THE DISTRIBUTION OF SUGAR IN NORMAL HUMAN BLOOD.

BY MICHAEL SOMOGYI.

(From the Laboratory of the Jewish Hospital of St. Louis, St. Louis.)

(Received for publication, April 23, 1928.)

In a previous paper (1) we reported that the non-fermentable substances of blood, which reduce alkaline copper solutions, are not evenly distributed between the formed elements and the plasma, the former containing about 5 times as much of “reducing non-sugars” as t.he latter. From this it follows that the ratio, corpuscle sugar plasma sugar ’

derived from apparent sugar (t.otal reduction)

values, must be substantially different from the one based upon “true sugar” values. Since all investigations concerning the matter heretofore dealt only with apparent sugar values, we made it our t,ask to obtain a more accurate image of the distribu- tion of blood sugar by the determination of true sugar in the corpuscles and in the plasma.

Determination of True Suga,r.

The true sugar was obtained by the indirect method as in our earlier work; vix., as the difference between apparent sugar and reducing non-sugars. Having recognized that the precipitation of proteins in t.he presence of yeast in no way enhances the removal of the sugar-as stated also in a recent paper by Benedict @j---we reverted to the older practice, employed by Rona and Takahashi (3), and recently by Folin and Svedberg (4), of fermenting the deproteinized filtrates. Thus we determine the apparent sugar and the reducing non-sugars in portions of one and the same filtrate. This technique yields values from 3 to 4 mg. per cent lower than our original procedure, a discrepancy which we are unable to explain definitely. At first thought we attribut.ed the decrease to dilution by the water content of the yeast, but ex-

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118 Sugar in Normal Human Blood

periments showed this not to be the case. If one and the same batch of yeast is used for consecutive determinations of the reduc- ing non-sugars in two portions of a blood filtrate, the second de- termination, where dilution does not enter, yields no higher values than the first. Even more convincing than this were experiments with arabinose; the reduction exerted by solutions of this non- fermentable sugar was not lowered by treatment with yeast, show- ing that the dilution is entirely negligible. Apparently very little water is retained after centrifugation between the yeast cells, and the intracellular moisture obviously does not transude into the outer solution. From t,he fact that in a few pathologic specimens, after fermenting the whole blood, we found reduction values far in excess of the apparent sugar, we infer that some enzymes of the blood may produce non-fermentable reducing substances in con- tact even with washed yeast; this effect may prevail also in some normal specimens, only to such a small extent that it is hardly discernible in individual samples. Although the slight difference affects our results in no appreciable measure, we are inclined to accept as more accurate the lower values obtained from depro- teinized filtrates.

Procedure.

The procedure is as follows: Into a Pyrex test-tube measure 6 to 7 cc. of a 20 per cent suspension of washed yeast; centrifuge and discard the supernatant water, invert the tube t,o drain for some seconds, and by means of a strip of filter paper remove the moisture adhering to the walls of the test-tube. Introduce 12 to 14 cc. of the deproteinized filtrate to be fermented, stir up the yeast with a glass rod, and allow to stand at room temperature for about 10 minutes with occasional inversions of the test-tube to prevent settling of the yeast. (Should the room temperature be unusually low, place the test-tube in a beaker containing water of 25-30”.) Transfer the contents to another Pyrex test-tube without moistening it,s walls above the final level, and centrifuge. Decant the supernatant fluid at once. This procedure without filtration yields over 10 cc. of perfectly clear sugar-free solution, sufficient for two determinations of its reduction value.

Such quantities of sugar as are ordinarily encountered in blood

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111. Somogyi

filtrates are completely fermented at room temperatures within 5 minutes, but prolongation even for several hours of the contact between yeast and filtrate has no ill effect upon the results. On protracted incubation at body temperature, however, even the most carefully washed yeast is prone to yield non-fermentable reducing substances. Their amount is usually sma,ll, yet measur- able at the end of 30 to 60 minutes.

The careful washing of the yeast must again be stressed as an essential phase of this technique. The impurities contained in commercial yeast represent a variable, complex source of error, and are probably responsible for the major part of the obscurity and confusion that has surrounded the problem of “residual reduction.” A recent report by Bigwood and Wuillot (5) seems to bear out this contention. They determined the non-ferment- able reducing substances in plasma after fermenting their speci- mens with unwashed commercial yeast for 30 minutes at body temperature, using blanks-carried through identical conditions- to correct their results. They report values between a trace and 26.1 mg. per cent in terms of glucose. These figures are undoubt- edly wrong, and the main source of error is probably the action of plasma enzymes upon the soluble impurities of the yeast.

Regarding the application of lcaolin, Rona and Takahashi (3) reported in 1911 that deproteinized blood filtrates, treated re- peatedly with kaolin subsequent to fermentation, were opti- cally neutral and contained no reducing substances; the non- fermentable reducing substances were obviously adsorbed by the kaolin in this procedure. Benedict (2) recently investigated the effect of kaolin, and found it to adsorb about 2 mg. per cent of the non-fermentable reducing substances. Our values are from 5 to 10 mg. per cent, the difference between Benedict’s results and ours probably being due to the fact that we are using copper re- agents of maximum reduction value. Paradoxically, the Folin- Svedberg technique, in which kaolin is employed, yields in many instances appreciably higher reducing non-sugar values than our procedure. A plausible explanation of this is that a fraction of the reducing impurities introduced with the yeast is left behind, which exceeds the fraction of reducing non-sugars adsorbed by the kaolin.

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120 Sugar in Normal Human Blood

Sugar Content of Corpuscles.

In 1885 Otto (6), the first investigator concerned with the dist#ribution of sugar in blood, asserted that the corpuscles are entirely devoid of sugar, the whole of the blood sugar being confmed to the plasma. Corroborated by Abderhalden (7), this view was generally accepted until in 1909 Rona and Michaelis (8), and since then many other workers, demonstrated that the corpuscles of human blood always contain more or less ferment- able sugar. But soon afterwards Bang and his coworkers (9, lo), and later, in 1919 Falt,a and Richter-Quittner (ll), and quite recently Glassmann (12) offered evidence of the contrary effect, upholding Otto’s conception. None of these authors attempted to explain this remarkable conflict of experimental results, but the conclusions of Falta and Richter-Quittner were a few years later summarily dismissed as erroneous in a report from Falta’s own laboratory (13).

An inquiry into the cause of this controversy directed our attention to the fact that the authors asserting that the corpuscles are free of sugar, washed the corpuscles with several changes of saline in order to remove the adhering plasma preceding the determination of their sugar content. Examining the effect of such washings we ascert,ained that erythrocytes, mixed with 10 volumes of saline but for a minute, loose practically their entire sugar content to the fluid medium, and the reducing substances still present in them after centrifugation are-except for 3 to 5 mg. per cent-non-sugars. (Ege and Hansen (14) assumed on the ground of similar experiments that this latter reduction value represents a rather firmly “combined” fraction of the corpuscle sugar, while another fraction which is readily detached from the cells by rinsing, is loosely adsorbed upon t.he surface of the cells.) From this it is evident that the washing of the corpuscles is the error responsible for the false conclusion that the corpuscles are free of sugar.

The small amount of intercellular plasma, left with the cor- puscles after centrifugation, has no measurable effect upon the result in the determination of corpuscle sugar. But there is a serious source of error, especially in the case of specimens con- taining no anticoagulants. In defibrinated blood the sum of

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M. Somogyi 121

plasma sugar and corpuscle sugar is 5 to 10 mg. per cent below the sugar content of the whole blood as determined immediately before it is centrifuged. This discrepancy is due to glycolysis which-under the given circumstances-occurs in the main at the expense of the corpuscle sugar. Consequently, we accepted in the course of the present work the indirect determination of the corpuscle sugar as correct. The sugar is determined in the whole blood and in the plasma or the serum from which, with the knowledge of the corpuscle volume, the corpuscle sugar is cal- culated according to the practice followed by most investigators. As a control, the direct determination of the corpuscle sugar was also carried out.

E$ect of Anticoagulants.

The experiments reported in this paper were all performed on defibrinated blood samples. The comparison of our results with those of Folin and Berglund (15), and of other authors who had taken every possible precaution to preclude alterations in the distribution of the blood sugar (collection of the blood in paraffined tubes without anticoagulants, etc.), proves to our satisfaction that defibrination does not alter the distribution. Nor have moderate amounts of anticoagulants any effect upon it, as we found identical distribution ratios in several portions of the same specimen, whether the coagulation was prevented by defibrination or by the addition of 0.1 to 0.15 per cent of potassium oxalate or sodium citrate. However larger quantities of these anticoagulants, which render the plasma appreciably hypertonic and cause the corpuscles to shrink, exert a marked influence upon the distribution of the sugar.

Sodium fluoride, which inhibits the action of yeast,, must not be used in experiments involving the determination of true sugar values.

Determination of Distribution of Sugar.

Taking into account the foregoing considerations our ultimate course in the determination of the distribution of sugar is as fol- lows: 20 to 25 cc. of blood are drawn from an arm vein of the subject. Promptly after defibrination two portions of 2 cc. each are measured out and deproteinixed according to the Folin-Wu

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122 Sugar in Normal Human Blood

method, while the remainder is being centrifuged at 3000 R.P.M. for 10 minutes. After syphoning off the serum, 5 cc. of the cor- puscles are laked in 24 cc. (4.8 volumes) of water, and precipitated by the addition of 10 cc. (2 volumes) of 0.75 N sulfuric acid and 11 cc. (2.2 volumes) of 10 per cent sodium tungstate. For the determination of apparent sugar in plasma, two 1 cc. portions are deproteinized at 1: 10 dilution, while for fermentation a 1:4 filtrate is prepared from 4 to 5 cc. of plasma, as the amount of the reducing non-sugars a.t 1:lO dilution is too low to be determined by the method employed throughout these experiments (16).

The apparent sugar values for corpuscles, as given in Tables I and II, are calculated from blood sugar and serum sugar. The reducing non-sugars of the corpuscles, too, represent calcu- lated values, which are 3 to 5 mg. per cent higher than obtained by direct determination. The reason for this discrepancy is twofold; first, unlike the case of the apparent sugar, here the diluting effect of adhering plasma is appreciable (5 per cent of admixed plasma causes a decrease of almost 2 mg. per cent), and second, we find that the reducing non-sugar values slowly decline if blood or corpuscles are allowed to stand for some length of time, the loss mounting occasionally to 30 to 40 per cent after 4 t,o 5 hours incubation. Consequently, for corpuscles we accepted the calculated values of the reducing non-sugars as more accurate, although the difference between these and the directly determined values is too small to affect the results materially.

Table I presents the distribution of the sugar in blood specimens obtained from thirty-six healthy adults, thirty-two men and four women. As can be seen, the apparent sugar values conform with the generally accepted view of a nearly equal distribution of the blood sugar between the corpuscles and the plasma, the

ratio, ““$lau~~s~a~ being on the average 110 : 100 (Frank (17),

Bailey (18), Schmid (19), Folin and Berglund (15), John (20), Wiechmann (21), Ege and Hansen (14), and several others). But the actual ratio, based upon true sugar values, is quite different, to wit : the average is 77: 100, with considerable deviations on either side; 70: 100 may be considered the lowest ratio, a few high values approaching unity. High ratios are relatively in- frequent as only in one-fourth of the cases were they above 80 : 100.

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TABLE I.

Distribution of Sugar and of Reducing Non-Sugars in Human Blood.

Speri- men No

Mg. in 100 cc. of:

Serum. COP ,uscles. Serum. COP

pus&s. Serum. FW

bpparent sugar.

Far true sugar.

1 117 102 42 8 75 94 1.15 0.80 2 122 107 45 7 77 100 1.14 0.77 3 107 80 44 8 63 72 1.34 0.87 4 114 110 36 7 78 103 1.04 0.76 5 130 110 42 7 88 103 1.18 0.86 6 111 109 39 7 72 102 1.02 0.71

7 107 93 40 8 67 85 1.15 0.79 8 81 73 38 8 43 65 1.11 0.66

9 102 94 42 8 60 86 1.08 0.70 10 114 104 39 9 75 95 1.10 0.79 11 103 103 36 7 67 96 1.00 0.70 12 101 82 39 8 62 74 1.23 0.84 13 119 110 39 8 80 102 1.08 0.78 14 112 117 38 8 74 109 0.96 0.68 15 103 99 39 9 64 90 1.04 0.71

16 143 143 45 8 98 135 1.00 0.73 17 111 82 40 7 71 75 1.35 0.95 18 100 100 38 9 61 91 1.00 0.68 19 151 149 45 7 106 142 1.01 0.75 20 125 121 40 8 85 113 1.03 0.75 21 114 107 41 7 73 100 1.07 0.73 22 107 98 43 7 64 91 1.09 0.70 23 148 146 43 7 105 139 1.01 0.76 24 103 89 43 7 60 82 1.16 0.73 25 108 101 38 8 70 93 1.07 0.75 26 110 86 37 7 73 79 1.28 0.92 27 118 111 34 7 84 104 1.06 0.80 28 145 137 38 8 107 129 1.06 0.83 29 112 99 36 9 76 90 1.13 0.84 30 105 100 37 6 68 94 1.05 0.73 31 110 100 39 8 71 92 1.10 0.77 32 118 89 36 8 75 81 1.25 0.93 33 110 103 38 10 72 93 1.07 0.77 31 98 94 35 8 63 86 1.04 0.73 35 117 102 42 8 75 94 1.15 0.80 36 118 106 42 9 76 97 1.11 0.78

-i- Apparent sugar. Reducing non-sugars.

Lowest ............ Highest ........... Average. ..........

34 45 40

6 10

8

123

. _

-

Corpuscle sugar. Serum sugar

0.96 1.35 1.10

0.66 0.95 0.77

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124 Sugar in Normal Human Blood

The distribution seems to be unaffected by the nutritional state of t.he subjects, as blood specimens with high sugar content, drawn after meals, exhibit variations within the same range as those obtained before breakfast. As to the constancy or variabil- ity of the distribution in one and Dhe same subject, we do not pos- sess sufficient data to justify a conclusion; in a few persons we found considerable variations.

The ratio, corpuscle sugar plasma sugar ’

then, is an illusory figure if based

upon apparent sugar values. According to Table I its average value is over 40 per cent higher than the average actual ratio. The discrepancy between these two values is, however, variable and is determined by two factors. A simple equation will be helpful in the analysis of the role of these factors:

c+5n EC-.----- P+n

where R is t,he distribution ratio calculated from apparent sugar values, c is the true sugar in corpuscles, p the true sugar in plasma, n represents the reducing non-sugars in plasma, and 5 n, with a good approximation, the reducing non-sugars in corpuscles.

If in this equation n gradually diminishes, to the same extent

the value of R approaches 5, that is t,he actual ratio of distribution;

if n finally becomes zero, as with Benedict’s latest reagent (2), or is separately determined by our method and ruled out by

subtraction, t.hen R = 5, the actual ratio of distribution. The

calorimetric methods of the Folin-Wu type yield lower values for n than the methods involving iodometric titrat)ions, hence they furnish somewhat lower R values.

The other factor that is apt to distort the true ratio of distri- bution, and to cause variations in its value to a still great,er extent, is the alterat.ion of the blood sugar level. For a given analytical method n may be considered as nearly constant. Now, if the blood sugar level, and consequently both c and p rise, the discrep-

c + 5n ancy between ~

p+n and % gradually diminishes, and finally

-at very high blood sugar levels-the difference becomes almost,

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M. Somogyi 125

negligible. If, on the other hand, the values of c and p decline, the lower they become, the more prominent will be the disguising effect of n. Finally in extreme instances, where the blood sugar would approach zero value-e.g. as the result of glycolysis or of insulin hypoglycemia-the reducing substances still present in

both corpuscles and plasma would lead to a ratio of R = 5;,

which would mean that the corpuscles contain 5 times as much sugar as the plasma.

A few examples in Table II illustrate the significance of this point, although they are far from representing extreme cases.

TABLE II.

Description of specimen.

1 Diabetic blood .......................... 2 Normal blood ........................... 3 Blood from Fasting.. ................... 4 normal

! 30 min. after glucose ........

5 subject. 3 hrs. after glucose ..........

Apparent True algar. *“gSr.

Mg. in 100 cc. of:

---- 304 358 266 3% 84 64 45 5:

112 99 76 9( 184 184 148 17! 77 59 41 5(

--T--- o- 2b-t _,1 Pa gr” 88 u

L3 El z g; 0. F$g 5% *a cg r.3 Qrn & -- 1.85 0.76 1.310.77 1.130.84 1.00 0.85 1.310.82

If the distribution of the sugar in Specimens 1 and 2 is compared on the basis of the apparent sugar values, the relative sugar content

of the corpuscles in Specimen 2 appears to be (1.31 - 0.85) 100 =

0.85 54 per cent higher than that in Specimen 1, while the true sugar values plainly demonstrate that their ratios of distribution are practically identical. Without the knowledge of the true sugar values one is prone to arrive at the conclusion that the capacity to combine with sugar is impaired in the corpuscles of diabetic blood (Rona and Sperling (ZZ), Loewi and collaborators (23), and others).

Specimens 3, 4, and 5 show the changes of the sugar in the

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126 Sugar in Normal Human Blood

blood of a healthy man after the ingestion of 120 gm. of glucose. The apparent sugar values would indicate a decline of 13 per cent in the relative sugar content of the corpuscles as the sugar level goes up, and again an increase of 31 per cent as the sugar reaches a low level. Such figures are conducive to the assumption that in alimentary hyperglycemia, too, the distribution of the blood sugar is changed, inasmuch as the relative sugar content of the plasma becomes greater than at normal sugar levels (Hober (24), Wiechmann (21), and several others). The true sugar values, however, evince the fact that the actual ratio of distribution is practically the same in all of the specimens.

These findings suggest the desirability of a revision of some conclusions and theories derived from changes observed in the distribution of the blood sugar.

SUMMARY.

The distribution of the sugar between the corpuscles and the plasma in normal human blood was determined upon the basis of true sugar values. It is demonstrated that. the ratio, corpuscle sugar

plasma sugar , as obtained from true sugar values, differs sub-

stantially from the ratio derived from apparent sugar values. This is an instance in which the substitution of apparent sugar for true sugar entails serious errors and misconceptions.

A modification is presented of the author’s technique for the determination of reducing non-sugars by means of washed yeast.

BIBLIOGRAPHY.

1. Somogyi, M., J. Biol. Chem., 1927, lxxv, 33. 2. Benedict, S. R., J. Biol. Chem., 1928, Ixxvi, 457. 3. Rona, P., andTakahashi, D., Biochem. Z., 1910, xxx, 99. 4. Folin, O., and Svedberg, A., J. Biol. Chem., 1926, lxx, 405. 5. Bigwood, E. J., and Wuillot, A., Compt. rend. Sot. biol., 1927, xcvi, 414. 6. Otto, J. J., Arch. ges. Physiol., 1885, xxxv, 467. 7. Abderhalden, E., Z. physiol. Chem., 1898, xxv, 67. 8. Rona, P., andMichael@ L., Biochem. Z., 1909, xvi, 60. 9. Lyttkens, H., andsandgren, J., Biochem. Z., 1910, xxvi, 382.

10. Bang, I., Biochem. Z., 1912, xxxviii, 166. 11. Falta, W., and Richter-Quittner, WI., Biochem. Z., 1919, C, 148. 12. Glassmann, B., 2. physiol. Chem., 1926, clviii, 113.

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M. Somogyi 127

13. Hiigler, F., andueberrack, K., Biochem. Z., 1924, cxlviii, 150. 14. Ege, R., and Hansen, K. M., Acta med. Scan& 1927, Ixv, 279. 15. Folin, O., and Berglund, H., J. Biol. Chem., 1922, Ii, 213. 16. Somogyi, M., J. Biol. Chem., 1926, lxx, 599. 17. Frank, E., 2. physiol. Chem., 1910, lxx, 129. 18. Bailey, C. V., Arch. Int. Med.;1919, xxiii, 455. 19, Schmid, F., Compt. rend. Sot. biol., 1922, lxxxvii, 1367. 20. John, H. J., Arch. Int. Med., 1923, xxxi, 555. 21. Wiechmann, E., 2. ges. Med., 1924, xii, 462. ezp. 22. Rona, P., and Sperling, M., Biochem. Z., 1926, clxxv, 253. 23. Loewi, O., Klin. Woch., 1927, vi, 2169. 24. HBber, R., Biochem. Z., 1912, XIV, 207.

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Michael SomogyiNORMAL HUMAN BLOOD

THE DISTRIBUTION OF SUGAR IN

1928, 78:117-127.J. Biol. Chem. 

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