Pyridine nucleotide metabolism in stored human erythrocytes

CLINICA

CCA

CHIhlICA ACT.4

4861

PYRlDINE

NUCLEOTIDE

METABOLISM

IN STORED

HUMAN

BRYTHROCYTES

AKIRX

OAIi\CHI,

Depavtwent (Received

I.

C.\ROLTN

of Physiology, Septernbcr

When

B. SCOTT

Cwiversity

AS,, DxVID

of Illinois

L. FORD

at the Medical

Centrv, Chicago,

Ill. (C.S.A

.)

27, 1971)

erythrocytes

were stored

in acid-citrate-dextrose

medium,

NADH

content increased up to 4 weeks and then decreased whereas NADPH declined slowly over an g-week period. No significant change was observed in NAD+ or in NADP+. 2. Essentially similar nucleotide patterns were observed when stored erythrocytes were washed and incubated for 2.5 h at 37” in a glucose-containing Tris-Ringer’s medium before enzymatic analysis. The NADH rise appeared to be arrested at a time when the ability of these cells to produce lactate began to decrease. 3. Cells suspended in glucose-free media contained less NADH than cells incubated with glucose, but the difference became smaller as erythrocytes stored for longer periods were used. At 6 weeks, no difference was detected. On the other hand, NADPH was always lower and NADP+ higher when cells of any storage age were incubated without substrate. 4. After 8 weeks of storage, incubation of washed erythrocytes with inosine caused NADH and lactate production to increase and NADf to decrease. 5. These results indicate that pyridine nucleotide levels generally reflect the metabolic state of stored human erythrocytes. There was no indication that these compounds are directly implicated in the development of the storage lesion.

INTRODUCTION

\Vhen human erythrocytes are stored in acid-citrate-dextrose (ACD) medium at 5”, progressive metabolic changes occur1 which may be responsible for rendering these blood cells unsuitable for transfusion. As yet, however, a specific metabolic change has not been clearly associated with loss in transfusion viability. In a previous study with washed erythrocytes incubated in vitro at 37’, we noted that NADH and NADf contents were different when cells were used which had been stored for two weeks instead of for a few days2. Since these compounds plav vital metabolic roles and are present in small concentrations that might easily be modified, a systematic study of these compounds during prolonged cold storage seemed in order. This report is concerned with such a study. Clin. Chzvn. Acta,

37 (1972) 351-358

352

OMACHI

ct cd.

Pyridine nucleotide levels in incubated as well as nonincubated erythrocytes were found to undergo modification during 8 weeks of storage. The nature of the variations was explored by measuring lactate production and by incubating erythrocytes in substrate-free and inosine-containing media. In general, the results suggest that pyridine nucleotide levels characterize the metabolic state of lmman erythrocytes under various

conditions,

dicating

primary

includinff involvement

cold storage.

No evidence

of these compounds

was obtained,

in the development

however,

in-

of the storage

lesion. METHODS

The basic procedures normal,

have been described

adult males in ACD (National

Institutes

before 2$3. Blood was collected of Health,

Formula

from

A). After a bag

was opened, the same blood was not used again if the elapsed period was greater than two weeks. For studies of erythrocytes stored for ZI days or longer, outdated blood from the Blood Bank Service of the University of Illinois Research Hospital was employed. A given sample of blood was centrifuged for 5 min at 8oog and 5”, and the plasma and buffy coat were removed. Extracts were prepared from a portion of the packed red cells for analysis of pyridine nucleotides. The remaining erythrocytes were washed 3 times with one volume of a Ca 2+-free , glucose-containing Ringer’s medium buffered with tris(hydroxymethyl)aminomethane (Tris) to read pH 7.4 at 37”. A fourth wash was carried out with the same medium except that it now contained Ca2i (Tris-Ringer’s medium). It was estimated from the original hematocrit and the fluid volumes added that at least 99.9% of the plasma was cleared by the overall washing procedure. The washed, loosely packed cells (I m1) were resuspended in Tris---Ringer’s medium (z ml) to give a hematocrit of 2576, and the suspension was incubated for 2.5 h at 37” in a Dubnoff shaker cycled at roo/min. Cell extracts were also prepared at the end of an incubation for pyridine nuclootide analysis. Reduced and oxidized nucleotides were extracted by the methods of Lowry and associates4 and Burch and associates5, respectively. The procedure of Gupta and colleague@,’ was used in measuring NADH and NAD+ . This method involves a coupled oxidoreduction between ethanol and lactaldehyde producing acetaldehyde and propanediol, in the presence of horse liver alcohol dehydrogenase (Boehringer) The reaction is driven by rate-limiting amounts of either NAD+~ or NADH and the acetaldehyde, which is flushed out of the reaction vessel with N, into a semicarbazide solution, is measured at 224 rnp as the scmicarbazone. As previously described”, NADPH and NADP+ were evaluated after digestion of the appropriate extracts with alkaline phosphatase (Worthington, BAPSF) which cleaved these compounds to NADH and NADT, respectively. The actual figures for NADPH and NADP+ were therefore obtained by difference, i.e., by subtracting the separately measured NADH and NAD-+ values from the values determined in the digested extracts. The pyridine nucleotide data were expressed in terms of grams of hemoglobin since cells stored in ACD become swollen with timel. Hemoglobin was determined by the cyanhemoglobin method8. Lactate measurements were performed with lactic dehydrogenases on perchloric acid extracts of initial and final blood suspensions. Lactate production was calculated Cliff.

Chip%. ,4&a, 37 (1972)

jjI-3jX

PYRIDINE

WUCLEOTII)ES

IN ERYTHROCYTES

353

from initial and final values, as it was shown in separate experiments tate production

takes place under the conditions

used in the experiments

described

that a linear lac-

of our study. The inosine and adenine

in Table I were obtained

from Calbiochem

and Sig-

ma, respectively. RESULTS

I~~~.Lfluence qf stoqgc on pyuidinc nucleotide batcrl evythrocytes Pyridine nucleotide concentrations

and lactate conted in cold-stored

Figs. I, 2 and 3 by the open circles. Nucleotide

in, 3zottilzcuhated and inew erythrocytes

concentrations

are shown in

were also determined

in

each experiment after erythrocvtes were washed and incubated at 37” for 2.5 11, and these values are represented by the closed circles. In general, the patterns of change were not greatly different in the two cases. Thus, in both groups NADIR rose steadily during storage, reached a peak, and then declined toward initial levels (Fig. I). In incubated

cells, the rise appears to have been arrested at an earlier time, i.c. at

2 weeks,

instead of at 4 weeks as observed in nonincubated cells. After 4 weeks, the two curves were different from each other according to the analysis of variance (p < 0.01). In the incubated

cells, NAD

decreased

during the first

2 weeks

as

NADH rose and, subse-

quently, the oxidized nucleotide appeared to rise as the reduced nucleotide declined. This reciprocal variation was not evident in the nonincubated cells. XADPH in both incubated and nonincubat~d cells showed a tendency to fall with storage duration after the first few days (Fig. 2). KADP+ appeared to increase correspondingly with time except for an initial transitory change in nonincubated cells. Lactate production by erythrocytes incubated at 37’ was greater in experiments conducted after z weeks of storage instead of on the day of collection (Fig. 3). After 2 weeks, the production of lactate decreased progressively. Iti.@uence

ofstorage

on the fiyriditle

7vucleotide yesjhcvfse to the absczcr

ofmedium

glucose

In a previous study2, no change in NAD- level was found when cells stored for o-z days were incubated in glucose-free media even though NADH decreased under these circumstances. On the other hand, when cells previously stored for z weeks were subjected to the same test, an increase in the NAD+ level was noted. In the present studv,KAD+content did not show anychange when cells preserved for longer durations were tested in the same manner (Fig. 4). Thus, for reasons which are not clear at this time, a NAD+ increase in the absence of glucose was only observed in cells that had been stored for 2 weeks. An unexpected rest& in this experiment was the progressively smaller difference in NADH content between erythrocytes suspended with and without glucose, as storage time was increased. In fact, no difference was detected after 6 weeks. This observation appears to provide an independent assessment of glycolytic function in cold-stored erytbrocytes and seems to be in general accord with previous work. Thus, severe depression in glucose utilization10t11 and in ATP restoration10 have been reported after 3 to 6 weeks of storage, when ACD blood was rewarmed to 37O. In contrast to the h_ADH response, the NADPII decrease and NADPi- increase which occurred in the absence of medium glucose were generally observed during 8 weeks of storage (Fig. 4). Clin. Chin.

Ada,

37 (197~)

3jr---jgS

OMACHI et al.

354 NAD*

225

100

NADP’

50

NADPH

0

io DAYS

40 OF

60

0

STORAGE

2b DAYS

40 OF

60

STORAGE

Fig. 1. NXDH and NA4D~-content as a function of cold storage. Measurements were made directI!on stored cells (open circles) or on cells that were washed and incubated in a b’lucose-containin::l~ Tris-Ringer’s medium at 37O for 2.5 h (closed circles). Mean values (n = 4) arc bracketed by two SEs. Nonincubated and &bated groups were compared with the paired t test. *p -: o.or. **p < 0.05. The following comparisons were also significant accordin, 0 to the nonpaircd t test: Nonincubated cells: NADH: Day o us. Day z8*, Day 28 us. Day 60*+ Incubated cells : NADH: Day o us. Day IJ*, Day 1‘3 vs. Day ho* NAI>+: Day 2 vs. Day I+**. Fig. 2. S.SDPH and NADP+ content as a function of cold storage. SW legend under L:ig. I. The following comparisons were significant accordin, 0 to the nonpaired t (*t, -.: 0.01. **p .. . 0.05) : Nonincubated cells: N.-ZDPH: Day 1 vs. T)ay Go* NXDP+: 11;~~ rS vs. Ilay (lo**.

The influence

of inosine

and adenine

stored cells is well documented12. For rewarmed in the presence of inosine, presence of adenine causes an even observations (Table I). In our study,

in rejuvenating

the metabolism

of cold-

example, when blood stored for 6 weeks in ACD is lactate production is increased and the additional greater lactate outputl”. Our data confirm these moreover, the NADH level increased in the pres-

ence of inosine although the added presence of adenine caused no further change. ~111 increase in ??ADPH and a decrease in NADP+ was also observed. The latter alterations were greater with inosine than with glucose, suggesting that glucose h-phosphate is formed more readily from the riboside than from glucose in these K-week-old cells. u1scuss10s

Pyridinc nwleotide covztent i?s xonincubated aped incubated evythrocytes The concentrations of the reduced nucleotides, NADH and KADPH, were found to vary when human erythrocytes were preserved at 5” over an g-week period. The changes observed were so small, however, that they could easily have escaped detection in earlier attempts to discern differencesIS. No significant change was noted in the oxidized nucleotides, as reported previous11 713,14.There was also no large alteration in total pyridine nucleotide, i.c,. the sum of P\TAD*-,NADH, NADP+ , and NADPH, which suggests that the turnover of these compounds is relatively slow since ATP, which is required for NAD and NADP synthesis, decreases significantly during prolonged storagelp11,15.

PI-RIDINE KUCLEOTIDES IN ERYTHROCYTES

355

NADPH

I

0

1

I

20 DAYS

40 OF

60

STORAGE

lactate production by erythrocytes incubated at 373 after different periods of cold storage. See legend under Fig. I. The dashed line refers to a second set of cells that were washed 3 cstra times before the incubation in order to reduce the initial lactate level. Lactate production by cells washed more thoroughly was different from that by normally mashed cells over the first 4 weeks, according to the analysis of variance (p < 0.05) ; this difference could have been related to a slightly higher pH (0.1 pH unit) in the former group of cells. In both groups, Day 60 values were significantly different from Day 14 values (p < O.Oj). In a larger series with normally washed cells, Day 14 values (32 = 14) was different from Day o values (S = 6) at the j”/(, probability level,

according

to the nonpaired

t test.

Fig. 4. Pyridine nucleotide content of erythrocytcs and glucose-containing (closed circles) Tris-Ringer’s See legencl uncler Fig. I, *t) < 0.01, * *fl < o.oj.

incubated at 37’ in glucose-free (open circles) media, after different periods of cold storage

Nucleotide concentrations in erythrocytes that were washed and incubated at 37O for 2.5 h were essentially the same as that seen in nonincubated erythrocytes. This suggests that pyridine nucleotide metabolism was generally similar in the two cases even though glycolytic rates were undoubtedly dissimilar due to the difference in temperature. The actual situation may be more complex since nucleotide changes can occur when glycolytic rate is increased, as will be discussed below.

Although it is generally believed that glycolysis is progressively inhibited during cold storage of human erythrocytes, we observed that lactate production by washed cells incubated at 37” actually increased during the first z weeks of storage (Fig. 3). As discussed earlier2, this may have been due to Pi accumulation1+11p15 which could have stimulated glycolycis by activating hexokinase16 or phosphofructokinaselTT1a. After z weeks, the ability to produce lactate declined steadily due presumably to a decline in activity of a particular enzyme or enzymes (see below). In contrast, Deloecker and Prankerdlg have reported a constant decrease in lactate production from the day of collection. Since their cells were incubated in Pi buffer, it would appear that they Cl&. Chiw. A&z,

37 (1972)

3jI-358

were measuring

glycolysis

under conditions

of maximal

Pi activatioIil”,l:,“.

Our results

indicate, therefore, that lactate production is not necessaril)? depressed during tllc first few weeks of storage even though maximal glycolytic capacity may bc declining from the first day of storage. The NADH level appeared to reflect the glycolytic state in these incubated erythrocytes. Thus, lactate production (Fig. 3) and NADH content (Fig. I) both increased up to z weeks, following which the decrease in lactate production was associated with a cessation of the NADH rise and a subsequent decline in the level of this nucleotide. Another example of this relationship was seen when inosine was added to a suspension of S-week-old cells which utilized glucose poorly (Table I). In tllc presenccx

of the nucleoside, lactate production was augmented in conjunction with an elevation in NADH content. These changes with inosine are plausible since its degradation within the erythrocyte can give rise to phosphoglyceraldehyde, themetabolism of which can lead to formation of both NADH and lactate. It should be noted in these instances that NADH content would not have been altered if a molecule of NADH synthesized at the phosphoglyceraldehyde dehydrogrnase (PGDH) step were simply utilized at the lactic dehydrogenase (LDH) step for each molecule of lactate formed. The NADH rise indicates, therefore, that formation of reduced nucleotide exceeded its utilization as lactate formation was increased. ‘IX5 can happen because some of the glucose or inosine carbon may be converted to 2,_3-Inosine diphosphoglycerate (2,3-DPG) instead of to lactate under these condition9. addition, in fact, is known to raise 2,3-DPG level+. With decreased gly-colysis, the NADH level falls because formation camlot keep pace with utilization. The latter can take place in conjunction with such continuously occurring events as methemoglol:in reduction22. In general, therefore, a change in NADH content can be associated with a change in glycolytic rate because the rates of formation and utilization of this nucleotide are not necessarily affected equally3”.

PTRIDINE

XVCLEOTIDES

IN ERYTHKOCYTES

In the case of the nonincubated, NADIR content

(Fig.

I)

cannot

357

cold-stored

be easily associated

erythrocyte,

the early increase

with an elevated

in

rate of lactate

production, since the slow increase in lactate content during the early weeks of storage appears to occur at a constant rate 51”+za.Nevertheless, it seems possible that NADH formation could increase under theseconditionsof reduced metabolism as a consequence of Pi accun~ulation1~11~15. The latter change could cause more substrate to be formed for the PGDH

reaction

by stimulating

certain

enzymes

such as pl~ospl~ofructokinase17~1R

allosterically. A concurrent event as this time is the degradation of 2,3-DPG to pyruvate which causes NADH to be utilized. The constant lactate production indicates, however, that NADH is being recycled continuously so that the elevated NADH level could be maintained even as 2,3-DPG is being broken down. Ultimately, glycolytic inhibition develops and NADH would he expected to decline as formation rate decreases below utilization rate. .%ccording to our data, this occurs at about 4 weeks which is about 2 weeks after z,3-DPG has virtually disappearedX5*ls.

It-is well known that when blood stored for several weeks at 5” is rewarmed to glucose is utilized poorlyl”T1l whereas inosine addition causes significant lactate 3Y> production*@. We have observed similar results in our incubation studies with washed erythrocptes, including an elevation in NADH in the presence of the riboside (Table I). These observations indicate that the intermediate steps between PGDH and LDH are not significantly depressed as a cons-quence of cold storage. In addition, NADPH was elevated in the presence of inosine which show.; that the steps involved with ribose phosphate formation from this nucleoside, the conversion of the pentose phosphate to glucose h-phosphate, and the oxidation of the latter in conjunction with NADPH generation are also functional. There was also some indication that the NADPH level could be raised by the addition of glucose (Fig. 4, Table I) suggesting that glucose 6phosphate formation from this substrate can take place although this reaction could be inhibited somewhatz4p25. Our results suggest, therefore, that a glycolytic block may exist between the phosphofructokinase and PGDH steps. Since phosphofructokinase appears to be particularly affected by cold storage 26**8, it would appear that the same metabolic block may persist after these cells are restored to ph~sioI~~gica1 temperatures. The pllospllo~luconate pathway was not completely inhibited by 8 weeks of storage according to our nucleotide data. NADPI-I generation could have been depressed to a certain extent, judging by the decline in content of this nucleotide with storage time (Fig. 2) and by the smaller difference in NADPH between cells incubated with and without glucose (Fig. 4). This result is consistent with previous observations iildicating that the enzymes in the pllospllogluconate pathway are not seriously depressed during cold storage”j. It may be emphasized, finally, that the pyridine nucleotide changes observed during prolonged storage were not irreversible, since nucleotide variations were observed following inosine addition to S-week-old cells. This indicates that the developmcnt of the storage lesion cannot be easily explained on the basis of a permanent loss in pyridine nucleotide f~lnction.

CliTZ. ChiWt. ACta, 37 (1972) 351-35S

358 ACKXOWLEDGMENT

This research and HE-14641.

was supported

33. s. RlIXAlCARlI

AX,)

S. MIK.~RAMI,

in E. T~EUTSCH

and Thvombocytes,

H.

~~SHIK.~WA,

Gem-g

et al.

Thieme

by U.S. Public

Hiochim. (RdS.), \ycrlag,

Biophys.

Mrtaboliswz Stuttgart,

Health

Service

Grants

HE-1354;

Acta, 99 (196.5) ~7j. Pl~wzcabilzt+ I? lX;r,I’throc_vfr2&

and Mrtnbranr 1968,

p.

IO.