• Kynurenine is a marker of osmotic fragility, and its levels are reproducible within a donor across donations.

  • Polymorphisms in SLC7A5, ATXN2 are associated with kynurenine levels in stored RBCs, Hgb increments, and in vivo hemolysis upon transfusion.

Abstract

In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

1.
Yoshida
T
,
Prudent
M
,
D'Alessandro
A
.
Red blood cell storage lesion: causes and potential clinical consequences
.
Blood Transfus
.
2019
;
17
(
1
):
27
-
52
.
2.
Francis
RO
,
D’Alessandro
A
,
Eisenberger
A
, et al
.
Donor glucose-6-phosphate dehydrogenase deficiency decreases blood quality for transfusion
.
J Clin Invest
.
2020
;
130
(
5
):
2270
-
2285
.
3.
Roubinian
NH
,
Reese
SE
,
Qiao
H
, et al
.
Donor genetic and nongenetic factors affecting red blood cell transfusion effectiveness
.
JCI Insight
.
2022
;
7
(
1
):
e152598
.
4.
Donovan
K
,
Meli
A
,
Cendali
F
, et al
.
Stored blood has compromised oxygen unloading kinetics that can be normalized with rejuvenation and predicted from corpuscular side-scatter
.
Haematologica
.
2022
;
107
(
1
):
298
-
302
.
5.
D'Alessandro
A
,
Kriebardis
AG
,
Rinalducci
S
, et al
.
An update on red blood cell storage lesions, as gleaned through biochemistry and omics technologies
.
Transfusion
.
2015
;
55
(
1
):
205
-
219
.
6.
D'Alessandro
A
,
D'Amici
GM
,
Vaglio
S
,
Zolla
L
.
Time-course investigation of SAGM-stored leukocyte-filtered red blood cell concentrates: from metabolism to proteomics
.
Haematologica
.
2012
;
97
(
1
):
107
-
115
.
7.
Pertinhez
TA
,
Casali
E
,
Baroni
F
,
Berni
P
,
Baricchi
R
,
Spisni
A
.
A comparative study of the effect of leukoreduction and pre-storage leukodepletion on red blood cells during storage
.
Front Mol Biosci
.
2016
;
3
:
13
.
8.
Unruh
D
,
Srinivasan
R
,
Benson
T
, et al
.
Red blood cell dysfunction induced by high-fat diet: potential implications for obesity-related atherosclerosis
.
Circulation
.
2015
;
132
(
20
):
1898
-
1908
.
9.
D'Alessandro
A
,
Fu
X
,
Reisz
JA
, et al
.
Ethyl glucuronide, a marker of alcohol consumption, correlates with metabolic markers of oxidant stress but not with hemolysis in stored red blood cells from healthy blood donors
.
Transfusion
.
2020
;
60
(
6
):
1183
-
1196
.
10.
D'Alessandro
A
,
Fu
X
,
Reisz
JA
, et al
.
Stored RBC metabolism as a function of caffeine levels
.
Transfusion
.
2020
;
60
(
6
):
1197
-
1211
.
11.
Bertolone
L
,
Roy
MK
,
Hay
AM
, et al
.
Impact of taurine on red blood cell metabolism and implications for blood storage
.
Transfusion
.
2020
;
60
(
6
):
1212
-
1226
.
12.
Stefanoni
D
,
Fu
X
,
Reisz
JA
, et al
.
Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers
.
Transfusion
.
2020
;
60
(
6
):
1160
-
1174
.
13.
Nemkov
T
,
Stefanoni
D
,
Bordbar
A
, et al
.
Blood donor exposome and impact of common drugs on red blood cell metabolism
.
JCI Insight
.
2021
;
6
(
3
):
e146175
.
14.
D'Alessandro
A
,
Fu
X
,
Kanias
T
, et al
.
Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity
.
Haematologica
.
2021
;
106
(
5
):
1290
-
1302
.
15.
Kanias
T
,
Lanteri
MC
,
Page
GP
, et al
.
Ethnicity, sex, and age are determinants of red blood cell storage and stress hemolysis: results of the REDS-III RBC-Omics study
.
Blood Adv
.
2017
;
1
(
15
):
1132
-
1141
.
16.
Tzounakas
VL
,
Kriebardis
AG
,
Georgatzakou
HT
, et al
.
Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells
.
Free Radic Biol Med
.
2016
;
96
:
152
-
165
.
17.
Hazegh
K
,
Fang
F
,
Bravo
MD
, et al
.
Blood donor obesity is associated with changes in red blood cell metabolism and susceptibility to hemolysis in cold storage and in response to osmotic and oxidative stress
.
Transfusion
.
2021
;
61
(
2
):
435
-
448
.
18.
D'Alessandro
A
,
Zimring
JC
,
Busch
M
.
Chronological storage age and metabolic age of stored red blood cells: are they the same?
.
Transfusion
.
2019
;
59
(
5
):
1620
-
1623
.
19.
Kleinman
S
,
Busch
MP
,
Murphy
EL
,
Shan
H
,
Ness
P
,
Glynn
SA
;
National Heart, Lung, and Blood Institute Recipient Epidemiology and Donor Evaluation Study REDS-III
.
The National Heart, Lung, and Blood Institute Recipient Epidemiology and Donor Evaluation Study (REDS-III): a research program striving to improve blood donor and transfusion recipient outcomes
.
Transfusion
.
2014
;
54
(
3 pt 2
):
942
-
955
.
20.
Josephson
CD
,
Glynn
S
,
Mathew
S
, et al
.
The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P): a research program striving to improve blood donor safety and optimize transfusion outcomes across the lifespan
.
Transfusion
.
2022
;
62
(
5
):
982
-
999
.
21.
D'Alessandro
A
,
Culp-Hill
R
,
Reisz
JA
, et al
.
Heterogeneity of blood processing and storage additives in different centers impacts stored red blood cell metabolism as much as storage time: lessons from REDS-III-Omics
.
Transfusion
.
2019
;
59
(
1
):
89
-
100
.
22.
Nemkov
T
,
Yoshida
T
,
Nikulina
M
,
D’Alessandro
A
.
High-throughput metabolomics platform for the rapid data-driven development of novel additive solutions for blood storage
.
Front Physiol
.
2022
;
13
:
833242
.
23.
Nemkov
T
,
Reisz
JA
,
Gehrke
S
,
Hansen
KC
,
D'Alessandro
A
.
High-throughput metabolomics: isocratic and gradient mass spectrometry-based methods
.
Methods Mol Biol
.
2019
;
1978
:
13
-
26
.
24.
Stefanoni
D
,
Shin
HKH
,
Baek
JH
, et al
.
Red blood cell metabolism in Rhesus macaques and humans: comparative biology of blood storage
.
Haematologica
.
2020
;
105
(
8
):
2174
-
2186
.
25.
Nemkov
T
,
Hansen
KC
,
D'Alessandro
A
.
A three-minute method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways
.
Rapid Commun Mass Spectrom
.
2017
;
31
(
8
):
663
-
673
.
26.
Reisz
JA
,
Zheng
C
,
D'Alessandro
A
,
Nemkov
T
.
Untargeted and semi-targeted lipid analysis of biological samples using mass spectrometry-based metabolomics
.
Methods Mol Biol
.
2019
;
1978
:
121
-
135
.
27.
Thomas
T
,
Stefanoni
D
,
Dzieciatkowska
M
, et al
.
Evidence of structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients
.
J Proteome Res
.
2020
;
19
(
11
):
4455
-
4469
.
28.
Moore
A
,
Busch
MP
,
Dziewulska
K
, et al
.
Genome-wide metabolite quantitative trait loci analysis (mQTL) in red blood cells from volunteer blood donors
.
J Biol Chem
.
2022
;
298
(
12
):
102706
.
29.
Page
GP
,
Kanias
T
,
Guo
YJ
, et al
.
Multiple-ancestry genome-wide association study identifies 27 loci associated with measures of hemolysis following blood storage
.
J Clin Invest
.
2021
;
131
(
13
):
e146077
.
30.
Guo
Y
,
Busch
MP
,
Seielstad
M
, et al
.
Development and evaluation of a transfusion medicine genome wide genotyping array
.
Transfusion
.
2019
;
59
(
1
):
101
-
111
.
31.
Delaneau
O
,
Coulonges
C
,
Zagury
J-F
.
Shape-IT: new rapid and accurate algorithm for haplotype inference
.
BMC Bioinformatics
.
2008
;
9
(
1
):
540
.
32.
Howie
B
,
Marchini
J
,
Stephens
M
.
Genotype imputation with thousands of genomes
.
G3 (Bethesda)
.
2011
;
1
(
6
):
457
-
470
.
33.
Zheng
X
,
Levine
D
,
Shen
J
,
Gogarten
SM
,
Laurie
C
,
Weir
BS
.
A high-performance computing toolset for relatedness and principal component analysis of SNP data
.
Bioinformatics
.
2012
;
28
(
24
):
3326
-
3328
.
34.
Aulchenko
YS
,
Struchalin
MV
,
van Duijn
CM
.
ProbABEL package for genome-wide association analysis of imputed data
.
BMC Bioinformatics
.
2010
;
11
(
1
):
134
.
35.
Perry
JA
,
Gaynor
BJ
,
Mitchell
BD
,
O’Connell
JR
.
An Omics Analysis Search and Information System (OASIS) for enabling biological discovery in the old order Amish
.
bioRxiv
.
Preprint posted online 3 May 2021
.
36.
Howie
HL
,
Hay
AM
,
de Wolski
K
, et al
.
Differences in Steap3 expression are a mechanism of genetic variation of RBC storage and oxidative damage in mice
.
Blood Adv
.
2019
;
3
(
15
):
2272
-
2285
.
37.
Churchill
GA
,
Gatti
DM
,
Munger
SC
,
Svenson
KL
.
The diversity outbred mouse population
.
Mamm Genome
.
2012
;
23
(
9-10
):
713
-
718
.
38.
Roubinian
NH
,
Plimier
C
,
Woo
JP
, et al
.
Effect of donor, component, and recipient characteristics on hemoglobin increments following red blood cell transfusion
.
Blood
.
2019
;
134
(
13
):
1003
-
1013
.
39.
Pang
Z
,
Chong
J
,
Zhou
G
, et al
.
MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights
.
Nucleic Acids Res
.
2021
;
49
(
W1
):
W388
-
W396
.
40.
Paglia
G
,
D'Alessandro
A
,
Rolfsson
O
, et al
.
Biomarkers defining the metabolic age of red blood cells during cold storage
.
Blood
.
2016
;
128
(
13
):
e43
-
e50
.
41.
DʼAlessandro
A
,
Yoshida
T
,
Nestheide
S
, et al
.
Hypoxic storage of red blood cells improves metabolism and post-transfusion recovery
.
Transfusion
.
2020
;
60
(
4
):
786
-
798
.
42.
Van 't Erve
TJ
,
Wagner
BA
,
Martin
SM
, et al
.
The heritability of hemolysis in stored human red blood cells
.
Transfusion
.
2015
;
55
(
6
):
1178
-
1185
.
43.
van 't Erve
TJ
,
Wagner
BA
,
Martin
SM
, et al
.
The heritability of metabolite concentrations in stored human red blood cells
.
Transfusion
.
2014
;
54
(
8
):
2055
-
2063
.
44.
Bonomini
M
,
Sirolli
V
,
Dottori
S
,
Amoroso
L
,
Di Liberato
L
,
Arduini
A
.
L-carnitine inhibits a subset of platelet activation responses in chronic uraemia
.
Nephrol Dial Transplant
.
2007
;
22
(
9
):
2623
-
2629
.
45.
van 't Erve
TJ
,
Doskey
CM
,
Wagner
BA
, et al
.
Heritability of glutathione and related metabolites in stored red blood cells
.
Free Radic Biol Med
.
2014
;
76
:
107
-
113
.
46.
Sinclair
LV
,
Neyens
D
,
Ramsay
G
,
Taylor
PM
,
Cantrell
DA
.
Single cell analysis of kynurenine and System L amino acid transport in T cells
.
Nat Commun
.
2018
;
9
(
1
):
1981
.
47.
Walker
AK
,
Wing
EE
,
Banks
WA
,
Dantzer
R
.
Leucine competes with kynurenine for blood-to-brain transport and prevents lipopolysaccharide-induced depression-like behavior in mice
.
Mol Psychiatry
.
2019
;
24
(
10
):
1523
-
1532
.
48.
Häfliger
P
,
Charles
RP
.
The L-type amino acid transporter LAT1-an emerging target in cancer
.
Int J Mol Sci
.
2019
;
20
(
10
):
2428
.
49.
D’Alessandro
A
,
Nouraie
SM
,
Zhang
Y
, et al
.
Metabolic signatures of cardiorenal dysfunction in plasma from sickle cell patients as a function of therapeutic transfusion and hydroxyurea treatment
.
Haematologica
.
2023
;
108
(
12
):
3418
-
3432
.
50.
Sullivan
KD
,
Galbraith
MD
,
Kinning
KT
, et al
.
The COVIDome Explorer researcher portal
.
Cell Rep
.
2021
;
36
(
7
):
109527
.
51.
Surendran
P
,
Stewart
ID
,
Au Yeung
VPW
, et al
.
Rare and common genetic determinants of metabolic individuality and their effects on human health
.
Nat Med
.
2022
;
28
(
11
):
2321
-
2332
.
52.
Meierhofer
D
,
Halbach
M
,
Şen
NE
,
Gispert
S
,
Auburger
G
.
Ataxin-2 (Atxn2)-knock-out mice show branched chain amino acids and fatty acids pathway alterations
.
Mol Cell Proteomics
.
2016
;
15
(
5
):
1728
-
1739
.
53.
Todd
JA
,
Walker
NM
,
Cooper
JD
, et al
.
Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes
.
Nat Genet
.
2007
;
39
(
7
):
857
-
864
.
54.
Wellcome Trust Case Control Consortium
.
Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls
.
Nature
.
2007
;
447
(
7145
):
661
-
678
.
55.
Issaian
A
,
Hay
A
,
Dzieciatkowska
M
, et al
.
The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality
.
Haematologica
.
2021
;
106
(
11
):
2971
-
2985
.
56.
Nemkov
T
,
Skinner
SC
,
Nader
E
, et al
.
Acute cycling exercise induces changes in red blood cell deformability and membrane lipid remodeling
.
Int J Mol Sci
.
2021
;
22
(
2
):
896
.
57.
Nemkov
T
,
Cendali
F
,
Stefanoni
D
, et al
.
Metabolic signatures of performance in elite world tour professional male cyclists
.
Sports Med
.
2023
;
53
(
8
):
1651
-
1665
.
58.
Nemkov
T
,
Skinner
S
,
Diaw
M
, et al
.
Plasma levels of acyl-carnitines and carboxylic acids correlate with cardiovascular and kidney function in subjects with sickle cell trait
.
Front Physiol
.
2022
;
13
:
916197
.
59.
Wu
H
,
Bogdanov
M
,
Zhang
Y
, et al
.
Hypoxia-mediated impaired erythrocyte Lands’ cycle is pathogenic for sickle cell disease
.
Sci Rep
.
2016
;
6
(
1
):
29637
.
60.
D’Alessandro
A
,
Nouraie
SM
,
Zhang
Y
, et al
.
In vivo evaluation of the effect of sickle cell hemoglobin S, C and therapeutic transfusion on erythrocyte metabolism and cardiorenal dysfunction
.
Am J Hematol
.
2023
;
98
(
7
):
1017
-
1028
.
61.
Bissinger
R
,
Nemkov
T
,
D'Alessandro
A
, et al
.
Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism
.
Kidney Int
.
2021
;
100
(
6
):
1227
-
1239
.
62.
D'Alessandro
A
,
Thomas
T
,
Dzieciatkowska
M
, et al
.
Serum proteomics in COVID-19 patients: altered coagulation and complement status as a function of IL-6 level
.
J Proteome Res
.
2020
;
19
(
11
):
4417
-
4427
.
63.
Recktenwald
SM
,
Simionato
G
,
Lopes
MGM
, et al
.
Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19
.
Elife
.
2022
;
11
:
11
.
64.
Thomas
T
,
Stefanoni
D
,
Reisz
JA
, et al
.
COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status
.
JCI Insight
.
2020
;
5
(
14
):
e140327
.
65.
Domizio
JD
,
Gulen
MF
,
Saidoune
F
, et al
.
The cGAS–STING pathway drives type I IFN immunopathology in COVID-19
.
Nature
.
2022
;
603
(
7899
):
145
-
151
.
66.
Galbraith
MD
,
Kinning
KT
,
Sullivan
KD
, et al
.
Seroconversion stages COVID19 into distinct pathophysiological states
.
Elife
.
2021
;
10
:
10
.
67.
D'Alessandro
A
,
Thomas
T
,
Akpan
IJ
, et al
.
Biological and clinical factors contributing to the metabolic heterogeneity of hospitalized patients with and without COVID-19
.
Cells
.
2021
;
10
(
9
):
2293
.
68.
Catala
A
,
Stone
M
,
Busch
MP
,
D'Alessandro
A
.
Reprogramming of red blood cell metabolism in Zika virus-infected donors
.
Transfusion
.
2022
;
62
(
5
):
1045
-
1064
.
69.
Huisjes
R
,
Bogdanova
A
,
van Solinge
WW
,
Schiffelers
RM
,
Kaestner
L
,
van Wijk
R
.
Squeezing for life - properties of red blood cell deformability
.
Front Physiol
.
2018
;
9
:
656
.
70.
Bouchla
A
,
Kriebardis
AG
,
Georgatzakou
HT
, et al
.
Red blood cell abnormalities as the mirror of SARS-CoV-2 disease severity: a pilot study
.
Front Physiol
.
2021
;
12
:
825055
.
71.
Talstad
I
,
Haugen
HF
.
The relationship between the erythrocyte sedimentation rate (ESR) and plasma proteins in clinical materials and models
.
Scand J Clin Lab Invest
.
1979
;
39
(
6
):
519
-
524
.
72.
Yang
YC
,
Walsh
CE
,
Johnson
MP
, et al
.
Life-course trajectories of body mass index from adolescence to old age: racial and educational disparities
.
Proc Natl Acad Sci U S A
.
2021
;
118
(
17
):
e2020167118
.
73.
Thomas
TA
,
Qiu
A
,
Kim
CY
, et al
.
Reticulocytes in donor blood units enhance red blood cell alloimmunization
.
Haematologica
.
2023
;
108
(
10
):
2639
-
2651
.
74.
Clement
CC
,
D'Alessandro
A
,
Thangaswamy
S
, et al
.
3-hydroxy-L-kynurenamine is an immunomodulatory biogenic amine
.
Nat Commun
.
2021
;
12
(
1
):
4447
.
75.
Mondanelli
G
,
Bianchi
R
,
Pallotta
MT
, et al
.
A relay pathway between arginine and tryptophan metabolism confers immunosuppressive properties on dendritic cells
.
Immunity
.
2017
;
46
(
2
):
233
-
244
.
76.
Powers
RK
,
Culp-Hill
R
,
Ludwig
MP
, et al
.
Trisomy 21 activates the kynurenine pathway via increased dosage of interferon receptors
.
Nat Commun
.
2019
;
10
(
1
):
4766
.
77.
Culp-Hill
R
,
Zheng
C
,
Reisz
JA
, et al
.
Red blood cell metabolism in Down syndrome: hints on metabolic derangements in aging
.
Blood Adv
.
2017
;
1
(
27
):
2776
-
2780
.
78.
Moriconi
C
,
Dzieciatkowska
M
,
Roy
M
, et al
.
Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease
.
Br J Haematol
.
2022
;
198
(
3
):
574
-
586
.
79.
Jagadeeswaran
R
,
Lenny
H
,
Vazquez
B
, et al
.
The abnormal presence of mitochondria in circulating red blood cells cause an increased oxygen consumption rate, ROS generation and hemolysis in patients with sickle cell disease
.
Blood
.
2017
;
130
(
suppl 1
). 2237-2237.
80.
Bruun-Rasmussen
P
,
Kragh Andersen
P
,
Banasik
K
,
Brunak
S
,
Johansson
PI
.
Intervening on the storage time of RBC units and its effects on adverse recipient outcomes using real-world data
.
Blood
.
2022
;
139
(
25
):
3647
-
3654
.
81.
Belpulsi
D
,
Spitalnik
SL
,
Hod
EA
.
The controversy over the age of blood: what do the clinical trials really teach us?
.
Blood Transfus
.
2017
;
15
(
2
):
112
-
115
.
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