Determination of Blood in Dried Formalin-Fixed Lung Tissue Spectrophotometric Method Involving Pyrrole Residues of the Porphyrin Ring Imanuel Bergman and Roy Loxley Safety in Mines Research Establishment, Department qf Trade and Industry, Shefield S3 7 H Q , England Inhaled dust retained in pneumoconiotic lungs leads to the formation of iron deposits and to other tissue changes. I t is of interest to study the dust and tissue changes quantitatively i n relation to chest radiographs before death. In general, lungs are available fixed i n formol-saline. Aliquots for analysis are best obtained by conversion of the lung into a dried powder. The analysis is complicated by the presence of blood, insolubilized by the fixation, and therefore not amenable to analysis by conventional methods. The present procedure utilizes a phenol digestion to remove the effect of the formalin fixation. A further digestion in hydriodic acid hydrolyzes the heme group into pyrrole fragments, which yield a stable color with p-dimethylamino-benzaldehyde in acetic acid.
IN FUNDAMENTAL STUDIES of pneumoconiosis, the analysis of pneumoconiotic lungs has provided results of great interest with respect to both the amount and composition of the retained dust and the related changes in the composition of the lung tissue. However, there are difficulties concerning the way in which the results of analytical determinations of this sort should be expressed; these arise partly because of the nature of the material available for analysis, which is usually tissue which has been fixed in formalin or formol-saline solution. Post-mortem lung material contains variable amounts of blood; this is rendered insoluble by the formalin treatment, and thus it becomes a n intimate part of the experimental material, If whole formalin-fixed lungs are available, they can be dried, ground, and weighed. The results of the analysis of aliquot samples can then be expressed in terms of total weight per lung; this figure is independent of the blood content. It may, however, be argued that for components of lung tissues, as well as for dust, concentration rather than total quantity is the relevant parameter. Otherwise a large man breathing large amounts of dusty air into large lungs would appear, from the analyses, to be more affected by pneumoconiosis than would a smaller man. For the analyses to provide meaningful figures for concentration per unit weight of lung tissue, the blood content must be known. Another instance where knowledge of the blood content is important is where only a small portion of lung is available for analysis. Even if it is otherwise a representative sample, and its weight is known in relation to the rest of the lung, its blood content may not be the same as that of the rest of the lung. Blood contains about 15 grams of hemoglobin and about 7.5 grams of other materials, such as serum proteins, in every 100 ml. Hemoglobin should therefore be a good measure of the contribution of blood to the dry weight of tissue. Hemoglobin contains iron, but the latter cannot be used as a n index of the blood content of tissue, as other iron-containing materials such as hemosiderin or ferritin may be present. Pneumoconiotic lungs have been shown to contain nonheme iron such as hemosiderin (1). (1) I.
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Bergman, A i m Occup. Hyg., 13, 163 (1970).
Hemoglobin is conveniently determined in fluid blood by the color it gives in various diluting media. However, such determinations cannot be carried out on formalin-fixed tissue, because the blood is insoluble in the media. A qualitative test for dried blood involved heating with acetic acid containing sodium chloride. The hemoglobin molecule is split and the porphyrin moiety gives characteristic crystals of hemin chloride. Extraction with hot acetic acid was therefore tried as a method of determination of hemoglobin in dried tissue. Extraction of unfixed dried blood or tissue with hot glacial acetic acid gave stable colored solutions of hemin, whose absorption spectra could be accurately related to hemoglobin content. Unfortunately, extraction of formalin-fixed blood or tissue gave low yields of hemin in solution, and highly colored residues. If acetic acid containing ammonium acetate was used, the formalin-fixed tissue went into solution even though the temperature of extraction was no higher (2). It was not possible to estimate the hemin content accurately from absorption spectra of the latter solution, because hemin became unstable when heated in the presence of ammonium acetate, and because the dissolution of the tissue resulted in a n interfering color. A procedure was developed for the determination of dried blood in finely ground tissue, fixed or unfixed, by suspending it in a mixture of fluids designed to match the blood-containing particles of tissue both in refractive index and in density. However, this procedure could not be used for the lungs of pneumoconiotic coal miners, because the coal dust present masked the color of the hemoglobin. The treatment of hemin with hot hydriodic acid splits the porphyrin ring into four substituted pyrroles (Figure 1) and corresponding carboxylic acids (3). The development of methods for the determination of hydroxyproline in lung tissue resulted in a procedure well suited t o the determination of a t least one of the related pyrroles by color formation with Ehrlich's reagent (acid p-dimethylamino-benzaldehyde) ( 4 , 5). These two processes were combined t o give a new method for the determination of hemin. Its applicability to the determination of the blood content of lung tissue is based on the assumption that the contribution of myoglobin and, for instance, cytochrome pigments to the color yield is likely to be relatively small. The reductive fission of hemin, or of the hemoglobin in dried unfixed blood tissue, with hydriodic acid was first tried in glacial acetic acid a t 100 "C. Precautions were taken t o prevent the distillation of the pyrroles from the reaction mixture. It was expected that the products of the reaction would be four different pyrroles or related carboxylic acids (see Figure 1). Three of these have a t least one unsubstituted a-position, and would therefore react easily with Ehrlich's reagent. Opsopyrrole was expected to be particularly reactive, since it has both a-positions free. A colored material (2) (3) (4) (5)
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
I. Bergman, ANAL. CHEM.,38,441 (1966). H. Fischer and A. Treibs, Aizr?. Chem., 450, 132 (1926). I. Bergman and R. Loxley, ANAL. CHEM., 35, 1961 (1963) 1. Bergman and R. Loxley, A m l y s t , 94, 575 (1969).
OPSOPYRROLE
H
f2: M
HAEMOPYRROLE
H
CRYPTOPYRROLE
H
PHYLLOPYRROLE
M>T$-M
H
M = -CH3 E = -CH2 CH3 Or
-CH2 CH2 COOH
Figure 1. Pyrroles produced by action of hydriodic acid on hemin
x : mp Figure 2. Absorption spectra of blank and solution corresponding to 40 p g of hemin per 10 ml in a 1-cm cuvette
was in fact produced within minutes of the addition of Ehrlich's reagent and was stable for many hours at room temperature. However, the color of the iodine produced by the hydriodic acid interfered with the spectrophotometry. A number of reducing agents were tested, and sodium sulfite was found to be suitable for the suppression of the iodine color. This procedure gave a colored product with unfixed blood or tissue, but gave very little color with formalin-fixed blood or tissue, despite the fact that these materials dissolved in the hydriodic acid-acetic acid mixture. It was concluded that formaldehyde might be released by the digestion process, and might theri block the relevant a-positions on the pyrrole fragments, Phenol reacts strongly and irreversibly with formaldehyde. When it was added to the digestion mixture, formalin-fixed blood or tissue also gave pyrroles that reacted with Ehrlich's reagent. Eventually a procedure was developed in which the formalin-fixed tissue was first digested in hot phenol and then in a mixture of acetic and hydriodic acids.
water bath, stoppered after 15 sec, and heated for a further hour. The tube is then cooled and the contents are transferred to a standard flask and made up to 25 ml with glacial acetic acid. With pneumoconiotic lung digests, the dust may interfere with subsequent spectrophotometry, so the solution is centrifuged to allow an aliquot to be taken from the supernatant. Color Development. One milliliter of the acetic acid solution is pipetted into a test tube. A 1 % aqueous sodium sulfite solution is added dropwise from a graduated pipet or buret until the iodine color fades. A further 0.1 ml of solution is then added; the volume added should be less than 1 ml, and enough water is added to make volume added up to 1 ml. After 3 minutes, 8 ml of the Ehrlich's reagent (5 p-dimethylamino-benzaldehyde in glacial acetic acid) are added. The absorbance at 547 mp is recorded in a 1-cm cuvette. Color development is normally complete within 3 minutes and the color is stable for several hours at least. The amounts of sample suggested for the procedure (40 pg hemin or 1.6 mg blood in the 1-ml aliquot) should give an absorbance of about unity in a 1-cm cuvette-i.e., a molar absorptivity relative to hemin of 192 X IO3 liter cm-1 mole-'.
EXPERIMENTAL
RESULTS AND DISCUSSION The amounts and concentrations of the various reagents used in the procedure were varied about the recommended levels by i.5 %. No significant variations in the color yield were observed. As the phenol used in the preliminary digestion of formalin-fixed material is somewhat hygroscopic, the effect on the color yield of adding water prior to the phenol digestion stage was tested. The addition of up to 0.5 ml of water had no effect. The most concentrated solution of hydriodic acid conl'eniently available (sp gr 1.94) gave the highest color yield. Reagent bottles of hydriodic acid used for more than a few months tended to give low yields, possibly because of the accumulation of iodine or of water, so relatively fresh hydriodic acid was always used in these experiments. The addition of 0.1 and of 0.5 ml of water prior to the hydriodic acid digestion reduced the color yield by 1 . 2 z and 6.8z,respectively. Acetic acid was found to be a relatively good solvent for the digestion of lung (2), and one in which hemin was relatively stable. It gave no consistent increase in the color yield when added to the hydriodic acid reagent, but was thought likely to increase the efficiency of the hydriodic acid digestion by its action on the tissues surrounding accumulations of blood. Figure 2 shows the absorption spectrum of the colored
All the chemicals used were of AnalaR grade where available. Absorbances were measured on a Cary Model 11 recording spectrophotometer in cuvettes of path length up to 10 cm. The treatment of ethanol or formalin-fixed lungs to give dried powders from which representative samples can be taken has been described by the authors (5). There were three sources of human erythrocytes: whole blood, heparinized erythrocytes, and citrated erythrocytes washed free of citrate. No significant differences were observed. Samples of these materials were freeze-dried or dried in an oven at 105 "C,in some cases after fixation in formol-saline. Some freeze-dried unfixed samples were fixed in formolsaline and then freeze- or oven-dried. All analytical values have been corrected for the moisture content of the material in question by heating a portion for eighteen hours at 105 "C. Digestion of Blood-Containing Tissue. About 1.0 mg of hemin or 40 mg of dried blood or tissue is weighed into a 30-1111 glass-stoppered test tube. A sample of the same material is taken for a moisture determination. About 4 grams of phenol are added and the tube is placed into a boiling water bath. After a delay of about 15 sec to allow the air to expand, the tube is stoppered and heated for 1 hour. Two milliliters of glacial acetic acid and then 2 ml of hydriodic acid (sp gr 1.94) are then added. The tube is replaced in the
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10. AUGUST 1971
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product of the reaction between a hemin digest andp-dimethylamino-benzaldehyde in acetic acid, in relation to the spectrum of a reagent blank. A sample of hemin of iron content 8.45% was taken as a basic standard for the present work. The molar absorptivity of the final color with respect to the hemin was 192 X l o 3 liter cm-' mole-', and with respect to the iron was 194.5 X lo3 liter cm-l mole-'. The iron contents of the various blood samples were assumed to be entirely due to the presence of hemoglobin. This meant that the color yield could be related to the iron content irrespective of the presence of serum or of the additional material owing to formalin fixation. Freeze-dried human whole blood, heparinized red cells, or red cells citrated and then washed free of citrate, all gave molar absorptivities of about 165 X l o 3 liter cm-1 mole-', based on their iron contents-ie. about 85 % of the hemin figure. When these freeze-dried materials were heated overnight at 105 "C, the color yield dropped by about 2 0 z and on further heating continued to drop by about 6 % of the previous value per day thereafter. The yield also dropped when the materials were stored some months at room temperature. The formalin-fixed lungs for which blood content values were to be determined, had been dried at 105 "C for one to two days. Their color yield might well have been affected by this treatment. A number of the lungs were heated at 105 "C for several more days. Some of these lungs were moistened with four times their weight of water or formolsaline during heating. The color yields were not significantly affected. This suggested that formalin fixation might prevent the drop in color yield which resulted when freeze-dried blood was heated. This theory was reinforced when it was found that the color yield of an unfixed freeze-dried human lung was affected by heating at 105 "C in ; same way as the freeze-dried blood samples.
.
However, when various blood samples were fixed in formolsaline, their color yields were just as sensitive to heat as they were before fixation. Formalin fixation alone was evidently not enough to protect the crucial blood component from inactivation by heat. It appeared that there might be a material present in the lung tissue which protected the crucial blood factor, but only after formalin fixation. A human lung was minced and fixed in AnalaR formolsaline, and the entire suspension was freeze-dried. The color yield of this material fell less than 10% during one day of heating at 105 "C and less than 3 % of the previous value per day thereafter. Another portion of the minced, fixed lung was dried at 105 "C for two days with the entire formolsaline suspension. This time the color yield fell by only 3 % when heated for a further day, and about 0.3 of the previous value per day thereafter. The lungs whose blood contents were to be determined had been dried in a vacuum oven for one to two days at 105 "C and so were unlikely to have lost even as much as 15% of their hemin color yield. Such an effect is not serious, especially in the context of biological variations, when the hemin iron values are used in conjunction with total iron values to calculate the nonhemin iron in pneumoconiotic lungs; the hemin iron figures for a series of eighty coal-workers' lungs were on average less than a tenth of the total iron figures. The other main use of hemin analyses in the present work was as a correction to allow analytical values for dust, for instance, to be referred to blood-free lung tissue. The average computed whole-blood content of a series of coalworkers' lungs was about 20%, so the possible 15% coloryield loss owing to oven-drying was not likely to be serious in this context either. RECEIVED for review December 30, 1970. Accepted April 27, 1971.
Direct Potentiometric Measurement of Hydrogen Ion Concentrations in Sodium Chloride Solutions of Fixed Ionic Strength G. R. Hedwig and H. K. J. Powell Department of Chemistry, Uniuersity of Canterbury, Christcliurch, New Zealand The calibration of the cell glass electrode ' , H + (aq)~calomel electrode as a [H+} probe in the p H range 2.0 to 10.3 and at I = 0.04, 0.10, 0.15, and 0.20M (NaCI) is described. Calibration is effected against dilute HCI solutions and the buffer solutions ethylenediamineethylenediammoniumchloride and sodium acetateacetic acid, all of known [H+]. Plots of pH' (measured pH) against p[H+] are colinear for the three systems and coincident, within experimental error, for each ionic strength. The relationship pH' = (0.9951 =t 0.0003) (0.088 =t0.002) was observed. From this calip[H!] bration, [H+] can be accurately determined from pH' for NaCl solutions at these ionic strengths. I n contrasi, calibration against standard buffers and conversion of pH' to p[H+] by use of the Davies equation for mean ionic activity coefficients involves significant assumptions concerning residual liquid junction potentials and activity coefficients. The two approaches are shown to give quite different results for [H+] measurements. The described method of calibration i s applied to the determination of the protonation constants for 1,5,8,12-tetraazadodecane.
+
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THEREIS WIDESPREAD use of glass electrodes for the measurement of solution pH. The electrode is commonly used in a cell with liquid junction and its response to solution pH is determined relative to response to one or more standard buffer solutions of defined (conventional) hydrogen ion activity ( I ) . For the standard buffer, the emf of the cell glass electrodel 1 solution~KC1(saturated), Hg2C12(S),Hg(1) is given by E,
=
E"
=
E"
+ E,, + ELJ - ( R T . I ~ ~ A + ) / F + E,, + ELJ+ (2.303 RT.pH,)/F
(1)
where E,, and EL^ are, respectively, the asymmetry potential of the glass membrane and the liquid junction potential for the cell, and E" is the emf of the reference electrode with KC1 (satd) chosen as the standard state. (1) R. G. Bates, "Determination of pH Theory and Practice," John Wiley and Sons, New York, N. Y . , 1964, p 31.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
