Fluorometric analysts of glycolytic intermediates in human red blood cells

BIOCHEMICAL MEDICINE 8, 123-134 ( 1973)

Fluorometric Analysts of Glycolytic Intermediates

in Human Red Blood Cells1

H. NIESSNER AND E. BEUTLER

CitV of Hope Medical Center, Dwrte, California 91010

Received October 11, 1972

Within the past 10 yrs, defects affecting most of the enzymes of gly- colysis have been discovered in patients with nonspherocytic congenital hemolytic anemia. In many instances, a clear-cut difference in the activity of defective enzyme has been demonstrated under in vitro assay conditions. It has also become apparent, particularly in the case of pyruvate kinase, that functionally important lesions affecting glycolytic enzymes may not be uncovered by the usual in vitro assays. The level of metabolic intermediates in the erythrocyte may reflect a functional de- rangement that is not apparent once the assays are carried out under in vitro conditions. Thus, the block in the metabolic stream will result in accumulation of intermediates that are formed prior to the defective step, while intermediates distal to the metabolic lesion may be decreased in concentration. The analysis of such data, using “cross-over” plots has been described by Minakami ( 1) and by Williamson (2).

The levels of gIycolytic intermediates are very low, however, and, as we .shall show, they quickly change when the blood sample has been removed from the body. Minakami (3) measured the levels of many red cell intermediates using spectrophotometric techniques. However, the low levels found strain at sensitivity of spectrophotometry so that the levels of several intermediates are close to the noise level of even values of sophisticated recording ultraviolet spectrophotometers. Fluorometric techniques seem to be more suitable for such determinations.

In their pioneering work on the glycolytic pathway, Lowry and co- workers measured all glycolytic intermediates in brain by fluorometric methods (4). Subsequently, several groups ( 5-8) adapted these methods for the investigation of red cell metabolites. However, detaiIed proce- dures have been reported only by Segel et al. (B), and normal values in fresh red cells only by Oski (6).

1 Suppxted in part by NIH Grant HE 07449, and by grants from the Max

Kade Foundation.

123 Copyright @ 1973 by Academic Press, Inc.

All rights of reproduction in any form reserved.

124 NIESSNER AND BEUTLER

We have now made some substantial modifications for the determination of red cell intermediates, giving special attention to the technique used for drawing blood and producing filtrates; these factors were found to be very important, particularly with respect to FDP levels. Further- more, to determine the feasibility of shipping samples, extracts were stored after deproteinization for 48 hr at room temperature, at O’C, and at -20°C.

MATERIALS AND METHODS

All enzymes, coenzymes, and substrates were obtained from Sigma Chemical Co., St. Louis, MO. Fluorometric measurements were made in a Turner Fluorometer, Model 111 (G. K. Turner Associates, Palo Alto, CA). The instrument was fitted with temperature control door, through which 37°C water was circulated. Regulation of temperature was es- sential because of the change in fluorescence of pyridine nucleotides with changing temperature. The “1X” sensitivity setting was sufficient for all readings. A Corning 7-60 primary and a Corning 3-72 secondary filter were used. The course of the reaction was recorded on a Varian aero- graph, Model 20 strip-chart recorder.

Four methods of preparing blood are compared. In method 1. no tourniquet was used, and 2 ml of blood were drawn from the antecubital vein into a plastic syringe containing 8 ml of ice cold 4% PCA. The blood and PCA were mixed thoroughly and left on ice for at least 10 min. The exact amount of blood in the syringe was determined by weighing the syringe before and after drawing the blood and calculating the volume based on a specific gravity of 1.06. In method 2, a tourniquet was applied; otherwise the technique was the same as in method 1. In method 5, a tourniquet was also used, and the blood was mixed with heparin (10 U/ml whole blood) in a glass tube and 2 ml were added immediately to 8 ml of ice cold 4% PCA. After mixing thoroughly, the mixture was allowed to stand in ice for 10 min. In method 4, blood was taken without a

a Abbreviations: ADP, adenosine 5’-diphosphate; ATP, adenosine 5’-triphosphate; DHAP, dihydroxyacetone phosphate; FDP, fructose I,&diphosphate; F6P, fructose-

g-phosphate; GAP, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde-3-phosphate

dehydrogenase; G6P, glucose-6-phosphate; GGPD, glucose-6-phosphate dehydrogenase; GPI, glucose6phosphate isomerase; HK, hexokinase; LDH, lactic dehydrogen-

ase; NAD, nicotinamide-adenine dinucleotide; NADH, nicotinamide-adenine dinucleo-

tide, reduced form; NADP, nicotinamide-adenine dinucleotide phosphate; NADPH, nicotinamide-adenine dinucleotide phosphate, reduced form; PCA, perchloric acid; PEP, phosphoenol pyruvate; PFK, phosphofructokinase; 2-PGA, 2 phosphoglyceric acid; 3-PGA, 3-phosphoglyceric acid; PGK, 3-phosphoglyceric phosphokinase; PK,

pyruvate kinase; TPI, triosephosphate isomerase; Tris, tris (hydroxymethyl ) amino-

methane.

INTERMEDIATES IN RED CELLS 125

tourniquet, mixed with heparin and stored for 10 min at 0°C before deproteinization.

In studying stability of the extract, blood was drawn by method 1, but after removal from the precipitate, the PCA extract was stored for 2 days, unneutralized or neutralized, at different temperatures. Only unneutralized extracts were stored at room temperature. For the investigation of storage at 0°C 4 ml of blood were drawn in a syringe containing 16 ml PCA. After deproteinization, one part was neutralized before storage, another part was stored unneutralized. In studying storage at -20°C 6 ml of blood were drawn into a plastic syringe containing 24 ml of PCA. After deproteinization one part of the extract was neutralized for the immediate measurement of the intermediates, a second part also was neutralized but frozen stored, and finaIIy a third part was unneutralized frozen stored.

Blood samples from anemic patients with reticulocytosis were obtained using method 1.

In all groups, the samples that had been mixed with PCA were centrifuged for 10 min at 12,000g at 4°C and 7 ml of this supernatant solution were neutralized with 1 M &CO, using methyl orange as indicator. The volume was adjusted to 10 ml, and the samples were centrifuged for 15 min at 27,000g. In each case, a microhematocrit determination of an aliquot of whole blood was also carried out.

Fluorometric assays were carried out in 75 X lo-mm glass tubes. After each addition, the contents of the tube were mixed with a “Vortex” mixer, Parafilm was not used since it fluoresces,

A. Assays for G6P and F6P

Tris-HCl buffer, pH 8.0, 1.0 M KADP, IO mM

/3-mercaptoethanol 7 M Extract

Hz0

A baseline was obtained

GBPD (150 U/ml)

Fluorescence was recorded until a

stable reading was obtained. GPI (1,500 U/ml)

Fluorescence was recorded until a stable reading was obtained.

CBP, 50 pM


G6P, 50 PM standard


0.500 ml

0.400 ml 0.005 ml

1.200 ml 1.900 ml

0.005 ml

0.005 ml

0.010 ml

0.020 ml

126 NIESSNER AND BEUTLEH

The change in fluorescence after addition of GGPD represents the amount of GFP

in the sample, while the change after the addition of GPI represents the amount of

F6P in the sample.

B. Estimation of GAP, DHAP and FDP

Tris-HCl buffer, pH 8.0, 2 M NAD, 10 mM

Na-arsenate, pH 8.0, 300 mM p-mercaptoethanol, 14 M Extract

Fluorescence was recorded until

the base line was stable (approxi-

mateyy 10 mm).

GAPDH (550 U/ml)


constant, slow rate way obtained.

TPI (24,000 U/ml)

Fluorescence was recorded until a constant, slow rate was obtained.

Aldolase (99 U/ml)


constant, slow rate was obtained.

FDP standard, 50 pM Fluorescence was recorded until a


FDP standard, 50 hM Fluorescence was recorded until a


0.20 ml

0.80 ml

0.80 ml

0.005 ml 2.2 ml

0.015 ml

0.005 ml

0.010 ml

0.010 ml

0.020 ml

The decrea-e in fluorescence after addition of GAPDH represents the concentra-

tion of GAP in the sample. The change in fluorescence after the addition of TPI

represents a concentration of DHAP in the sample. The increase in fluorescence after addition of aldolase represents twice the concentration of FDP in the sample.

C. Estimation of 3-PGA.

K-phosphate buffer, pH 7.4, 100 mM ATP, 10 mM MgCL, 100 mM

,&mercaptoethanol, 14 M NADH, 0.2 mM Extract

Hz0 Fluorescence was recorded until the

base line was stable (approximately 10 min ) .

GAPDH ( 550 U/ml)

Fluorescence was recorded until a constant, slow rate was obtained,

PGK (3200 U/ml)


0.50 ml 0.40 ml

0.04 ml 0.005 ml

0.08 ml 0.50 ml 2.48 ml

0.005 ml

0.005 ml


The addition of GAPDH did not result in any change in fluorescence. The decrease

in fluorescence after addition of PGK represents the concentration of 3-PGA.

D. 2-PGA and PEP.

K-phosphate buffer, pH 7.4, 0.5 M 0.50 ml ADP, 10 mM 0.40 ml MgcfI, 0.1 M 0.04 ml

NADH. 0.2 mM 0.16 ml

Hz0 1.40 ml

Extract 1.50 ml

LDH, 150 U/ml 0.005 ml

After the LDH reaction was complete (approximately 5 min) additional O.Ol-ml aliquots of NADH were added until addition of NADH caused an increase of fluores-

cence, which was no longer followed by any substantial decrease of fluoroescence. In

this way, a’1 excess pyruvate was reduced.

Fluorescence was recorded until the baseline was stable (approximately

15 min).

PK ( 3700 U/ml)


constant, slow rate was obtained. Enolase (410 U/ml )



2-PGA standard, 50 pM


constant, slow rate was obtained. 2-PG.4 standard, 50 pM


0.005 ml

0.010 ml

0.010 ml

0.020 ml

The change in fluorescence after addition of PK represents the amount of PEP,

while the change after the addition of enolase represents the amount of 2-PGA in

the sample.

E. Estimation of pyruvate

K-Phosphate buffer, pH 7.0, 0.1 M NADH, 0.2 mM Extract

Hz0

Fluorescence was recorded until the

base line wa8 stable (approximately 10 min). LDH (300 U/ml)


constant, slow rate was obtained. Pyruvate standard, 50 pM



0.500 ml

0.080 ml 0.200 ml

3.22 ml

0.005 ml

0.010 ml

I28 NIESSNER AND BEUTLER

Pyruvate standard, 50 pM Fluorescence was recorded until a constant, slow rate was obtained.

0.020 ml

The change in fluorescence after addition of LDH represents the anlount of

pyruvate. Lactate was estimated spectrophotometrically with LDH.

All standards were kept frozen as 10 mM stock solution. The concentration of each standard was measured spectrophotometrically using the same type of procedure described above for fluorometric analysis, and using a mM extinction coefficient of 6.22 for NADH and for NADPH.

A blank extract was prepared by adding 1.6 ml water to 8.0 ml 4X PCA. If any change in fluorescence occurred because of the addition of enzymes, the data were corrected for the changes. However, the changes were negligible in most of the experiments, The differences between the concentrations of the glycolytic intermediates in methods 2, 3 and 4, and also in the extracts stored at room temperature and 0°C compared to method 1, were tested for statistical significance by Student’s t test. The levels of the intermediates in the frozen stored extracts were compared to the values measured on the freshly prepared extract.

RESULTS

The means and the standard deviations of the levels of the glycolytic intermediates and the number of subjects investigated by each method are shown in Tables 1 and 2. Lactate and pyruvate concentrations are expressed in nmole/ml whole blood; the levels of the other intermediates are given in nmole/ml red cells.

The effects of different techniques used for drawing blood and producing filtrates are shown in Table 1. Using a tourniquet for taking blood caused an increase of lactate levels whereas pyruvate slightly decreased. There was also apparently a slight increase in the amount of 3-PGA present. Using a tourniquet and mixing with heparin before deproteinization caused a slight increase of lactate and of FDP. After storage of heparinized blood for 10 min at 0°C the most significant change was the increase of FDP. An increase of 3-PGA and DHAP also occurred, whereas G6P, F6P and pyruvate decreased.

The mean values of glycolytic intermediates in red cells of three anemic patients with reticulocytosis are listed in the right column of the table. One subject suffered from severe, recently treated iron deficiency; the other two are patients with sickle cell anemia. The reticulocyte count ranged from 4.S58-6.058. The levels of all glycolytic intermediates were increased, most of them significantly.

Changes of the concentrations of the glycolytic intermediates during


TABL

E 2

CHAN

CES

IN

LEVE

LS

OF

GLY

COLY

TIC

INTE

RMED

IATE

S IN

RE

D CE

LLSQ

Unn

eutra

lized

U

nneu

tralia

ed

Neu

traliz

ed

Unn

eutra

lized

N

eutra

lized

ex

t.rac

t st

ored

ex

tract

st

ored

ex

tract

st

ored

Bl

ood

extra

ct

froze

n ex

tract

. fro

zen

for

48 h

rs a

t. fo

r 48

hrs

fo

r 48

hrs

im

med

iate

ly

stor

ed

for

stor

ed

fot

room

te

mpe

ratu

re

at

0°C

at

0°C

de

prot

eini

zed

48 h

rs

48 h

rs

(N

= 4)

(N

=

4)

(N

= 4)

(N

=

3)

(IV =

3)

(IV

=

3)

G6P

73

.8

f 20

.5d

32.2

zk

6.9

32

.0

+ 7.

6 34

.9

* 0.

8 32

.7

+ 2.

9 32

.R+

1 R

F1

F6

P 11

.7

+_ 3

.1

10.4

12

.5

8.9

_+ 2

.3

9.8

zk 0

.8

9.1

+ 1.

7 9.

9 *

1.1

2 FD

P 1.

7 rf-

0.3

2.

2 *

0.5

1.7

* 0.

4 1.

5 4

0.4

1.7

rt 0.

2 1.

3 +

0.4

E D

HAP

5.

8 f

l.O*

11.6

+

2.5

11.5

+

3.2

11.4

rt

2.2

11.0

i

1.3

10.1

*

:I..?

3-

PGA

44.6

f

10.3

48

.5

k 7.

6 44

.6

+ 4.

6 43

.5

+ 2.

2 46

.9

+ 3.

5 :3

9 9

f 3.

6 2-

PGA

6.6

+ 1.

2 6.

1 ;f:

1.3

5.

7 &

1.7

9.1

+ 3.

7 6.

0 +

1.9

6.3

+ 0.

9 $

PEP

7.2

i l.Z

d 11

.9

& 1.

7 14

.9

F 1.

9 13

.1

It 1.

4 11

.3

+ 2.

1 12

.2

+ 0.

X K

Pyru

vate

55

.7

+ 24

.2

63.0

+

34

34.1

*

43.9

84

.2

+ 23

.4

87.4

&

32.4

36

.1

& 49

$

Lact

ate

1038

.0

* 50

0.0

1015

*

395

992.

8 +_

377

.8

1361

k

501

1180

*

33s

1170

ir

%L’

L

Lact

atej

pyru

vate

18

.7

f 3.

3d

17.2

+

4.6d

16

.2

f t5

.S

14.1

&-

2.2

xl

-

-

0 JZ

xpre

ssed

in

nmol

es!m

l re

d ce

lls (

nmol

es/m

l wh

ole

bloo

d fo

r la

ctat

e an

d py

ruva

te)

durin

g st

orag

e of

lm

neut

raliz

ed

or

neut

raliz

ed

est r

acts

at

diffe

rent

t.e

mpe

ratu

res.

Fo

r st

.atis

tical

si

gnifi

canc

e th

e di

ffere

nce

betw

een

the

conc

entra

tions

in

ext

ract

s,

siore

d al

ro

om

tem

- pe

ratu

re

and

0°C

wer

e co

mpa

red

to m

etho

d 1

(Tab

le

I, fir

st.

colu

mn)

. Th

e le

vels

of

the

inte

rmed

iate

s in

th

e fro

zen

stor

ed

extra

cts

were

c.

ompa

red

t,o th

e va

lues

m

easu

red

on t

he f

resh

ly

prep

ared

ex

t,rac

ts

(Tab

le

2, f

ourth

co

lum

n).

6 0.0

5 >

> 0.

01.

p c

0.01

> p

> 0.

001.

d p

> 0.

001.


storage of PCA extracts for 48 hr at different temperatures are shown in Table 2. During storage of,‘the unneutralized PCA extract for 48 hr at room temperature, a striking increase of G6P was found. F6P and lactate also showed an increase, while DHAP and PEP decreased significantly. l>ue to a decrease of pyruvate and increase of lactate, the lactate/pyr- rrvate ratio was significantIy raised.

Storage of unneutralized extracts at 0°C caused only a slight increase of the lactate/pyruvate ratio. During storage of neutralized extracts at 0°C the most remarkable change was the loss of pyruvate; in two extracts uo pyruvate at all was measurable. Similar changes were found during storage of frozen neutralized extracts. Unneutralized extracts, stored at -20°C maintained all of the intermediates at base-line levels.

DISCUSSION

Fluorometric analysis of red cell metabolic intermediates was found to be tedious, but to yield reproducible results. In carrying out these studies, we found a filter fluorometer to be preferable to the use of a spectrofluorometer. The principal reason for this is probably the fact that the amount of light passing a filter, although less pure than that passing a diffraction grating or prism, is considerably more intense. Thus, less electronic amplification is required. The double-beam design of the fluorometer used compensates for changes in intensity of the light source, variation in line voltage, or photomultiplier drift. Therefore, sensitivity remained constant over long periods of time.

All reactions were usually complete within 10 min. This included the assays of FDP and DHAP; the GAPDH system, containing arsenate (19), seems in this respect to be superior to the ,a-glycerophosphate dehydrogenase system employed by others (4, 6, 8). Moreover, systems containing NAD have the advantage that uncertainties imposed by the non- enzymatic oxidation of NADH (3) cannot occur.

No reaction was usually observed after addition of GAPD in the assay for GAP. The changes never exceeded 0.04 times full scale on the recorder. ‘4 measurement with a higher sensitivity was not possible because of the level of background fluorescence. Therefore, no values are given for GAP.

Adding increments of NADH to the assay medium for PEP and 2-PGA until no further decrease in fluorescence occurs provides an adequate final NADH concentration in the system, independent of the pyruvate content in the sample. Due to the relatively high NADH concentration, we obtained rapid PK and enolase reactions even in filtrates containing high revels of pyruvate.


TABLE 3 LEVELS OF GLYCOLYTIC ~TERMI~DIATW IN RKD Csn~s~

G6P F6P FDP DHAP GAP 3PGA PPGA PEP Pyruvate Lactate

Minakami (3) (N = 5)

27 * 2.4 11 5 2.5 5 * 0.9

12 * 3.7 4 & 1.5

48 + 16.1 7 +_ 1.7

12 + 0.9 71 If: 17.7

1190 * 180

Oski (6) Oski (6) Normal adults Reticulocytosis

(N = 10) in’ = 11)

24.8 + 9.8 38.6 + 11.7 5.4 * 1.0 9.3 k 3.2 4.6 + 1.0 5.3 & 1.4 4.9 f 3.5 9.4 + 3.9 2.6 k 0.7 1.9 A 2.7

61.2 + 12.4 63.8 + 17.2 4.3 f 1.8 5.0 rt 2.4 8.8 + 2.6 11.3 i 1.9

73.5 * 33.1 77.3 & 40.3

n Expressed in nmoles/ml red cells (nmoles/ml whole blood for lactate and pyruvate) obtained on five normal adults spectrophotometrically by Minakami et a/. (3) and on 10 normal adults and 11 patients with reticulocytosis fluorometrically by Oski (6).

Our results are in good agreement with the concentrations of glycolytic intermediates in fresh red cells, reported by Minakami and co-workers (3) and Oski (6) (Table 3), except for the significant lower FDP values in our investigations. One hundred percent of FDP added to blood could be recovered in our system, indicating that poor recoveries were not responsible for the results obtained. The difference in the FDP values that we obtained can be explained by the different length of time after taking blood until deproteinization. Merely heparinizing blood for a few moments before deproteinization (method 3) caused a 30% increase of the FDP levels; storage of heparinized blood at 0°C for only 10 min before adding PCA raised the FDP concentration more than 300% (method 4). The importance of immediate deproteinization has already been pointed out by Minakami et ~2. (3). Storing heparinized blood for 1 hr at O’C, he obtained an FDP increase from 5-17 and 6-22 nmoIe/ml red cells. However, the blood for his normal values “was deproteinized at least 5 min after removal from the body.” In the investigations of Oski ( 6), the blood was also heparinized before it was transferred into a tube with PCA. The sensitivity of FDP values to manipulations during prep- aration of the extract is also demonstrated by the investigations of Gerlach et al. ( lo), Bartlett ( ll), and Feig and co-workers (7). Incu- bating some samples for 60 min, Gerlach measured FDP values of 430 nmole/ml red cells by paper chromatography. In Bartlett’s investigations, the FDP values in washed red cells, estimated chromatographically on anion-exchange columns, ranged from 60-120 nmole/ml red cells. 111


three-times washed red cells, Feig measured FDP values of 90 nmole/ml red cells.

The prompt increase of FDP in red cells after removal from the body can be explained by a striking pH shift, observed in heparinized blood at 0°C ( 12). This causes an increase of PFK activity ( U-15)) which results in elevated concentrations of FDP. In accordance with that explanation, the hexose-monophosphates showed a decreasing tendency from method I to methods 3 and 4 in our investigations. It should be emphasized that the temperature has a striking effect on the pH (12, 16). The fact that Il4inakami (3) found storage of heparinized blood at 37°C caused only a slight rise of FDP, whereas the FDP values after storage at 0°C were significantly increased, is probably explained by this pH effect.

Compared to the values obtained on adults with a mean reticulocyte count of 7.1 by Oski ( 6)) in our investigation, despite lower reticulocyte counts, the hexosemonophosphate levels were raised more. Furthermore, we found in contrast to Oski (6) a significant increase of FDP, DHAP, SPGA, and PEP. That the FDP and DHAP levels are less elevated than G6P and F6P is in good agreement with the reported slighter increase of PFK and aldolase activity, compared to the strikingly increased H-1 activity in samples with high reticulocyte counts (3, 17).

?r4inakami (3) has already pointed out the lability of metabolites in extracts at high or even at neutral pH. To determine the feasibility of shipping samples, extracts were stored for 48 hr after deproteinization. and neutralized or unneutralized at different temperatures. Even at low pH, storage at room temperature causes changes of metabolites, such as a striking increase of G6P and a loss of DHAP and PEP. The increase in levels of G6P and F6P is best explained by the hydrolysis of the acid labile l-phosphate group of glucose-1-6-diphosphate and fructose-l-6- diphosphate.

During storage at 0°C or at -20°C the most striking change was the pyruvate loss. In some extracts, no pyruvate at all was measurable. Furthermore, especially in comparison to the unneutralized stored extracts in the same groups, a slight loss of FDP and 2-PGA occurred. This finding might be an explanation for the significant lower 2-PGA values found by Oski (6) compared to the concentrations reported by Minakami (3) and to our values, because in Oski’s investigations neutralized extracts were left to stand overnight at 4°C.

Storage of unneutralized extracts frozen or at 0°C caused no sigiiifi- cant change of glycolytic intermediates. Only a slight loss of 2PGA occurred.

These studies show that reproducible values for red cell intermediates may be obtained by suitable fluorometric techniques using a filter &io..


rometer. They emphasize the extreme lability of red cell intermediates in shed blood. It is suggested that samples drawn for the determination of such intermediates should be collected without the use of a tourniquet directly into a syringe or tube containing ice cold PCA. Storage of blood for even as brief a period as IO min produces substantial alterations of some of the intermediates, particularly of FDP. Furthermore, these studies make it clear that shipping of samples is feasible without changes of the glycolytic intermediates when the PCA extracts are unneutralized and kept refrigerated or frozen.

SUMMARY

Methods for the fluorometric determination of glycolytic intermediates in red cells are described. Special attention was given to the technique used for drawing blood and producing filtrates. These factors were found to be very important, particularly with respect to FDP levels. Further- more, to determine the feasibility of shipping samples, extracts were stored for 48 hr at different temperatures. No changes of the glycolytic intermediates were found when the PCA extracts after deproteinization were unneutralized and kept refrigerated or frozen.

REFERENCES

1. MINAKAMI, S., in “Metabolism and Membrane Permeability of Erythrocytes and

Thrombocytes” (E. Deutsch, E. Gerlach, and K. Moser, Eds.), 1st Interna-

tional Symposium, Vienna, p. 10, 1968. 2. WILLIAMSON, J. R., Biology 6, 117 (1970).

3. MINAKAMI, S., SUZUKI, C., SAITO, T., AI\‘D YOSHIKAWA, H.. J. Biochem. 58, 543

(1965).

4. LOWRY, 0. H., PASSONEAU, J. V., HASSELBERGER, F. X., AND SCHULTZ, D. W.,

J. Biol. Chem. 239, 18 ( 1964).

5. ROSE, I. A., AND WARMS, J. V. B., J. Biol. Chem. 241, 4848 (1966). 6. OSKI, F. A., Pediatrics 44, 84 (1969).

7. FEZ, S. A., SHOHET, S. B., AND NATHAN, D. G., Clin. Invest. 50, 1731 ( 1971).

8. SEGEL, G. B., FEE, S. A., BAEHNER, R. L., ANI) NATHAN’, D. G., 1. Lab. C&I.

Med. 78, 969 ( 1971). 9. WAHBURG, O., AND CHRISTIAN, W., Biochem. Z. 303, 40 (1939).

10. GERLACH, E., FLECKENSTEIN, A., GROSS, E., AND Likmm, K., Arch. Gesamte

Physiol. 266, 528 ( 1958). 11. BARTLETT, G. R., J. Biol. Chem. 234, 449 ( 1959).

12. BEUTLER, E., AND DUROS, O., Trunsftcsion 5, 17 (19653. 13. ~~INAKAXI, S., SAITO, T., SUZUKI, C., AND YOSHIKAV-A, H., Hiochem. Biophgs,

Res. Commun. 17, 748 ( 1964). 14. MINAKA~II, S., AXD YOSHIKAWA, H., Biochem. Biophys. Res, Corrrmrm. 18, 345

(1965). 15. MINAKAMI, S., AND YOSHIKAW.I, H., J. Biochem. 59. 145 ( 1966 ). 16. ROSEXTHAL, T. B., J. Biol. Chcm. 173, 25 ( 1948).

17. OIIYAhIA, JI.. .4p\‘I) ~fIXAKAhl1, s.. J. ~ior~~,,r. 61. It):3 ( 196'7 ).

Documents

Fluorometric analysts of glycolytic intermediates in human red blood cells