June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

Plasma vitamin K-dependent protein C as early diagnostic marker
of feline chronic kidney disease
1

2

2

3

4

2

Hiroto Maeda , Yuiko Suzuki , Kokoro Masuda , Yoshio Kodera , Toshifumi Watanabe , Kazuyuki Sogawa
Maeda Veterinary Hospital, 1‒4‒8 Yamatemachi, Tomakomai, Hokkaido 053‒0851, Japan
Department of Biochemistry, School of Life and Environmental Science, Azabu University,
1‒17‒71 Fuchinobe, chuo-ku, Sagamihara, Kanagawa 252‒5201, Japan
3
Center for Disease Proteomics, Kitasato University School of Science,
1‒15‒1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252‒0373, Japan
4
DVMs Animal Medical Center Yokohama, 2‒2 Sawatari, Kanagawa-ku, Yokohama, Kanagawa 221‒0844, Japan
1

2

Abstract

Chronic kidney disease (CKD) is a common disorder and cause of death in cats. In the classiﬁcation proposedby the International Renal Interest Society (IRIS), stage 1 and 2 CKD are diﬃcult to diagnose accurately using markers,
in comparison with normal control cats. We recently described a simple and highly reproducible tandem mass tag (TMT)
labelling method for identifying potential disease-marker candidates among low-abundance plasma proteins. In the current
study, plasma samples were obtained from 90 normal control cats and 50 cats with CKD (stage 1). To identify new plasma
biomarkers for CKD, six plasma samples (three from normal control cats and three from CKD stage 1 cats) were extractedwith SSA lectin magnetic beads, diﬀerentially labelled with TMTs, digested with trypsin and subjected to analysis using
LC-MS/MS. Sialylated Vitamin K-dependent protein C was identiﬁed as a protein with lower levels in CKD stage 1 catscompared to normal control cats. An ELISA of plasma sialylated vitamin K-dependent protein C showed within-run (4.3‒
4.9%) and between-day (4.3‒5.2%) reproducibility. Plasma sialylated vitamin K-dependent protein C levels measured withthis assay were signiﬁcantly greater in normal control cats than in CKD stage 1 cats (1.40±0.13 vs. 1.07±0.11 AU/mL, p＜
0.001). These results indicate that sialylated vitamin K-dependent protein C may be useful as a complementary marker withplasma creatinine, BUN and SDMA for detection of CKD stage 1 in cats.
Key words: chronic kidney disease, feline, plasma, vitamin K-dependent protein C

Introduction

2)tion of survival and improvement of QOL . However, there

Feline renal diseases are increasingly common in veteri-are only a few reports on this area in clinical veterinary

1)3)nary practice . It is important to diagnose and identify the

medicine .

pathological basis of renal dysfunction accurately at an

In daily clinical practice, plasma creatinine (p-Cre) andearly stage, since early diagnosis and treatment can delay
progression of renal dysfunction, which leads to prolonga-

serum symmetric dimethylarginine (SDMA) are used as
markers based on the stage classiﬁcation criteria of the
4)International Renal Interest Society (IRIS) . However,many of these markers have low speciﬁcity for renal func-* Corresponding author
Kazuyuki Sogawa

tion and there is a blind area without abnormal values,

Department of Biochemistry, School of Life and Environmental Science, Azabu University, 1‒17‒71 Fuchinobe, Chuo-ku,

despite development of mild renal dysfunction. Therefore,

Sagamihara, Kanagawa 252‒5201, Japan
Tel: ＋81‒42‒769‒1924, Fax: ＋81‒42‒769‒1924

it is especially important to evaluate the transitional period

Received: February 6, 2024. Accepted: April 3, 2024.

to identify a mild decrease of renal function in the early

Epub May 10, 2024.

phase using the available markers in clinical practice. In

DOI: 10.24508/mms.2024.06.004

stage 1 there may not be any reduction in GFR or surrogate

from a normal status to Stage 1 in the IRIS classiﬁcationfor early diagnosis of renal function. However, it is diﬃcult

E-mail: sogawa@azabu-u.ac.jp

27June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Magnetic bead sample preparation using the Glycoprotein Isolation Kit

markers of GFR, despite ongoing active disease in the kidney.
We recently described a simple and highly reproducible
tandem mass tag (TMT) labelling method for identifying
potential disease-marker candidates among low-abundanceParamagnetic non-porous particles coupled with a sambucus sieboldiana lectin ligand (MB-SSA; Bruker Dalton-

8)
plasma proteins . In this study, we used the lectin and tandem mass tag (TMT) method to generate comparative pro-ics, Bremen, Germany) were used to process the plasma
samples. Binding, washing and elution solutions were pre-tein proﬁles of plasma samples from normal control catspared according to the manufacturerʼs instructions. MB-SSA

and CKD stage 1 cats. Plasma levels of candidate proteinsbinding solution (10 μL) and plasma (5 μL) were transferred
to a 0.2‒ mL thin-walled tube. A homogenous magnetic-par-were compared to evaluate their potential to discriminate
between the normal control cats and CKD cats. Novelticle solution (10 μL) was added, mixed, and incubated for
5 min. The tubes were placed in an 8×12-well magnet-

plasma biomarkers were identiﬁed that may be superior to

ic-bead separator (MBS; Bruker Daltonics) for 30 sec formagnetic ﬁxation of the MB-SSA particles. The supernatantclassical markers for early and speciﬁc detection of CKD
stage 1 in cats.

was aspirated and the tubes were removed from the MBS

Materials and Methods

device. Washing solution (100 μL) was added and carefully

Animals

mixed with the magnetic beads. The tube was placed back

Plasma samples from 50 cats with stage 1 CKD and 10

in the MBS device and moved back and forth sequentiallycats with stage 2 CKD brought to Maeda Veterinary Hospital between January 6, 2017 and February 20, 2018 (Ameri-

between adjacent wells on each side of the magnetic bar of

can Shorthair: 5, Chinchilla: 2, Mix: 53; 29 males and 31
females; age 2‒13 years) were used in the study. Plasma

supernatant was aspirated. This washing procedure was

samples from 90 cats aged 2‒13 years (Scottish Fold: 4,

bound molecules were eluted by incubation with 10 μL
MB-SSA elution solution for 1 min, before the eluate was

the device. After ﬁxation of the magnetic beads for 30 s, therepeated three times. After the ﬁnal washing step, theAmerican Shorthair: 2, Mix: 84; 43 males and 47 females)
were used as normal control cats. Cats clinically diagnosed
as normal based on serum biochemistry and ultrasonogra-

collected using the MBS device.

phy tests were deﬁned as normal cats, and cats showing

Tandem mass tag (TMT) labelling

clinical signs of CKD in blood chemistry were deﬁned as

Reduction and alkylation were performed as describedthose with stage 1 CKD. Criteria for normal urinalysis wereurine speciﬁc gravity (USG) ＞1.030, urinary protein to cre-previously . A TMT sixplex Isobaric Label Reagent Set(Thermo Scientiﬁc, Rockford, IL, USA) was used accord-

atinine ratio (UPC) ≤ 0.2, and a negative bacteriologic urine

ing to the manufacturer's instructions. Tubes containing the7)

5)culture . Diagnosis of CKD was made prior to inclusion in

diﬀerent isobaric chemical tags (0.8 mg each) were added

the study, based on clinical and laboratory ﬁndings. After5)
stabilization, cats were classiﬁed into 2 groups with p-Cre

to 41 μL of anhydrous acetonitrile and dissolved for 5 minwith occasional vortexing at room temperature. TMT solu-of ＜1.6 and 1.6‒2.8 mg/dL in stages 1 and 2, based on thetion (20 μL) was added to each tube and allowed to react at

IRIS guidelines. Diagnosis of stage 1 CKD was made if
p-Cre was ＜1.6 mg/dL, urine concentrating ability had

room temperature for 60 min. A volume of 4 μL of 5%

been lost, other disorders related to decreased urine concen-

was incubated for 15 min to quench the reaction . Samples
were ﬁnally pooled and lyophilized. Analyses were per-hydroxylamine was added to each sample and the mixture
7)

trating ability were excluded, and ultrasonographic changes
or absent renal corticomedullary diﬀerentiation) . Samples

formed for samples of 20 μL of glycoprotein-isolated
plasma from normal control cats (n＝3) (TMT6-126, 127

were centrifuged at 1,710×g for 10 min (Model 6200;

and 128) and stage 1 CKD cats (n＝3) (TMT6-129, 130 and

Kubota, Tokyo, Japan) and then stored at −80°
C for further

131). Plasma samples were randomly drawn.

were consistent with CKD (e.g., small kidney size and poor
6)use. The ethics committee for Maeda Veterinary Hospital
approved the protocol (2016-02). All owners gave signed

Protein identiﬁcation and quantiﬁcation by LC-MS/
MS analysis

informed consent to participation of their animal in the

Trypsin-digested peptides were injected into a trap col-study.
28

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

umn (C18, 5 μm, 0.3×5 mm) (IonPac; Thermo Scientiﬁc,

IL, USA) at 0.5 mg/well and incubated for 1 day at 4°C.

Rockford, IL, USA) and an analytical column (C18, 3 μm,

The plate was washed three times with PBS containing
0.05% Tween-20 (PBST). The microtiter plate was coated0.075×120 mm) (C18 packed emitter column；Nikkyo
Technos, Tokyo, Japan) attached to an HPLC system (Ulti-

with 20% NOF102 containing 10% sucrose for 1 day at 4°C.

mate 3000; Thermo Scientiﬁc). The ﬂow rate of the mobile

ELISA conditions

phase was 300 nL/min. The solvent composition of themobile phase was programmed to change in 120 min
cycles, with varying mixing ratios of solvent A (2% aceto-

After washing the microtiter plate with PBST, 100-μL aliquots of 10-times diluted plasma samples were added innitrile and 0.1% formic acid) to solvent B (90% acetonitrile

duplicate to wells. The plates were incubated at room temperature for 1 h and then washed three times. HRP-conju-

7)

and 0.1% formic acid), as described previously . Puriﬁed
peptides from HPLC were moved to a hybrid ion-trap Fou-gated SSA lectin (Vector Laboratories, Burlingame, CA,rier transform mass spectrometer (LTQ-Orbitrap XL;
Thermo Scientiﬁc) and analyzed using previously estab-USA) in PBST (100 μL) was added to each well and thelished parameters . A database search engine (Proteome

plate was incubated at room temperature for 30 min. The
plate was washed three times and then 100 μL of TMB solu-

Discoverer; Thermo Scientiﬁc) was used to identify andtion (FUJIFILM, Osaka, Japan) was added. After incuba-quantify proteins from the mass, tandem mass and reportertion at room temperature for 10 min, 100 μL of stop solution

ion spectra of peptides. Peptide mass data were matched by

was added and the absorbance at 450 nm was measured.

searching the UniProt database (https://www.uniprot.org/
proteomes/?query＝*) using the search parameters: peptide

Other procedures

8)

Creatinine was measured enzymatically with creatinine
deiminase using a Fuji DRI-CHEM Slide CRE-PIII kit

mass tolerance＝2 ppm and fragment tolerance＝0.6 Da, withthe enzyme parameter set to trypsin, allowing up to one
missed cleavage variable modiﬁcation, methionine oxidation, and cysteine alkylation. The minimum criteria for pro-

(FUJIFILM, Osaka, Japan). Blood urea nitrogen (BUN)
was measured using a N-Assay BUN-L Nittobo D-Type kit

tein identiﬁcation were ﬁltered with Xcorr vs. charge state(Nittobo Medical, Tokyo, Japan). Plasma SDMA was mea-

and a false discovery rate (FDR) ＜1%.

sured using a Cat SDMA ELISA Kit (MyBioSource, San
Diego, CA, USA). Serum levels of vitamin K-dependent

ELISA
Immunization and establishment of hybridoma celllines

protein C was determined by enzyme-linked immunosorbent assays according to the manufacturerʼs instructions(ELISA Kits for vitamin K-dependent protein C, LifeSpan

Synthetic peptides of 20 amino acids (vitamin K-depen-

Biosciences. Seattle, WA, USA).

dent protein C N peptide; FUJIFILM, Osaka, Japan) wereused for immunization of BALB/c mice. Hybridoma cell
9)line vitamin K-dependent protein C N-01 was established

Statistical analysis

and antibody isotypes were determined using a Mousetistics 19 (SPSS, Chicago, IL, USA). Numerical data arepresented as the mean±standard deviation (SD). Diﬀer-

Statistical analysis was performed using IBM SPSS Sta-

Monoclonal Antibody Isotyping Test Kit (AbD Serotec,

ences in non-parametric data between two groups were analyzed by Mann-Whitney U-test, three groups were analyzed

Kidlington, United Kingdom). To obtain pure monoclonal
antibodies on a large scale, BALB/c mice were initiallystimulated with 1.0 mL pristine (Sigma-Aldrich, St. Louis,

by Steel-Dwas test, with P＜0.05 considered signiﬁcant.
Receiver operating characteristic (ROC) curves were con-

MO, USA) and then inoculated 2 weeks later. Monoclonal
9)

antibodies were puriﬁed as described elsewhere .

structed to assess the sensitivity, speciﬁcity, and areas under
the ROC curves (AUCs).

Immobilization of antibodies on a polystyrene microtiter
plate

Results

The anti-vitamin K-dependent protein C N peptide antibody dissolved in PBS buﬀer was dispensed into a 96-well

Identiﬁcation of candidate proteins as markers of
CKD stage 1
Clinical characteristics and biochemical variables of thepolystyrene microtiter plate (Thermo Scientiﬁc, Rockford,29June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Establishment of an ELISA for sialylated vitamin
K-dependent protein C
Range, dilution analysis and detection limit

normal control cats and CKD groups are shown in Table 1.
To identify new plasma biomarkers for CKD, six plasma
samples (three normal control cat and three CKD stage 1)

A standard curve was drawn based on the colorimetricintensity of diluted recombinant cat vitamin K-dependent

were alkylated with iodoacetamide, digested with trypsin,
diﬀerentially labelled with TMTs, pooled and subjected toanalysis using LC-MS/MS (Fig. 1). The three normal con-protein C (Cusabio Technology) to establish the relationship of intensity with the sialylated vitamin K-dependent

trol cat samples were labelled with TMTs with reporter ionsat m/z＝126, 127 and 128, and the three CKD stage 1 samples were labelled with TMTs with reporter-ions at m/z＝protein C concentration (Fig. 2). The assay had a working
range of 0‒10 AU/mL and gave linear results from 0 to

129, 130 and 131. After comparing the proﬁles of proteins
by LC-MS/MS using an LTQ Orbitrap XL mass spectrome-

10 AU/mL (y＝0.021×−0.0018, r ＝0.9993, p＜0.0001). The
detection limit was estimated by assaying the zero concen-ter, 18 sialylated glycoproteins with unique peptide

tration eight times, and was deﬁned as the sialylated vitamin K-dependent protein C “zero” concentration＋3SD. The

2

sequences were found with higher levels in normal control

limit was found to be 0.18 AU/mL.

cats compared to CKD stage 1 cats (Table 2). Among these
proteins, hemoglobin subunit alpha and vitamin K-depen-

Within-run and between-run reproducibility

dent protein C had plasma levels elevated more than two-

The precision of the assay was determined using two

fold in all three normal control cats compared to the CKD
stage 1 cats. Vitamin K-dependent protein C was selectedsialylated vitamin K-dependent protein C concentrations of
0.5 and 2.0 AU/mL. Within-assay CVs were determined

for further analysis.

with eight replicates of each sample. Between-assay CVs
Table 1.

Clinical characteristics and biochemical variables of normal control cats and CKD cats
Variables

Normal control cats

CKD groupStage 1

Stage 2

p-ValueAge (Mean±s.d.)6.8±3.6

6.7±3.3

6.8±3.6

0.825

Sex (male, female)

90 (43, 47)

50 (24, 26)

10 (5, 5)

0.793

Blood urea nitrogen (mg/dL) (Mean±s.d.)p-Creatinine (mg/dL) (Mean±s.d.)24.1±5.7

25.6±6.8

26.8±5.90.714

0.96±0.23

1.20±0.23

1.99±0.24

0.015

Symmetric dimethylarginine (mg/dL) (Mean±s.d.)5.9±0.8

6.4±1.0

10.9±1.6

0.009

Urine speciﬁc gravity (Mean±s.d.)1.053±0.006

1.051±0.005

1.045±0.016

0.618

Urinary protein to creatinine ratio (Mean±s.d.)0.13±0.04

0.15±0.05

0.17±0.06

0.024

Fig. 1.

Identiﬁcation of candidate proteins using TMT labelling and LC-MS/MS analyses.
30June 2024

Table 2.

Medical Mass Spectrometry Vol. 8 No. 1

Plasma sialylated glycoproteins detected at higher levels in normal control cats compared to CKD stage 1 cats inproteome analysis

Database
Accession
No.

ID

Coverage(%)

TMT

Number ofmatching
peptidesNormal control cats

Stage 1 CKD cats

126

127

128

Average

129

130

131

Average

0.268

0.233

0.226

0.242

0.206

0.134

0.152

0.164
0.161

M3WPG6Alpha-2-macroglobulin
Apolipoprotein A-I

12.41

4

0.298

0.240

0.167

0.235

0.236

0.108

0.139

M3WG78ATP synthase subunit beta

8.00

3

0.212

0.158

0.133

0.168

0.277

0.106

0.113

0.166

4.69

6

0.260

0.134

0.307

0.234

0.180

0.164

0.133

0.159

2.86

2

0.357

0.164

0.154

0.225

0.282

0.122

0.121

0.175

M3W3E7M3W7M4 Attractin
M3WB05

Bifunctional purine biosynthesis protein ATIC

3.46

6

M3VZY7

Ceruloplasmin

5.63

4

0.230

0.142

0.268

0.213

0.230

0.136

0.142

0.169

M3WKP2Clusterin

16.63

13

0.391

0.132

0.263

0.262

0.140

0.157

0.140

0.146

M3VU42Complement factor I

2.31

2

0.394

0.162

0.227

0.261

0.106

0.263

0.219

0.196

P14450Fibrinogen alpha chain(Fragment)

100

1

0.191

0.191

0.245

0.209

0.198

0.160

0.175

0.178

P14469Fibrinogen beta chain

5.43

3

0.217

0.237

0.251

0.235

0.270

0.214

0.156

0.213

M3WB06

Fibronectin

4.24

8

0.194

0.155

0.270

0.207

0.297

0.181

0.131

0.203

P07405

Hemoglobin subunit alphaPhosphatidylinositol-glycanspeciﬁc phospholipase D

18.44

2

0.480

0.336

0.198

0.338

0.167

0.130

0.133

0.143

3.93

3

0.487

0.114

0.220

0.274

0.122

0.130

0.224

0.159

P49064

Serum albumin

31.58

19

0.251

0.177

0.130

0.186

0.278

0.131

0.128

0.179

M3WEV9Transthyretin
UDP-glucose 4-epimerase

8.84

2

0.169

0.121

0.188

0.159

0.118

0.189

0.116

0.141

2.66

2

0.152

0.208

0.217

0.192

0.251

0.174

0.099

0.174

Q28412

Vitamin K-dependent protein
C

14.21

3

0.280

0.291

0.289

0.287

0.132

0.140

0.137

0.136

M3W9I5

Vitronectin

5.47

2

0.337

0.343

0.388

0.356

0.236

0.197

0.186

0.206

M3WEG4

M3WFT7

Interference
Interference was assessed in samples containing 1.0 AU/
mL sialylated vitamin K-dependent protein C. Potential interference materials were added to urine at various concentrations. There was no substantial interference from hemoglobin(up to 5000 mg/L), free bilirubin (up to 207 mg/L), ditaurobilirubin (up to 204 mg/L), chyle (up to 1400 formazine turbidity units, equal to 1176 mg/L triglyceride), ascorbic acid (upto 500 mg/L), and rheumatoid factor (up to 500 U/L).

Fig. 2.

Recovery test

Standard curves for plasma sialylated vitamin
K-dependent protein C in the ELISA.

To evaluate recovery in the ELISA, 0.5 and 2.0 AU/mLof recombinant Cat vitamin K-dependent protein C were

Colorimetric intensity and plasma vitamin K-dependent
protein C concentration were related in the range of0‒10 AU/mL. Four concentrations of plasma sialylated
vitamin K-dependent protein C were determined by
ELISA.

added to pooled plasma (1.6 AU/mL). The percentage
recovery ranged from 94.3 to 102.7%.

Sialylated vitamin K-dependent protein C levels are
decreased in plasma of cats with CKD

were determined based on assays performed on 5 diﬀerentdays (two replicates of each sample per day). The within-run

Plasma vitamin K-dependent protein C levels andsialylated vitamin K-dependent protein C levels were mea-

CV was 4.3‒4.9% and the between-run CV was 4.3‒5.2%.
31

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Medical Mass Spectrometry Vol. 8 No. 1

sured in stage 1 CKD cats (n＝50), stage 2 CKD cats (n＝mL and an AUC of 0.989, with sensitivity of 97.5% and10) and normal control cats (n＝90). Plasma vitamin K-de-

speciﬁcity of 97.5%.

pendent protein C values for normal control cats, stage 1

Discussion

CKD cats and stage 2 CKD cats were 3.36±0.32 μg/mL,

In this study, the lectin and TMT method was used to

3.46±0.31 μg/mL and 3.53±0.30 μg/mL, respectively (Fig.
3). Plasma sialylated vitamin K-dependent protein C was

detect 18 proteins in plasma from CKD stage 1 cats andnormal control cats. Sialylated vitamin K-dependent protein

signiﬁcantly higher in normal control cats than in stage 1

C was identiﬁed as the protein with the largest diﬀerence inlevels between the two groups. The vitamin K-dependent

CKD cats (1.40±0.13 vs. 1.07±0.11 AU/mL, p＜0.001),
and in stage 1 CKD cats compared to stage 2 CKD catsprotein C is synthesized in the liver in a single-chain form,(1.07±0.11 vs. 0.82±0.14 AU/mL, p＜0.001) (Fig. 4). ROC
curves were constructed to evaluate the cut-oﬀs for plasma
sialylated vitamin K-dependent protein C, plasma creati-

and through several proteolytic steps is secreted into blood
primarily as a two-chain form, containing a light chain andnine, BUN, and SDMA for distinguishing stage 1 CKD catsa heavy chain linked via a single disulﬁde bond . Sialic

from normal control cats. The AUCs were 0.989 for plasma
sialylated vitamin K-dependent protein C, 0.698 for plasma

acids are derivatives of the negatively charged acidic sugar

creatinine, 0.554 for BUN, and 0.568 for SDMA. These
results suggest that plasma sialylated vitamin K-dependent

the sialic acid family have been discovered to date ;N-acetylneuraminic acid, N-glycolylneuraminic acid, andprotein C may be a better diagnostic stage 1 CKD bio-

deaminoneuraminic acid are representative members of thismarker than plasma creatinine, BUN and SDMA (Fig. 5).
The ROC curve for plasma sialylated vitamin K-dependent

family. Sialic acids usually form the terminal ends of the
carbohydrate groups of glycoconjugates. All sialyltransfer-protein C showed an optimal diagnostic cut-oﬀ of 1.22 AU/

ases characterized in animals to date, ranging from insects

10)

neuraminic acid. Over 40 naturally occurring members of
11)

Fig. 4.
Fig. 3.

Plasma vitamin K-dependent protein C levels innormal control cats and stage 1 and stage 2 CKD
cats.

Plasma sialylated vitamin K-dependent protein C
levels in normal control cats and stage 1 and stage
2 CKD cats.

Plasma sialylated vitamin K-dependent protein C wassigniﬁcantly higher in normal control cats than in stage 1
CKD cats (1.40±0.13 vs. 1.07±0.11 AU/mL, p＜0.001;
Mann‒Whitney U-test) and in stage 1 CKD cats compared
to stage 2 CKD cats (1.07±0.11 vs. 0.82±0.14 AU/mL,
p＜0.001).

Plasma vitamin K-dependent protein C values for normal
control cats, stage 1 CKD cats and stage 2 CKD catswere 3.36±0.32 μg/mL, 3.46±0.31 μg/mL and 3.53±
0.30 μg/mL, respectively.

32

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Fig. 5.

Medical Mass Spectrometry Vol. 8 No. 1

Plasma sialylated vitamin K-dependent protein C is more eﬀective than plasma creatinine, BUN and SDMA for early
detection of stage 1 CKD.
ROC analyses were performed for serum levels of plasma sialylated vitamin K-dependent protein C, plasma creatinine, BUN and
SDMA in stage 1 CKD and normal cats. The AUCs were 0.989 for plasma sialylated vitamin K-dependent protein C, 0.698 forplasma creatinine, 0.554 for BUN, and 0.568 for SDMA.

with CKD have signiﬁcantly higher FVII, FVIII, ﬁbrinogen18)
and D-dimer levels and a signiﬁcantly lower level of pro-to mammals, feature a type II transmembrane topology andusually localize to the Golgi apparatus. The enzyme isgrouped into 4 families according to the type of carbohy-tein C. However, it has not been reported in the sialylated
vitamin K-dependent protein C. Similar results were obtained

drate linkage they synthesize: β -galactoside α 2,3-sialyltransferases (ST3Gal-I-VI), β -galactoside α 2,6-sialyltrans-in this study, with plasma sialylated vitamin K-dependent

ferases (ST6Gal-I and -II), GalNAc α 2,6-sialyltransferases(ST6GalNAc-I‒VI), and α 2,8-sialyltransferases (ST8Sia-I‒

protein C found to be signiﬁcantly higher in normal control
cats (1.40±0.13 AU/mL) than in stage 1 (1.07±0.11 AU/mL)

12)VI). Sialic acid was added by these enzymes . During
coagulation, plasma vitamin K-dependent protein C is acti-

and stage 2 (0.82±0.14 AU/mL) CKD cats.

presence of protein S, activated protein C (APC) functions

In this study, we showed that plasma sialylated vitamin
K dependent protein C is a potential biomarker for CKD. Various attempts have been made to identify new plasma biomark-as an anticoagulant by proteolytic degradation of factors Va

ers for CKD, and some molecular targets have been suggested

11)

vated by a thrombin/thrombomodulin complex , and in the

12)

5)

and VIIIa , the only protein substrates hitherto identiﬁed
for vitamin K-dependent protein C. Several animal models,

as diagnostic or prognostic markers . The AUC for plasma
sialylated vitamin K-dependent protein C was 0.989, with sen-including canine, bovine and feline vitamin K-dependent

sitivity of 97.5% and speciﬁcity of 97.5%. These results suggest that plasma sialylated vitamin K-dependent protein C may

protein C, have been used to examine the physiologicalmechanisms of vitamin K-dependent protein C, and spe-be complementary to plasma creatinine, BUN and SDMA as acies-speciﬁc eﬀects have been found for the anticoagulant

marker for detection of stage 1 CKD in cats. We note that the

13,14). Thus, in squirrel

study is limited by the number of samples, and evaluation ofmonkeys, human APC has an anticoagulant eﬀect, but nomore samples from multiple facilities is required. Longitudinalfollow-up of cats in stage 1 CKD is also important to identify

and ﬁbrinolytic functions of APC
15)

proﬁbrinolytic activity , whereas human APC shows boththose with true CKD, which is progressive, and those recover-16)

anticoagulant and ﬁbrinolytic responses in cats .
A decrease in vitamin K-dependent protein C has been

ing from AKI when samples were taken.

reported in renal disease in humans, but not in cats. Patients

Acknowledgmentswith CKD commonly have blood coagulation disorders, andseveral tests can be used to measure procoagulant biomark-This work was supported by a Grant-in-Aid for Scientiﬁc

17)

ers in CKD . Compared to normal control cats, patients

Research (KAKENHI C: 22K07406).
33

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Conﬂict of Interest

et al: Development of a novel fragments absorbed immunocapture enzyme assay system for tartrate-resistant acid

The authors declared no potential conﬂicts of interest

phosphatase 5b. Clin Chim Acta 376: 202‒212, 2007.

with respect to the research, authorship, and/or publicationof this article.

11) Varki A: Diversity in the sialic acids. Glycobiology 2: 25‒

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