a beaker (No. 1), by gently tapping the inverted funnel. The cotton-wool was then removed from the funnel, and the upper one-third, containing the remainder of the dust, was cut off and added to the dust in beaker No. 1. The remainder of the cotton-wool was placed in a second beaker (No. 2); 2¹⁄₂ c.c. of hot nitric acid was dropped on the dust in beaker No. 1 from a pipette, a little water added, and the whole heated. The solution was filtered into a 50 c.c. Nessler glass, and the liquid remaining in the cotton-wool also removed by squeezing the wool with a glass rod against the side of the beaker. The cotton-wool in beaker No. 2 was similarly extracted with 2 c.c. of nitric acid, and the solution added to that remaining in beaker No. 1. The liquid was heated, the cotton-wool macerated in it and filtered as before. The wool was then washed with hot water about three or four times, and the liquid filtered into the Nessler glass. A number of standards were then made up by running into Nessler glasses, from a burette, varying amounts of standard lead solution covering a fair range. Usually five standards were made up, containing 0·5, 0·8, 1·0, 1·2, and 1·4 c.c. lead solution, depending on the volume of the air aspirated and the quantity of lead expected in the known weight of dust found. To each standard 4¹⁄₂ c.c. nitric acid was added, and 5 c.c. of the caustic soda solution and 4 c.c. of the sugar solution were run into all the six solutions—i.e., one test and five standards—from pipettes. It was invariably found that the test was coloured faintly yellow, and if this is not allowed for in the standards high results are obtained. Hence a drop or two of the coloured solution (see solutions required) was added to the standards placed on white paper till they matched the test. Lastly, to the contents of each of the six glasses was added 4 c.c. of sulphuretted hydrogen solution, and the liquid in each made up to the 50 c.c. mark, and the whole well stirred. Usually it was found that the colour of the test came somewhat deeper than that of one standard, and a drop or two of lead solution was added to the standard till its colour matched that of the test. The elaborate method of making up a number of standards was adopted because it was found that any other way gave high results. In the way described many trial experiments were made, and they were invariably correct within half a drop of the standard solution.”
The estimation of the quantity of lead present in organic fluids, with the disturbing influence of the presence of other metals, is a factor which always complicates the use of the sulphuretted hydrogen colorimetric estimation. It is almost impossible, when dealing with fæces, with blood, or, on the other hand, with artificial digests containing bread and milk, to eliminate the disturbing influence of iron. Further, if steps are taken to remove the iron and other metals, so much loss takes place in the manipulations necessary that the results arrived at are not satisfactory. Whenever dealing with organic matter, such as urine and fæces, it is best to make a blank test with a similar quantity of the substance under examination obtained from other sources, and to subtract the error found due to iron, when a rather closer approximation may be arrived at.
So far as can be estimated, the minimal quantity of lead required to produce poisoning is 0·005 gramme per kilogramme of body weight; but, on the other hand, persons who have swallowed much larger doses than this have exhibited no symptoms of poisoning. There is every reason to suppose that lead absorbed through the lung produces a maximum toxic effect, and, from the estimation of the quantity of lead found in the body after death, it is highly probable that exceedingly minute quantities of lead have, when absorbed over long periods, produced changes not only by their actual presence in the tissues, but also have set up degenerative changes which progress even after the elimination of the metal from its local position.
Histological Examination.
—In addition to the chemical examination of tissues from a person who has died of suspected lead poisoning, it is of the highest importance to make histological examinations, as the naked-eye appearance of post-mortem examination is frequently insufficient to give any clue to the cause of the poisoning. Moreover, in a large number of instances the necropsy may exhibit a number of signs of disease, such, for instance, as granular kidney, cirrhosis of the liver, and so forth, which are associated with diseases other than lead poisoning, and, in the absence of any present or past evidence of definite hæmorrhages found associated with the other lesions already mentioned, an ordinary autopsy must be inconclusive. It is true that such pathological conditions are consistent with poisoning by lead; and if the individual has been a lead-worker, it is easy, but frequently erroneous, to conclude that the symptoms owe their origin to the worker’s occupation. We are entirely in sympathy with the remarks of King Alcock[9], who says:
“I plead none the less for an impartial investigation of the symptoms presented by a lead-worker, before assigning full or even partial responsibility of the disease to the occupation. If any and every departure from the normal in a lead-worker is at once assigned—the occupation being known—to plumbism, early diagnosis naturally presents very few difficulties to the exponents of such methods. And however severely we may condemn in the abstract such a careless, unscientific attitude, the tendency has, in practice, to be reckoned with and combated. The balance of probabilities would possibly suggest that the occupation is, after all, responsible, in one sense or another, for the more usual illnesses classically associated with the poison; nevertheless, the attending practitioner is in duty bound to take into consideration, and to estimate the relation of, all the concurrent causes of such symptoms.”
On the extremely unsatisfactory position the certifying surgeon may find himself in at a coroner’s inquest King Alcock says:
“The problem, from a strictly scientific point of view, is complicated—one might almost say that the truth is stifled—by the fact that the ætiological relations of the symptoms of any suspended worker are swamped by the insistent legal relations under which he claims and receives compensation.
“When once a formal certificate of suspension has been issued, which has embodied a recognition of lead as a cause of certain existing symptoms, then it becomes almost hopeless ever to reopen the question of causation of these or other supervening troubles, be their origin independent of lead or not. The doctor, in a legal cross-examination, is, in scientific honesty, bound to admit at last the bare possibility of any fantastic chain of remote sequelæ; and his protests against the probabilities of such sequelæ are of no avail, as opposed to his own admission. The post, ergo propter, appeal carries the day easily.”
For the purpose of histological examination it is essential that portions of the brain, spinal cord, liver, kidney, and intestine, should be examined microscopically. The nervous tissue should be placed in formalin and Müller’s fluid, and a portion in alcohol for the examination of the fibres. The liver, kidney, etc., should be placed in 5 per cent. formaldehyde. The tissues are then treated by the ordinary histological methods, and sections prepared. With nervous tissue it is essential that those prepared for the examination of the cells should be made by the celloidin method; the others may be treated by imbedding in paraffin. The points to be sought for in the tissues are sufficiently indicated in the chapter on Pathology and Symptomatology, but may be briefly recapitulated:
In the brain, as well as in all the tissues, careful search should be made for minute microscopical hæmorrhages, and for evidences of old hæmorrhages in the form of small masses of fibrous tissue, etc. Parenchymatous degeneration, chromatolysis of nuclei, etc., nerve degeneration.
The arteries and veins should also receive close scrutiny, as the presence or absence of arteritis should be noted.
In the kidney particularly, search should be made for both interstitial and parenchymatous nephritis.
The liver frequently shows signs of microscopic hæmorrhage, and it is as well, in taking a portion of tissue for examination, to choose those portions which appear to be specially congested.
In the brain and spinal cord and nervous tissue, search is to be made for the same hæmorrhages as already noted. In addition, the condition of the nerve fibres should be noted, the presence or absence of periaxial neuritis, as well as degeneration of the axis cells, and the various ganglion cells both in the brain and spinal cord should be closely examined for chromatolysis and nuclear atrophy.
No evidence is afforded by micro-chemical tests of any of the sections thus obtained, except those of the lung. It may be possible in the case of the lung to determine the presence of lead granules in the alveolar cells, and attention should be paid to this. It is possible also that some evidence may be afforded by examination microscopically of the red bone-marrow.
The intestinal walls should be examined for evidence of lead particles.
If any dark staining, deep or superficial, be found in the intestine, it should be removed for chemical analysis. Necrotic areas of the intestinal wall should be sought for.
Hæmatology.
—For all practical purposes, the best stain for detection of basophile granules in the erythrocytes is Wright’s modification of Romanowski’s stain. This stain may be obtained in appropriate tablets, and may be prepared immediately before use, although a stain which has been standing for ten days or a fortnight gives much better results than a quite new stain. The stain consists of a solution of polychrome methylene blue, together with eosin in methyl alcohol, and the method of procedure is as follows:
Blood is obtained by a small puncture, and slides smeared and allowed to dry. Immediately on drying the slip is flooded with the stain, and allowed to remain for two minutes. This causes fixation. At the end of the two minutes the stain is diluted with an equal volume of distilled water, and allowed to remain on for a further three minutes. At the end of this time the stain is poured off, and the slip washed in distilled water for another three minutes, or until the characteristic purple-violet appearance is produced. It is better to examine such films with an oil-immersion lens, the oil being placed directly upon the films, and not covered with a cover-slip, as the action of Canada balsam tends to decolorize the blue. If such specimens are required to be kept, the oil may be washed off with xylol. It is possible to observe basophile staining with a good sixth, but an oil-immersion lens gives much the best result. The typical staining produced by this means gives darkish bodies scattered about the red corpuscles, staining sometimes deeply as the nuclei of the white corpuscles. In other cases the appearance is like that of fine dust scattered throughout the cell. In addition to these two forms, the whole red cell may take on a slight generalized lilac tint, the normal cells remaining free from granules, and stained red by the eosin. Search of 100 fields of the microscope should be made, and if no basophile granules are found in such fields it is unlikely that they will be found.
Basophile staining is not more pathognomonic of lead poisoning than of any other form of anæmia, but may be regarded as a highly important confirmatory diagnostic sign.
A differential count of the leucocytes present may be also made on the same film in which basophile staining is observed; 300 should be counted at least. In a typical case of lead poisoning it is found that diminution in the polymorphonuclear leucocytes has taken place with a corresponding increase of the lymphocytes, and possibly also the large mononuclears, and probably a slight increase in the number of eosinophiles.
This hæmatological method of diagnosis is of the utmost importance in lead poisoning. A differential count such as is given on [p. 137], showing a large diminution in the polymorphonuclears, an increase in the lymphocytes, evidence of changes in the red cells, consisting of basophile staining, alteration in the shape of individual cells, poikilocytosis, with vacuolation, is strong presumptive evidence of lead absorption.
To complete the hæmatological examination, the hæmoglobin should be estimated. This is best performed with Haldane’s instrument—an exceedingly simple one to use. The estimation of the number of red cells and white cells present is useful, but does not by any means give such valuable information as does the differential count and search for basophile granules.
Blood-Pressure.
—Several methods are available for the estimation of the blood-pressure. The pressure may be roughly estimated as too high or too low by means of the finger. The presence of thickening of the arteries may be also estimated in this way, but for determining the absolute blood-pressure it is necessary to use one or other of the instruments on the market. The estimation of blood-pressure is an important point in relation to the suspected presence of arterio-sclerosis, and should be performed wherever possible. Sphygmographic tracings may also be taken. Such a tracing in a case of typical poisoning gives a peculiar form of curve, which, however, may be present in alcoholism and heavy work, and arterio-sclerosis of many types.
Urine Examination.
—In suspected cases of lead poisoning the examination of the urine may reveal the presence of lead. In addition, albumin is frequently present, especially in the early stages of kidney inflammation. The ordinary tests for albumin should be carried out, and it is also advisable to examine the urine spectroscopically, as at times hæmoglobin, methæmoglobin, hæmatoporphyrin, may be present in small quantities, each of which can be detected by means of spectroscopic examination. Blood is not common in the urine of lead-poisoned persons, although microscopically hæmorrhages undoubtedly take place in the kidney. These hæmorrhages are interstitial, and as a rule do not cause any blood-pigment to be passed in a quantity that can be determined. It is as well, however, to centrifugalize the urine, and examine the deposit for red blood-cells.
The presence of hæmatoporphyrin, as suggested by Steinberg[10], is probably due to hæmorrhages in the intestine, and its presence in the urine should be regarded with suspicion in a lead-worker.
Where a lead-worker is suffering from continued absorption of lead, even without the manifestation of other symptoms, a change has been noted in the acidity of the blood—namely, a loss of normal alkalinity. The estimation of the alkalinity or acidity of the blood direct is an exceedingly difficult process, but much information may be obtained by careful estimation of the acidity of the urine, and of the acidity of the urine in relation particularly to the phosphates.
Joulie[11] has pointed out the extreme value which may be obtained from a knowledge of the urinary constituents by the means of estimation of the acidity with suchrate of chalk. The reagent is made by slaking lime in such a way that the resulting compound is practically dry. A quantity of this—about 25 grammes—is then thoroughly shaken up with 10 per cent. solution of cane-sugar, allowed to stand, and the solution titrated against decinormal acid until it is of one-twentieth normal. The urine is then estimated directly, the suchrate is run into the 25 c.c. of urine until a faint white flocculent precipitate appears. The number of c.c. of the solution of suchrate is then noted, and multiplied by the factor of the solution. This gives the acidity related to the phosphate and other organic acid contents, and is similar to the method used to determine the acidity of wines.
The second estimation consists of estimating the phosphates present by means of a standard solution of uranium nitrate, using either potassium ferrocyanide or cochineal as an indicator. The specific gravity of the urine is also determined. The result is then expressed in terms of this specific gravity, or, rather, in the terms of the density of the urine in relation to distilled water, and the whole answer returned per litre. By this method it is not necessary to obtain a twenty-four hours sample of the urine, the urine passed first thing in the morning being taken for examination.
By using this density figure the quantity of acid and phosphate is expressed in relation to the density, the equation being—
The observed acidity The density per litre = Acidity per litre.
The phosphate content is expressed in the same manner, while the ratio of phosphate to acidity gives the ratio of excretion of phosphate to acidity.
There is in lead-workers a considerable diminution in the amount of phosphate excreted, and, as has been pointed out by Garrod and others, lead apparently produces alteration in the solubility of the uric acid content of the blood, and may therefore allow of its decomposition. Probably lead as a urate is stored up in the tissues. For further particulars of this method of the estimation of the urine, the reader is referred to “Urologie Pratique et Thérapeutique Nouvelle,” by H. Joulie.
An examination of the fæces of persons suspected of lead poisoning may often give definite results both of the presence of lead and hæmatoporphyrin. If small hæmorrhages have occurred high up in the intestine, the presence of hæmatoporphyrin in the fæces may result. The substance may be easily determined by means of the characteristic absorption bands. A quantity of fæces is taken and extracted with acid alcohol, and the filtrate examined spectroscopically. Urobilin bands are commonly present, and, particularly, where much constipation exists these bands are very well marked. There is no difficulty whatever, however, in distinguishing them from the characteristic bands of acid hæmatoporphyrin.
Examination of the Fæces for Lead.
—The moist method or chemical examination given above is the best one to apply for the determination of lead in the fæces. As has already been pointed out, lead is commonly excreted in the fæces, and, if only about 2 milligrammes per diem are being excreted by the fæces in a lead-worker, the quantity cannot be regarded as indicative of poisoning. One of us (K. W. G.) has at times found as much as 8 to 10 milligrammes of lead excreted in the fæces of persons engaged in a lead factory, and exhibiting no signs or symptoms whatever of lead poisoning. If, however, the quantity of lead in the fæces rises to anything above 6 milligrammes per diem, there is definite evidence of an increased absorption of lead, and if at the same time clinical symptoms be present, suggesting lead poisoning, such a chemical determination is of the first importance.
In estimating the presence of lead in fæces, it may be necessary to deal with the separation of iron, which may be precipitated as phosphate and filtered off, the quantitative estimation being proceeded with in the filtrate.
Lead is much more commonly present in the fæces of lead-workers than in the urine, and it is better to examine the fæces rather than the urine in suspected cases.