Quantitative Microchemistry in Clinical Laboratories ALBERT E. SOBEL, Pediatric
Research Laboratory and Division of Biochemistry, Jewish Hospital of Brooklyn, Brooklyn,
N. Y.
As s result of these early micromethods, the demand for estimations in clinical and experimental medicine grew by leaps and bounds, owing to the better understanding of their significance. Where, in the earlier days, one to three components were analyzed in blood, the demand rose in some cases for as many as twenty to thirty components in a given specimen. If these analyses were to be done in duplicate (as they should be for the sake of reliability) they might require as much as 100 to 200 ml. of blood serum, representing 250 to 500 ml. of whole blood. Naturally this was not desirable and in 8ome cases such analyses were done on specimens drawn a t various time intervals. Often a portion of these analyses was not done a t all, even though the clinical condition warranted it. From young children and especially newborn infants, only small amounts of blood may be obtained and there it was even more imperative to limit the amount taken for analysis below that used by the prevailing micromethods. For multiple determinations and for infants, therefore, methods for even smaller amounts of blood were desired. From this background a new form of clinical microchemistry is being developed which we may call “ultramicro”, since it deals with even smaller amounts of sample than the older micromethods. Only 0.01 to 0.2 ml. of blood is required for an analysis. The amount of the component to be analyzed in such samples is a t times less than 1 microgram. Quantitative ultramicrochemis try in the clinical laboratory was made possible in part by refinements in apparatus and techniques and in part by new reagents and new procedures. In titrimetric methods the refinements are based on (1) the capillary microburet which permits titrations with 0.010 to 0.100 ml. of liquid with a precision of 0.0001 or even 0.00002 ml., depending on the purpose for which it was designed, (2) more sensitive indicators for the visual observation of the end point, and (3) the use, for titration, of dyes like dithizone and dichlorophenolindophenol whose color change a t the end point is perceptible a t great
Development of quantitative ultramicrochemistry for use in the clinical laboratory i s discussed, with descriptions of refinements in apparatus and techniques, new reagents, and new procedures, I n titrimetric methods refinements are based on the capillary microburet, more sensitive indicators, and use of indicator dyes such as dithirone and dichlorophenolindophenol. In colorimetric methods refinements are based on the microcolorimeter and the photoelectric colorimeter. Precision and reliability have been improved.
T
H E use of micromethods in medicine dates back to the days of the discovery of the microscope. This made possible the visualization of small cells and, later, with the aid of a calibrated chamber, permitted estimation of the number of red and white cells present in a given volume of blood. As far back as 1870, the blood to be employed in a red cell count was measured in dilution pipets with a volume of the order of 0.002 to 0.005 ml. In such pipets the actual volume was not carefully calibrated, Instead, the ratio was calibrated, of one volume (designed to hold the blood) to a second volume (to which the blood was diluted). I n 1878, Gowers (3), using a 0.02-ml, capillary pipet for measuring out his samples, described a method for the quantitative estimation of hemoglobin on 0.02 ml. of blood, The amount of hemoglobin present in such volume is about 3 mg. However, micromethods for the estimation of the other components of blood, present in concentrations of 1 part in a thousand to 1 part in a million or even less, were more difficult and this problem was solved much later. The development of these methods was stimulated by the discovery that the composition of blo_od, especially of blood serum, changes in certain diseases-for example, sugar increases in diabetes, uric acid increases in gout, calcium decreases in a type of convulsion called tetany and increases in hyperparathyroidism, and bicarbonate increases in alkalosis and decreases in acidosis. In the early days these studies required a great deal of blood. As late as 1912 a reliable method for blood sugar required 100 ml. of blood ( 1 ) . With such methods the checking of blood sugar levels for a day or a “glucose tolerance” test would require 400 to 800 ml. of blood; for duplicate analyses 800 to 1600 ml. of blood would be required. Such amounts cannot be taken with safety even from a healthy adult, let alone a sick one, and for young children are entirely prohibitive. , Between 1912 and 1922, Europe saw the development of micromethods by Pregl and Emich which were mainly designed for the ultimate analysis of organic and inorganic compounds. In the same decade a similar and parallel development of micromethods took place in clinical chemistry. Here, American chemists were outstanding. A complete list of the principal investigators would be far too long to enumerate here. (The reader is referred to the excellent bibliography and critical discussion of methods by Peters and Van Slyke, 9, which include most of the important procedures developed during this period.) However, three important investigators worked along three main directions: (1) gasometric methods, D. D. Van Slyke and associates; (2) colorimetric methods, Folin and associates; (3) titrimetric methods, B. Kramer and associates. Their procedures were characterized by the use of the pipet and not the microbalance for measuring the original sample. They aimed a t simplicity and speed which was then, even as it is today, desired in a clinical micromethod, because the results must be not only reliable but fast if the patient is to benefit from such analyses. These early micromethods required from 1to 10 91. of blood which were analyzed for components present a t times to no more than 20 micrograms.
A.
15-ml. centrifuge tube Microceabiture tobe with Bat bottom C. Calibrated microcentrifuge tube. Glibretion mark In nanow upper portion D . Mkroaerrtion tube Rtted with rubber stopper containing glau inlet tube
E.
r 6 Figure 1
242
C
D
Microapparatus for Blood Analysis
April, 1945
ANALYTICAL EDITION
dilution. Thus, titrations may he carried out with solutions as dilute as 0.00005 N a n d still have a visual ohsewation of the end point. I n colorimetric methods the refinements are based on (1) the microcolorimeter which permits the visual matching of small volumes of colored solutions, and (2) the photoelectric colorimeter, using an almost monochromatic light with the aid of light filters or a diffraction grating or prisms. These instruments increase both the specificity and the sensitivity, so that more dilute color can he employed successfully. The gasometric clinical methods did not undergo such drastic changea because they were capable of analysing smaller amounts of blood almost at their inception. For most purposes they do not h d so much favor because of time-consuming features and expense when a great number of analyses are to be carried out. It is likely, however, that some gasometric device, possibly a modified Van Slyke or Warburg apparatus, will become part of the routine eauivment ultra.~ _ . of the clinical laboratory. employing micromethods. I n the course of organizing and directing the chemical leboratories of this hospita1;the prohlem of simplicity as well as the use of decreasing amounts of blood challenged the authnr. The additional goal wm to improve the precision and reliahility of such methods. The manner in which this was met may serve as an illustration of recent trends in clinical microtechnique. To improve reliability a rigid system of duplicates, reagent blanks, knowns, and recoveries of known amounts added to a portion of the sample t o be analyzed was introduced as a routine measure. To reduce quantities of materials used in most of the colorimetric methods all volumes were cut down in proportion. Employing the high-speed centrifuge for removing precipitated proteins, the aliquot of the filtrate used for actual analysis represented 66 to 90% of the original sample or even 100% (when the precipitate was washed, as in the estimation of free cholesterol, 15). I n the earlier micromethods filtration was used and the aliquot obtained from the filtrate for the actual analysis represented only 50% or less of the original sample. By employing a photoelectric colorimeter which measures the absorption of light of more dilute colors than the visual colorimeter, the volumes of blood were cut further. I n these methods the proportions of reagents to blood were changed, so as to produce a more dilute solution which would still give adequate color for the final measurement. A typical illustration is the estimation of inorganic phosphorus by the method of Fiske and Subharow (%).
243
~
ESTIMATION OF INORGANIC
PHOSPHORUS
ORIGINU METHOD. To 2.0 ml. of serum add 8.0 ml. of 10% trichloroacetic acid, and filter through filter paper. To 5.0 ml. of the filtrate in a 10.0-ml. volumetric flask add 1.0 ml. of molyhdate I1 reagent, mix, and add 0.4 ml. of reducing reagent. Mix &gainand make up to mark with water. At the end of 10 minutes, read ageinst standard in a colorimeter. The precision is *2% on 30 to 50 micronam8 of vhosohorus. ~~~~
~
I
~~
~~
tube graduaiid a t 2.0 ml. Add 0.2 ml. of molybdate 11, mix, and add 0.1 ml. of reducing reagent. Mix again and dilute t o mark vith water. At the end of 10 minutes read against standard in microcolorimeter. The precision is *2% on 6 t o 10 micrograms of phosphorus. METEODFoa PEOTOELECTFCIC COLORIMETER. To 0.1 ml. of
Dhotoelectrio colorimeter. setting the instrument to 0 with a r e ~~
~~
~~
~~
~
standard. The ;recision is * l %on 3 to 5 mkrograms of phosph0r"S. In a similar manner most of the colorimetrie methods were reduced to use to '/m of the original volumes employed.
. . ." .
Figure 2. Titration Assembly for Ultramicroquantities of Calcium with a Rohberg Capillary Buret
I n the case of titrimetric methods, by employing the capillary buret instead of the usual microburet most methods were reduced from 1/,. to '/m of the original amount. Further simplification was brought about by converting the methods to a direct aeidimetric titration procedure. The evolution of a few ultrsmieromethods may serve to illustrate the process.
I N D U S T R I A L A N-D E N G I N E E R I N 6 C H E M I S T R Y
244
acid solution of similar strength. [Figure 2 shows a modified Rehberg microburet ( I O ) while Figure 3 shows a modified Rehberg microburet with a reservoir a t top for filling and air interface between mercury column and acid (fg).] The.average error is -0.2% and the precision is *l.Z% on 7 to 11 micrograms of calcium. CONVERSION
OF KJELDAHL AND UREA N I T R O G E N TO ULTRAMiCRO SCALE
ORIGINALINDIRECT ACIDIMETRIC TITRA.TION METHODFOR UREANITROGEN (.W). Use 3 ml. of serum. Convert urea by urease to ammonium carbonate. After treatment with alkali, aerate the ammonia over to the receiving tube containing an excess of 0.01 N acid. Back-titrate the excess acid with standard base, using a microburet. The average error is -0.2% and the on 2333 to 4650 micrograms of urea nitrogen. precision is ~ 0 . 3 % The average error is +0.6rr/, and the precision is 10.3 on 233 to 1160 micrograms of urea nitrogen. DIRECTACIDIMETRIC MICROTITRATION METHODFOR UREA AND KJELDAHL IVITROGEN(20). Convert the urea of 1 m!. ai serum to ammonium carbonate. After treatment with alkali; aerate the ammonia over to the receiving serum tube containing an excess of boric acid. Titrate the dissolved ammonium borate directly with a 0.01 or 0.00714 N standard acid. Kjeldahl nitrogen can be similarly determined instead of using the usual microKjeldahl method after conversion to the ammonium salts with sulfuric acid. The average error is -0.3% and the precision is *0.3% on 1000 micrograms of urea nitrogen. The average error is -0.5% and the precision is +0.8y0 on 1000 to 2000 micrograms of Kjeldahl nitrogen. DIRECT ACIDIMETRIC ULTR-ICROTITRATIONMETHODFOR UREA AND KJELDAHLNITROGEN (16). Convert theureaof 0.1 mi. of semm or the Kjeldahl nitrogen of 0.01 ml. of serum to the ammoniumsaltin a small side-arm test tube (Figure 1D). Aerate the ammonia over, after treatment with alkali, to the receiving tube which contains boric acid and the indicator mixture of Ma and Zuazaga (8) in slightly different proportions. Titrate the trapped ammonia with standard acid, using a capillary microburet (see Figure 3). (The aeration setup is illustrated in Figure 4.) The average emor is +0.2% and the precision is ~ 0 . 6 7 for~ 10 micrograms of urea nitrogen. The average error is ~ 0 . 1 % and the precision is *1.3% for 10 micrograms of Kjeldahl nitrogen. EXTENSION
OF
Vol. 17, No. 4
the case of iodometric methods. Because of this sensitivity. ultramicroquantities at trace concentrations of 10-6 to IO-’ may he determined without the capillary microhuret. This is especially true for dithiaone which is used at dilutiom of 0.00005 N for the visual observation of the end point. The titrimetric extraction method with dithizone (@, using an ordinary microburet) replaced the spectroscopic method (8.9) of determining lead. The change improved the reliahility sud the precision of the method and eliminated the need for a skilled spectroscopist. This change can be better appreciated when it is realized that the normal concentration of hlood lmd is about 0.01 to 0.04 mg. per 100 &.-that is, 1 to 4 parts in 10,000,000 ($55). I n the titrimetric determination of vitamin C a visual end point is observed with O.OOFJ N dichlorophenoliudophenol. At this strength 0.15 to 0.45 ml. of the dye is required in the estimation of vitamin C present in 1.0 ml. of blood (4 to 12 micrograms of vitamin C). Far such titrations &n ordinary 1.0-ml. microburet can be used. With a capillary microburet more conceu-
DIRECT T I l R A T I O N M E T H O D S
The direct titration methods were extended to the estimation of total base of hlood. The total bases are electrodirtlyzed according to Keys (6) and trapped in boric acid instead of excess standard acid and then evaluated by a direct titration with a standard acid (20). Electrodialysis was shown to he a powerful tool for doing away with ashing in the analysis of alkalies and alkaline earths. Thus, the loss of cations due t o volatilimtiou wae avoided and interfering anions like phosphates were removed (If, 14, 17). An electrodialysis unit designed in the author’s laboratory is shown in Figure 5. For iodometric naibods the capillary microburet is not always necessary because o; %:,.esensitive end point with 0.001 or even 0.0005 N thiosulfate so!r::ion in the presence of starch. ESTIMATION OF CALG~JM (12). Isolate the calcium of 0.1 t o 0.2 ml. of serum as the carbonates ag described above. Dissolve the calcium carbonate in an excess of 0.Oi N hydrochloric acid, and evaluate the excess iodometrically by means of the reaction:
2Hf
+ KIOl (excess) + K I (excess) --+L + Ha0 + . . ... starch I, + 2Na&Oi Na&O, + 2NaI ---f
The average error is -0.3% and the precisian is *0.8% an 10 to 40 microgrsms of calcium. ESTIMATION OF POTASSIUM (4).- Separate the potassium in 0.1 to 0.2 ml. of serum by electrodialysis ( 1 0 and precipitate as the potassium cobaltinitrite. Treat the precipitate with excess ceric sulfate and evaluate the excess iodometrically by adding potassium iodide and titrating the released iodine with 0.0007 N thiosulfate. The average error is -0.3% and the precision is *0.8% on 35 micrograms of potassium (if). In the methods employing dyes for titration a sensitive end point may be obtained with even more dilute solutions than in
Figure 3. Titration Assembly for Ultramicroquantities of Urea, Kjeldahl, or Amino A c i d Nitroven SelFClling n o d i k d (10) R A b w u ~ i l b wbunt
A N A L Y T I C A L E D I T 1O.N
April, 1945
Mass production was developed by time-saving variations in t e c h n i q u e f o r example, the simultaneous distillation of 60 to 80 urea nitrogens or Kjeldahl nitrogens; the use ef thermostatically controlled ovens for evaporating solvents like deohal, ether, and drying precipitates; the use of self-filling and automatic burets for measuring r e w m t s and titrations; the use of syrinse pipets for automatic delivery of constant. volumes; and use of large angular centrifuge heads to eliminate balancing and permit simultaneous cent.rifuging of 64 tubes. CONCLUSION
Figure 4.
Microaeration Assembly for Ultramicroquantities of Urea, Kjeldahl, or Amino Acid Nitrogen
r
Figure 5.
Eloctmdialyrir Assembly for Total Bare Estimation caiclum, and magnriivm ions lor
0, weiiminaty rrparatlm 01 sodium, potassium.
rubraquonl anaiyiii
trated dichloroplienolindophenol is used unless still smaller amounts of vit,amin C are to he determined. Throughout, the initial g o d of simplicity and speed was never lost, sight of.’ To ensure simplicity the number of transfers was reduced by developing methods and microtubes such that the whole process could be carried out in one centrifuge tube. Figure lC, shows a calibrated microcentrifuge tube which is constructed on top like a volumetric flask. This WRS dwigned far colorimetric del.erminntion of potassium ( I S ) . I n tliis tube the potassium coba1i.init.rit.eis isolated, ashed, treated ll-ith reagents that are speeific for.robalt, and finslly made up to mark. It is also employed far est,imating magnesium colorimetrically after isolation of rhe magnesium ammonium phosphate. I n general, such tubes can be used for methods in nhich the isolation of a precipitate precedes a colorimetric measurement. The altering of &methodfor simplicity and speed is illustrated by comparing the new procedure for estimating ifee cholesterol to the earlier one used in the author’s laboratory. Three timeconsuming and painstaking Steps %!ere eliminated, thereby improving the reliability of the determination.
EARLIER METHODFOR FREECHOLESTEROL (6). Extract 0.5 mi. of serum with 25.0 ml. of alcohol-ether mixture, and evaporate the extract t,o dryness. Transfer the lipids in the residue quanbtstiveiy to a centrifuge tube with three 2.0-ml. portions of ether, and evaporate this extract to dryness. Then dissolve the residue in’95%alcohol and treat with a digitoninsolution. After 12 to 16 houn wash t,he DreeiDitated dieitonide and determine colorimetrically. When 200 to 400 micrograms of cholesterol.are added to the Lipid extract the average error, under routine conditions, IS -1.6% and the precisionis -3.8%. IMPROYED METHODFOR FREECHOLESTEROL (16). Extract 0.2 ml. of serum with 3.0 to 4.0 ml. of alcohol-acetone mixture, treat the extract with digitonin, and place,it in an incubator at 37“. After 3 houn wmh the precipitated digitonide and determine
. .
I
The last. chapter in this field has by no means been mitten. I3y the aid of new and improved instruments and more delicate methods simultaneous analyses for several components of one small sample will be possible. Furthcr developments in methods should see: (1) the routine use of the potentiometer and conductivity meier for determining the enti point nf n titration; (2) the development of the spectrophotometric colorimeter for smaller amounts of liquid; in addition, infr:n.eil, ultraviolet, and Ramnn yicatrosropy might he intmdiircd into clinical mdieinc: (3) the more successful use of the spwtwsrope for the simultaneous estimations of cations and trace elements; (4)the use of the polarograph for the simultaneous estimiLtians of a great numher of Components an 31 small amount of mnt.erial; ( 5 ) the employment of electrodialysis for the sepbration of cations, anions, and nonelectrolytes with subsequent estimations of these components by other methods; and (6) the use of the Tiselius electrophoresis apparatus for the simultaneous estimation of various protein fractions with the subsequent use af the sample for other analysis. W e may confidently look formrd to the day when n e will reach the ult.imate p a l of determining all eonst,itnents of blood on only one drop. LITERATURE CITED
(1) Bertrand, G., Bull. SOC. chim. Paria, 35, 1285 (1906). (2) Fiske. C. H.. and Subbarow, Y.. J . Diol. Chem.. 66,375 (1925). (3) Gowers, W. R.. Tinns. Clin. SOC.London, Lancet, 2, 882 (18i8). (4) Kaye. I. A,. IND. ENO.CHEM..ANAL.Eo.. 12, 310 (1940). (5) Kaye. I. A., J . Lab. Clin. Md..25. 996 (1940). (6) Keys, A,. J . Diol. Chem.. 114, 449 (1936). (7) Krarner, B.. and l’isdall, F. F., Ibid.. 47,475 (1921). (8) Ma, T. S., and Zuezaga, G., IND.ENO.CHEM.,ANAL Eo., 14, 280 (1942).
(9) Peters, J. P., and Van Slyke, D. D.. “Quantitative Clinical Chemistry”, Vol. 11. ,Methods, Baltimore. Willlmns & \VilkinsCo., 1932. (10) Rehhel-g. P. B.. Biochem. J.. 19, 270 (19?5). (11) Sohe1.A. E., IIanok. A,. and K r a m e ~B , . ,J . B i d . Chem., 144.363 (1942). (12) Sobel. A. E., and Knye, I. A,, IND.Exa. CHEM.,ANAL.ED.. 12, 118 (1940). (13) Sobel. A. E., and Krmer, B.. J . B i d Chem.. IW,561 (1933). (14) Sobel. A. E., Kraus. G., and K r m e r . B., Ibid.. 140,501 (1941). (15) Sobel. A. E., and Mayer, A. M., Ibid.. 157,255 (1945). (16) Sobel. A. E., Mayer. A. M., and Gottfried, S. P., Ibid., 156. 355 (1944). (17) Sobel. A. E., Rookenmaeher. M., snd Kramer. B..Ibid.. 152,255 (1944). (18) Sobel. A. E.. and Sklersky, S., Ibid., 122. 665 (1937-38). (19) Sobel, A. E., and Sobel, B. A,. Ibid.. 129.721 (1939). (20) Sobel. A. E., Yuska. H., and Cohen, J.. Ibid., 118,443 (1937). (21) Trevan, J., and Bainbridpe. H. W., Biochem. J., 20, 423 (1926). (22) Van Siyke, D. D., and Cullen. G. E., J . Bid. Chem.. 19, 211 (1914); 24, 117 (1916). (23) . . W d e r . I. B.. and Sobel. A. E., Proo. Soc. Ezpt. Biol. Med., 32. 719 (1935) (24) Wilkins, E. S.. Jr.. Willoughby. C. E., Kraemer. E. 0.. and Smith,F.L.. IND.ENO.CHEM..ANIL. ED..7.33 (1935). (26) Willoughby, C. E., and Wilkins, E. S.. JI., J . Bid. Chem.. 124, 639 (1938). PREBENTED before the Division of Analytical and Micro Chemistry, Symp~lsiumon Piaotioal Applioations of Microchemistry, at the 108th hlePfing o l t h e IYERICAN CREMICAL S o n ~ r l -New , Surk. &. S.
