the determination of true sugar in blood. · 2003-03-15 · true sugar in blood liminary...

Reprinted from THE JOURNAL OP BIOLOGICAL CHEMIRTRY Vol. LXXXII, No. 1, April. 1929

THE DETERMINATION OF TRUE SUGAR IN BLOOD.

BY EDWARD S. WEST, FREDERICK H. SCHARLES, AND VERNON L. PETERSON.

(Prom the Department of Biological Chemistry, Washington University School of Medicine, St. Louis.)

(Received for publication, December 17, 1928.)

It is generally appreciated that the “sugar” of blood, as determined in “protein-free” blood filtrates by any of the current reduction methods, includes reducing substances other than the true sugar. Furthermore the identity of all of the true sugar is perhaps not yet fully established as glucose. It is therefore to be expected that different copper or other reagents, all standardized as to their glucose equivalents, will yield each a different result when applied to such a mixture of gIucose (?) and other uniden- tified reducing substances as is contained in blood filtrate. That expectation is fully realized in the work of a number of investi- gators; it is indeed surprising that results on the same blood by different methods do not differ more than they do.

Assuming the true sugar of blood to be glucose, two essentially different plans may be followed in designing an analytical process for its determination. The simplest and most direct plan would obviously be to choose or construct some highly selective reagent which responds only to glucose, and fails to react with such other reducing substances as may be present in blood filtrates prepared by tungstic acid precipitation or similar precipitations now in use. The second plan would be the removal of the interfering reducing substances, leaving only the true sugar in the filtrates.

With a few exceptions the attempts to determine true sugar have so far followed the first plan. Two types of selective reagents have been used: the selective destruction of the true sugar by yeast fermentation or by glycoly- sis and its determination by the loss of reducing power toward sensitive reagents which react also with reducing “non-sugars;” and the construction of less sensitive copper reagents designed to be reactive only toward the true sugar. Results by the use of yeast should be decisive, but were not wholly satisfactory until Somogyi emphasized the importance of pre-

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True Sugar in Blood

liminary purification of the yeast suspensions and described the optimum conditions for their use. The earlier conflicting results with yeast are reviewed by Van Slyke and associates (1) and by Somogyi (2). By the Somogyi technique it seems probable that the true sugar may be determined with accuracy. The second type of selective reagent is represented by those of Benedict (3) and Folin (4), all of which however show the presence of reducing substances in blood filtrates after fermentation, and therefore give with unfermented filtrates results which though lower are perhaps only relatively less erroneous than are obtained with more sensitive reagents. To be sure Benedict reports data ( (3) Table I, p. 468) which show satisfactory determination (+ 3 mg. per cent) by his latest (1928) reagent of glucose (100 mg. per cent) added to previously fermented blood filtrates; though before the addition of the sugar the same fermented filtrates with the same reagent gave a reduction corresponding to from 6 to 14 mg. per cent. From this fact one may conclude either that the sum of non-fermented substances plus added sugar is not completely determined, or that in the presence of sugar (though not in its absence) the non-fermented substances are inactive toward the reagent, and that the results represent only the added glucose or the true sugar. The latter is Benedict’s interpretation, and may be the correct one in spite of the somewhat remarkable assumption that the presence of sugar somehow prevents the oxidation of the non-sugars. In this connection it may be noted that the diflerence between the reducing values before and after fermentation, presumably the true sugar, in the data reported by Benedict, are with Benedict’s reagent from 6 to 24 mg. per cent (average 15.6) lower than by the Folin-Wu reagent. Since both reagents are standardized against glucose, the decrease of reduction by fermentation should be identical unless (1) the true sugar is not glucose but some other sugar (or mixture) having different reducing power toward the two reagents or (2) unless the presence of other substances in the filtrates decreases the sen- sitiveness of the Benedict reagent toward the sum of true sugar and reducing non-sugars by an amount about equal to the reducing power of the non-sugars. It can hardly be supposed that the Benedict reagent is in- sensitive toward the non-sugars in view of the fact that when the true sugar is removed by fermentation the reagent is reduced by them, though only about half as much (in terms of glucose) as with the Folin-Wu reagent. It seems impossible at present to decide between these two possibilities.

For the second plan mentioned above, the removal of interfering substances preliminary to sugar determination, precipitation by mercury salts has proved effective. Introduced in 1887 by Johnson (5), who used mercuric chloride with urine, the process was improved by Patein and Dufau (6) who employed acid mercuric nitrate neutralized by alkali. The Patein- Dufau solution has been used with urine and blood by a number of later workers, Deniges (7), Benedict and Osterberg (8), Shaffer andHartmann (9), Ronzoni and Wallen-Lawrence (lo), Harned (II), and Bierry and Voskres- sensky (12), several of whom have shown that under certain conditions glucose is not lost and that the precipitation does not interfere with sub- sequent determination of sugar. Harned has compared results for blood


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West, Scharles, and Peterson 139

sugar by the Folin-Wu calorimetric method in filtrates from mercuric nitrate-bicarbonate precipitation with results in tungstic acid filtrates by the Folin-Wu and Benedict (1925) (13) methods. His results by the Folin- Wu reagent’ on mercury filtrates of twenty-six normal and diabetic human bloods are 12 to 28 mg. per cent lower than on the tungstic acid filtrates.

If all of the reducing substances other than true sugar are removed by mercuric nitrate precipitation, the results by any single copper reagent on such filtrates would agree with the loss of reducing power by fermenting tungstic acid filtrates. And similarly the decrease of reduction caused by mercury precipitation should agree with the residual reduction after fermentation. These hypothetical relations may be clearer from the following.

Tungstic acid filtrate -

(A)

mercury filtrate

@I

reducing substance - removed by Hg.

(0

Tungstic acid tungstic acid fil- fermentable reduc- filtrate - trate after fermen- = ing substances, true

tation sugar.

(A) CD) W

If the total observed reduction is expressed in terms of mg. per cent of glucose in the original blood and the loss on fermentation (E) taken as the most probable value for the true sugar, the question is, with any single copper reagent does B = E and C = D? According to the work of Somogyi (2) the value of D, the “reducing non-sugars” left after fermentation (with his modification of the Shaffer-Hartmann reagent), in 52 human bloods (whole bloods) averages 26 mg. per cent, the extremes being 31 and 23. By a slightly different reagent Dr. Somogyi informs us he finds an equally constant value at 20. From Benedict’s data ((3), p. 468) we find for the same fraction in ten bloods, by the Folin-Wu reagent 20 f 3 and by the Benedict reagent 11.7 f 3. Folin and Svedberg (14) report values in thirty-five diabetic bloods, with the Folin-Wu reagent averaging 20 f 5, and with the Folin reagent 9 f 4. As would be expected the glucose equivalent of the non-

1 Harned’s results by the Benedict reagent on fifteen tungstic acid filtrates averaged only 5 mg. per cent higher than the Folin-Wu results on mercury filtrates, indicating nearly correct results by the Benedict reagent on filtrates containing the non-sugar.


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True Sugar in Blood

sugars varies with reagents used and comparison must be limited to the same reagent. Taking 20 mg. per cent as the value of D (the non-fermentable reducing substance in tungstic acid filtrates) by the Folin-Wu reagent, we may compare Harned’s data on the decrease of reduction toward the same reagent caused by mercuric nitrate precipitation. His values range from 13 to 26 with an average of about 20 mg. per cent. The agreement indicates that the amount left by yeast approximately equals the amount precipitated by mercury salts though the variation of 13 to 26 is somewhat greater than found by Somogyi with his own reagent. The same comparison may be made of the Somogyi reagent value of 26 ZI= 4 with the results by that reagent following mercury precipitation recorded by Ronzoni and Lawrence. In a series of eight bloods the diflerence between results on tungstic acid and mercuric nitrate filtrates varies but little from 13 mg. per cent. The agreement in this case between 26 mg. of non-fermented and 13 mg. removed by mercury is less satisfactory and suggests that perhaps the precipitation of non-sugars by mercury was in- complete.

According to our experience in carrying out this comparison, discrepancies of this sort are not uncommon, and are perhaps due to unavoidable variations in the alkalinity of the solutions when bicarbonate or hydroxide is used for neutralization in the mercuric nitrate precipitation. The completeness of precipitation of the non-sugars varies with the reaction at which the precipitation takes place. We therefore sought means of obviating this diffi- culty in order that a practical procedure might be had, giving consistent and dependable results. With the slight modifications now introduced the mercuric nitrate precipitation yields filtrates, the reducing power of which, in practically all human bloods so far analyzed, agrees closely with the true sugar as indicated by loss of reducing power on yeast fermentation. As a rule B equals E in the above equations. The preparation of the mercury filtrates is little more laborious than the preparation of tungstic acid filtrates and requires less time and manipulation than Somogyi’s yeast method. The procedure is therefore recommended as a method for the direct determination of true blood sugar; direct, in the sense of preliminary removal of all interfering substances.

In some bloods, notably of pig and sheep, the mercury filtrates


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West, Scharles, and Peterson

contain a few mg. per cent more reducing power than corresponds to the fermentable sugar. The identity of these non-fermentable substances is not indicated by our data, but may benon-fermentable sugars such as appear to be present in many normal urines as suggested by Greenwald, Gross, and Samet (15) and Host (16), since it is to be expected that the same reducing substances present in urine are to be found also in blood, though not in the same relative amounts. The bloods of two human subjects, a case of hypertension with uremia and another of “malignant hypertension” with uremia have also given mercury filtrates in which the reducing power was several mg. per cent higher than corresponds to the true sugar by fermentation. This clue is being followed but so far we have found no other cases.

Procedure.

We shall describe two different methods of carrying out the mercury precipitation, in both of which neutralization is ac- complished by solid barium carbonate. The result of this method of neutralization is that a satisfactory reaction is automatically attained and variations of alkalinity which occur when NaOH or NaHC03 are used, are thereby avoided.

The first process utilizes the Patein-Dufau mercuric nitrate reagent, which in the second is substituted by a solution of mercuric sulfate in sulfuric acid. The latter possesses the distinct advantage in that the precipitation of non-sugars is if anything more complete, and that all of the constituents of the reagent are removed, leaving only the electrolytes of the blood in the filtrate. The sulfate as well as the mercury is removed by treatment with the barium carbonate. A decided advantage of the mercuric sulfate method from a practical standpoint is the rapidity with which it can be carried out, especially if the zinc method of removing mercury and mpid filter papers are used. In all respects we consider the mercuric sulfate method superior to the nitrate with the minor exception that it requires more barium carbonate for neutralization.

For determination of the reducing power of all blood filtrates a Shaffer-Hartmann copper reagent, as modified by Somogyi, has been used. The reagent is a second unpublished modification by


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142 True Sugar in Blood

Dr. Somogyi, of the following composition, with which a table of glucose values was constructed by him with pure glucose.

Sodium carbonate, anhydrous.. . . . . . . . 20 “ bicarbonate........................................ 23

Rochelle salt., . . . . . . . . . . . 23 Copper Sulfate, crystalline.. . . . . . . . . . . . 7 Potassium oxalate.......................................... 5

‘I iodide........................................... 10 “ iodate........................................... 0.8

Mercuric Nitrate Method.

Mercuric Nitrate Reagent of Patein and Dufau.-220 gm. of mercuric oxide are dissolved in small portions in 160 cc. of concentrated nitric acid. The solution is boiled, cooled, 60 cc. of 5 per cent sodium hydroxide added, diluted to 1000 cc., and filtrrrd through asbestos. It is preserved in a brown bottle.

5 cc. (1 volume) of blood are laked in 40 cc. (8 volumes) of water in a 125 to 150 cc. Erlenmeyer flask fitted with a rubbrr stopper. 5 cc. (1 volume) of the mercuric nitrate reagent arcs added slowly from a burette with shaking. The flask is stopper et I and vigorously shaken to break up the gel. Approximately :1 gm. of precipitated barium carbonate2 are added and the Has.; rapidly rotated for a few seconds until most of the CO2 has esc:apcd. The flask is then stoppered, shaken hard, and opened to release carbon dioxide. Shaking is repeated until there is no further pressure developed in the flask. Neutralization requires 2 or 3 minutes and is indicated by the absence of pressure in the flask after stoppering and shaking, the visible presence of an excess of barium carbonate, and the fact that red litmus paper moistened by the liquid is slowly turned slightly blue. The precipitated material is poured upon a rapid filter3 and the filtrate (16 to 18 cc.) collected in a 125 to 150 cc. Erlenmeyer flask. 1 drop of HzSO, (1: 1)

2 C.P. and pure “precipitated” BaC03. We have used the preparations from Mallinckrodt. The physical condition of the substance varies con- siderably, which affects its neutralizing power, especially in the case of mercuric sulfate precipitation. A very finely divided material is preferable

3 For the most exact work the filter papers should be washed with hot water and dried or tested for their content of extractable reducing substances as suggested by Benedict.


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and 0.3 gm. of anhydrous Na2S0,* are then added to the filtrate and the mercury precipitated by treating with H&S (moistened by bubbling through water) for a half minute. Excess hydrogen sulfide is blown off by a rapid current of air, likewise moistened by bubbling through water. This requires about 30 seconds. A drop of 10 per cent CuSO4 is added to insure the removal of the last traces of H2S and the solution filtered or centrifuged; if centrifuged the clear supernatant liquid is passed through a small filter to remove suspended particles. The preparation of the filtrate takes about 45 minutes. 5 cc. portions are accurately measured into 25 X 200 mm. test-tubes. 2 drops of phenol red (0.05 per cent in water) are added to each tube, followed by 0.5 N NaOH drop by drop from a medicine dropper until the red color of the indicator appears. Ordinarily 3 to 6 drops of the alkali are required to neutralize a 5 cc. portion of filtrate. 5 cc. of the Shaffer-Hartmann reagent are added and the sugar determination executed in the usual way.

Some points call for brief comment. An excess of barium carbonate above 3 gm. is of no consequence except that the volume of filtrate obtained is reduced. A larger quantity of filtrate, 5 to 8 cc., is obtained and the time required is possibly less when the precipitate is removed by centrifugation. 0.4 to 0.5 gm. of Na2S04 should be added to the filtrate in this case. 1 drop of the H2SOI is sufficient.

The quantity of filtrate obtained and the rate of filtration are increased by vigorously shaking the precipitated mixture with short quick jerks.

A large excess of sodium sulfate should be avoided because as shown by Somogyi (17) high salt concentration causes an increase in reduction values. The salt concentration of filtrates prepared as above outlined is close to 2 per cent and does not perceptibly affect the determinations with sugar concentrations under 100 mg. per cent. With concentrations of 200 mg. per cent the salt error is about +3 mg. per cent.

The pH of filtrates immediately after precipitation and filtration

* The Na&304 must not be added to the blood-barium carbonate mixture before filtration. When this is done barium carbonate reacts with Na&3~ forming Na2C03 which raises the pH of the solution into the distinctly alkaline region and erratic sugar values are obtained.


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was determined calorimetrically. In a series of seven filtrates prepared consecutively from beef blood six had a pH of 6.3 and one of 6.2. If the filtrates be prepared and allowed to stand exposed to the air for a time, irregular and higher pH values are found which are the result of the loss of carbon dioxide. After standing the pH may be above 7. It is likely that at the time of neutralization by barium carbonate the pH is rather less than 6.3 because a small amount of CO2 escaped before the tests were made. The sig- nificant fact is that a uniform pH is automatically obtained in the precipitation, thereby insuring uniform removal of interfering substances.

Sugar determinations on fermented mercury filtrates of beef blood, with and without added glucose, were made. The effect of adding the approximate quantity of salts found in mercuric nit.rate- barium carbonate filtrates to pure glucose solutions was also determined. The results show that the fermented mercury filtrates contained no reducing non-sugar or non-fermentable sugar detectable by the reagent used and that added glucose is quantitatively recovered when correction for the salt error ismade. For example 198 mg. per cent of glucose were added to the fermented mercury filtrate and 201 mg. per cent found. 197 mg. per cent of glucose were added to a salt solution (1.5 per cent NaN03 + 0.5 per cent Na2S04) and 200 mg. per cent found. The salt error, 3 mg. per cent, subtracted from 201 gives a recovery of 198 mg. per cent;the sugar added to the fermented filtrate. The salt error for a glucose solution of 63 mg. per cent was found to be about + 1 mg. per cent. These salt errors, certainly in the lower blood concentrations, are within the limits of experimental error for the method as applied to blood and no corrections have been made for them in the results of Tables I to III. They are probably partly balanced by similar errors in the Folin-Wu filtrates.

Mercuric Xulfate Method.

Mercuric Sulfate ReagenL4-The reagent consisted of a 30 per cent solution of mercuric sulfate in 10 per cent sulfuric acid. A

4 The reagent is best prepared by adding the sulfate, in small portions with shaking, to a volume of 10 per cent H2S04 equal to about 0.9 of the final volume. After all of the sulfate is dissolved the solution is dilutedto the required volume with more of the acid.


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slight precipitate of the basic sulfate generally forms from which the solution is decanted.

Procedure.

5 cc. of blood (1 volume) are laked in 50 cc. of water (10 volumes) and 5 cc. (1 volume) of acid mercuric sulfate added, with shaking, from a burette. The flask is stoppered and thoroughly shaken to break up the gel. 9 to 10 gm. of precipitated barium carbonate are added, the flask rotated for a few seconds until most of the carbon dioxide has escaped, and then stoppered and shaken. Carbon dioxide is released and the flask again stoppered and shaken. This is repeated until no further pressure develops in the flask and the mixture does not turn blue litmus red (neut,ral to litmus). If neutralization does not take place promptly, add 1 to 2 gm. of additional barium carbonate. The mixture is poured upon a rapid filter and traces of barium and mercury removed from the filtrate in either of two ways. One procedure is to add a drop of saturated sodium sulfate to the filtrate (20 to 22 cc.), and a pinch of zinc dust, followed by shaking for a few seconds. The zinc and trace of barium sulfate are filtered off on a rapid filter and the filtrate is ready for analysis. This method is recommended for use with the modified Shaffer-Hartmann reagent which we have employed. It has been found that the hydrogen peroxide formed in shaking the filtrates with Zn dust causes erroneous results upon heating with Shaffer-Hartmann reagents containing no iodide or insufficient quantities of it. The reagent of the composition given above is entirely unaffected by the peroxide and gives the same results with the zinc and hydrogen sulfide filtrates. It also gives the true sugar values on pure glucose solutions treated with either mercury and zinc or with hydrogen peroxide. Upon standing these zinc-treated filtrates develop a slight precipitation, presumably of zinc carbonate. It causes no trouble. The second method recommended for the removal of mercury is the usual treatment with hydrogen sulfide. 1 drop of concentrated sulfuric acid is added io the filtrate, moistened hydrogen sulfide passed through it for a half minute, and the latter removed by a current of moist air. 1 drop of 10 per cent CuSO4 is added and the precipitate filtered off on a rapid filter. The filtrate is neutralized before sugar determination in the manner


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indicated for mercuric nitrate filtrates. It is to be noted that the dilution is 1: 12 in the mercury sulfate filtrates instead of the customary 1: 10.

The mercury sulfate filtrates of normal blood are nitrogen-free. A study is being made of the nitrogen in these filtrates of pathological bloods.

Experiments demonstrated that the fermented mercury sulfate filtrates of normal bloods contain no reducing substances detectable by the sugar reagent used and that added sugar is quantitatively recovered from such filtrates. There is no salt error. For example, 199 mg. per cent of glucose were added to fermented mercury sulfate filtrate of beef blood and 200 mg. per cent found.

Table III gives sugar values obtained on mercury sulfate filtrates of various bloods in comparison with those by fermentation and in a few cases by mercuric nitrate.

DISCUSSION.

The data presented in Table I give a comparison of the sugar content of human blood as obtained directly on the mercury filtrates and by Somogyi’s fermentation procedure. It is assumed that the loss on fermentation represents the true sugar. With the exception of the bloods from two pathological subjects, the agreement between the true sugar values and those by direct determination is within the accuracy obtainable with the reagent. In most cases the results differ in either direction only 2 or 3 mg. per cent, in four cases out of twenty-six the mercury filtrate result is 5 mg. per cent higher. With the eleven normal bloods the average by the two methods is identical, and including the diabetic and nephritic bloods (excluding the two cases, Samples 21 and 28) the averages are 103 and 103.6 mg. per cent.

In the samples of Series 21 from a case of cardiac hypertension with high blood non-protein nitrogen we found an interesting exception. There was some reducing (or iodine-absorbing) substance present in the blood of this patient which was not precipitable by the mercury treatment and which was absent from the other bloods examined, with the possible exception of Sample 28. The fact that the patient of Series 21 was completely digitalized at the time all bloods were drawn suggested that possibly the digitalis was contributing directly or indirectly to the reducing


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TABLE I.

Human Blood.

Description of blood.

(3) :41 ;5) - w. w. w. m?. Per ?er Per Per :ent ent ent cent

1 Vormal human. .19 25 94 96 2 “ “ 93 19 74 75 3 “ “ 88 25 63 62 4 “ “ 98 21 77 75 5 “ ‘I 87 18 69 70 6 “ “ 87 19 68 66 7 “ “ 91 17 74 76 8 “ “ -02 18 84 85 9 I‘ “ 92 19 73 71

10 “ ‘I 92 16 76 77 11 “ “ 93 24 69 68 12 tiixed hospital. -58 19 39 42 13 “ “ -29 17 .12 12 14 I‘ “ 114 20 94 99 15 “ “ .28 21 .07 12 16 ‘I I‘ L.36 18 .18 18 17 Diabetic. .59 18 .41 46 18 ‘I nephritic (non-protein N 105) re-

ceiving insulin. to1 18 83 83 19 Nephritic, terminal (non-protein N 143). 137 25 .12 15 20 Diabetic (sugar before insulin 550) receiving

insulin. 124 24 too 99 21.9, Cardiac hypertension (non-protein N 95);

completely digitalized, blood glycolyzed 4 hrs. 47 41 6 24

21b Fresh sample July 19, 1928. 106 51 55 68 21c “ “ (non-protein N 110) July 20,

1928. 130 47 83 92 21d Fresh sample (non-protein N 207) July 22 t

1928. 116 4t 68 86 21e Sample in ice box overnight; July 23, 1928 ,

patient died. 123 4: 81 89 22 Terminal uremia, sample on ice 24 hrs. 38 3C 2 0 23 Diabetic. 288 1: 273 178 24 ‘I 238 2: 216 112

- - - *F. W. = Folin-Wu filtrate, F.W.Y. = Folin-Wu filtrate after yeast

fermentation. Sugar determinations were carried out on the filtrates in duplicate with the Shaffer-Hartmann reagent modified by Somogyi.

147

- - - -


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TABLE I-conchded.

25a 25b

26a 26b

27a 27b

28


(2)

Cardiac decompensation before digitalis. Completely digitalized (28 cc. digitalis for 6

days). Cardiac decompensation; not digitalized. Completely digitalized (25 cc. digitalis for 5

days). Cardiac decompensation; not digitalized. Completely digitalized (30 cc. digitalis for 5

days). “Malignant hypertension” with uremia

(non-protein N 250); died 2 hrs. after sample.

Average (Samples 21 and 28 omitted).

- -

l . s Fi i c

32

(3) - - mg. Per cent 117

118 100

128 123

107

354

l . b? SF

2% ‘G 2 -$Fj e:

(4)

ml. PW

cent

21

20 21

21 19

20

54

98 95 79 81

107 107 104 101

87 90

300 315 ~~ 103 103.6

substances of the blood and causing too high values by the mercury method. Tha,t this is not the explanation is shown in the results on Samples 25 to 27, determined both before and after digitalis treatment. IIere the fermentation and mercury procedures check in a normal way. The blood from a case of terminal uremia, Sample 22, contrasts strikingly with the blood of Sample 21. The mercury precipitation removed all reducing non-sugars from the glycolyzed blood and showed a sugar value of 0 (Somogyi reagent), which was quite in agreement with the true sugar content by fermentation. It appears from such findings that in some cases of retention fairly large quantities of reducing (or iodine-consuming) non-fermentable substances, not precipitable by mercury, are present in the blood. This point is being further invest.igated.

The data of Table II give results for the sugar and reducing non-sugars in blood of several animal species. The reducing non-sugars, on the average, are lower for pig blood than any ot,her blood examined, and yet the sugar content of pig blood, determined


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1 2 3

4

5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24


TABLE II.

Animal Blood.


(2)

w. Per xnt

86 88

w7. Per

cent

24 25

w. Pm xnt 62 63

24 88 22 66

w??. P@ cent

Beef blood. 62 “ “ 65 “ ‘I same as Sample 2, in ice box hrs. 62

Beef blood same as Sample 2, in ice box 48 hrs . 58

Rabbit blood. 114 Cat blood after ether anesthesia. 215 Pig “ 60

‘I ‘I 58 Same as Sample 8, in ice box 48 hrs. 58 Pig blood. 67

‘I “ 54 “ I‘ 63 “ “ 51 “ “ 58 ‘I “ in ice box 24 hrs. 40 ‘I “ “ “ “ 24 “ 44 “ “ 68 ‘I “ 72

Same as Sample 18, in ice box 11 days. 55 Sheep blood. 54

“ “ 49 “ ‘I 46 “ I‘ 39 ‘I ‘I 35

81 143 242 72 70 68 77 71 71 65 72 47 49

77 89

69

71 69

59

58 51

-

23 26 22 16 17 16 15

18 14 17 15 20 17 13 22 20 22 19 19 16 17

58 117 220 56 53 52 62 53 57 48 57 27 32 64 67 49 49 50 40 42 34

-

--

-

* F.W. = Folin-Wu filtrate, F.W.Y. = Folin-Wu filtrate after yeast fermentation. Sugar determinations were carried out on the filtrates in duplicate with the Shaffer-Hartmann reagent modified by Somogyi.


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by the mercury method, is generally several mg. per cent higher than the true sugar by fermentation. Mercury filtrates of some fresh pig bloods absorb a little iodine in acid solution, but this if included in the sugar results would be equivalent to no more than 2 to 3 mg. per cent of sugar in the Shaffer-Hartmann procedure. Mercury filtrates prepared from pig bloods kept in the ice box for some days contain much less iodine-absorbing materials but they

TABLE III.

Mercuric Sulfate Filtrates.

1 2 3 4 5 6 7 8 9

10

11

12 13 14

Blood.

Beef. C‘ I‘

Pig. “

Human, normal. “ cardiac decompensation. “ “ “ ‘I “ “

Hypertension with uremia (non- protein N 185).

“Malignant hypertension” with uremia (non-protein N 250).

Catarrhal jaundice. Cardiac decompensation. Congenital cystic kidneys (non-

protein N 168).

1

-

Sugar by iermenta-

tion.

Sugar, mercuric

sulfate filtrates.

%% :c: r;l% c”c”T 158 160* 213 215 253 252

56 54* 46 51 75 76

107 108 87 84 98 97

51

81 90 89

300 114 105

79

310 114* 108*

315

80*

* Mercury removed with zinc dust; in other cases removed with hydrogen sulfide.

still show about the same amount of sugar in excess of the fermentation methods as do filtrates of fresh blood. These facts seem to point to the conclusion that pig blood is unique among the animal bloods examined (with the possible exception of sheep blood) in containing appreciable quantities of an unknown reducing substance which is not precipitated by mercury and which is not destroyed by keeping the blood for a long time at low temperature.


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West, Scharles, and Peterson

It is interesting to speculate concerning the nature of these iodine-absorbing substances present in mercury filtrates of some pig bloods. It is improbable that thioneine and glutathione, both of which are present in pig blood (18), contribute to these substances because both compounds are precipitated by mercury. They resemble the iodine-consuming substances found in Folin- Wu filtrates cited by Somogyi (2) in that both disappear from blood on standing.

The data of Table III show that mercuric sulfate-barium carbonate precipitation of blood yields filtrates which give true sugar values as judged by those of yeast fermentation. In a few cases, Samples 5, 10, and 11, determinations were made on mercuric nitrate filtrates also. The results are identical within the limits of experimental error. Sample 11 shows a result 5 mg. per cent higher by the nitrate method than by the sulfate. This is probably due to salt error in the nitrate filtrates. Samples 10 and 11 are the same as Samples 21 and 28 of Table I and show the same exception to the sulfate method as to the nitrate.

The writers feel that the mercuric sulfate-barium carbonate technique, with zinc and the modified Shaffer-Hartmann sugar reagent, constitutes a procedure much superior to the nitrate method for the direct determination of true blood sugar. To summarize, it is much less laborious, very much faster, and probably a bit more accurate than the nitrate method because the salt error is ruled out. It is being found very satisfactory in these laboratories for the treatment of urine, hydrolyzed tissues, etc., in that no electrolytes which are not removed are introduced and there is little or no salt error in the sugar determinations. The case is quite the opposite with mercuric nitrate precipitation.

The writers are indebted to Mr. Earle Adler for technical as- sistance in carrying out many of the analyses. Mr. A. H. Dowdy also has aided in some of the determinations. To Dr. D. P. Barr of the Department of Medicine we wish to express our appreciation for his interest in the work and a generous supply of pathological bloods from Barnes Hospital. Dr. M. Somogyi has kindly given us many helpful suggestions and supplied some of the bloods used. To Professor Shaffer we owe much for valuable suggestions and criticism.


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SUMMARY.

A study has been made of the preparation of blood filtrates for sugar analysis by precipitation with mercuric nitrate followed by neutralization with barium carbonate. The neutralization pro- ceeds automatically and consistently to a pH of about 6.3. Fil- trates prepared from human blood by this procedure, both normal and diabetic, yield true sugar values as judged by those obtained with Somogyi’s yeast fermentation method. A case of cardiac hypertension with high nitrogen retention proved an exception to the usual close agreement. Cases of uremia and diabetes-nephritis gave true sugar values on mercury filtrates.

Some pig bloods contain a non-fermentable reducing substance (or substances) not completely removed by mercuric nitrate-barium carbonate treatment. Pig bloods also often contain some iodine- absorbing substance which apparently is neither glutathione nor thioneine.

A mercuric sulfate-barium carbonate-zinc technique is introduced for the preparation of filtrates for sugar analysis. It is simple, rapid, and probably a bit more accurate than the mercuric nitrate-barium carbonate method because the salt error is ruled out. It is especially recommended for the preparation of filtrates of urine, hydrolyzed tissues, etc., as well as blood, because no electrolytes from the precipitating agent remain in the filtrates.

Addendum.-Recently it has been found that Folin-Wu filtrates may be very simply treated by the mercuric sulfate-barium carbonate-zinc technique to give both true sugar and reducing non-sugar values.

Add 1 drop of I:1 H,S04 to 20 to 25 cc. of Folin-Wu filtrate and then about 0.5 gm. of solid mercuric sulfate, followed by vigorous shaking for 15 to 20 seconds. Considerable mercuric sulfate is converted into the basic sulfate and does not dissolve. This in no way interferes with the determination. Add 1 to 2 gm. of precipitated BaC03. Shake until most of the CO, has escaped, then stopper the flask, and shake until no further pressure develops and the mixture is neutral to litmus. If necessary add a little more BaC03. Filter, add I drop of saturated NaGSO solution, a pinch of Zn dust, shake, and filter. The sugar is determined in the filtrate by the modified Shaffer-Hartmann reagent cited above, which is unaffected by the peroxide contained in the Zn-treated filtrates. The sugar value obtained on the original Folin-Wu filtrate minus that on the mercury-treated filtrate represents the reducing non-sugar. This procedure is especially recommended for use when it is necessary to prepare Folin-Wu filtrates for a number of different determinations and it is desirable to estimate quickly and


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accurately true sugar and reducing non-sugar. It is also much less expen- sive than the direct treatment of blood with the mercuric sulfate solution, and some workers may prefer to prepare Folin-Wu filtrates first and then treat them as outlined. Filtrates prepared in this way contain some nitrogen but the reducing non-sugars are completely removed. Professor Shaffer has observed that mercuric acetate may be satisfactorily substituted for mercuric sulfate in the treatment of Folin-Wu filtrates. Urine and hydrolyzed tissues cannot be effectively precipitated with solid mercuric sulfate.

BIBLIOGRAPHY.

1. Hiller, A., Linder, G. C., and Van Slyke, D. D., J. Biol. Chem., 64, 625 (1925).

2. Somogyi, M., J. Biol. Chem., 76,33 (1927). 3. Benedict, S. R., J. Biol. Chem., 76, 457 (1928). 4. Folin, O., J. Biol. Chem., 67, 357 (1926). 5. Johnson, G. S., Proc. Roy. Sot. London, 62, 365 (1887). 6. Patein, G., and Dufau, E., J. pharm. et chim., 16,221 (1902); abstracted

in Chem. Zentr., 73,954 (1902). 7. Deniges, G., Ber. internat. Kongr. angew. Chem., 4, 130 (1903); cited in

Neuberg, C., Biochem. Z., 24, 426 (1910). 8. Benedict, S. R., and Osterberg, E., J. BioZ. Chem., 34, 195 (1918). 9. Shaffer, P. A., and Hartmann, A. F., J. BioZ. Chem., 46, 365 (1920-21).

10. Ronzoni, E., and Wallen-Lawrence, Z., J. BioZ. Chem., 74,363 (1927). 11. Harned, B. K., J. BioZ. Chem., 66,555 (1925). 12. Bierry, H., and Voskressensky, A., Compt. rend. Sot. biol., 98, 287 (1928). 13. Benedict, S. R., J. BioZ. Chem., 64, 207 (1925). 14. Folin, O., and Svedberg, A., J. BioZ. Chem., 70, 417 (1926). 15. Greenwald, I., Gross, J., and &met, J., J. Biol. Chem., 62,401 (1924-25). 16. Host, H. F., J. Metabol. Research, 4, 315 (1923). 17. Somogyi, M., J. BioZ. Chem., 70, 599 (1926). 18. Hunter, G., Biochem. J., 22, 4 (1928). Hunter, G., and Eagles, B. A.,

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Vernon L. PetersonEdward S. West, Frederick H. Scharles and

SUGAR IN BLOODTHE DETERMINATION OF TRUE

1929, 82:137-153.J. Biol. Chem.

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