Quantitative assessment of cyanide in cystic fibrosis sputum and its oxidative catabolism by hypochlorous acid

Article abstract of DOI:10.1016/j.freeradbiomed.2018.09.007

Rationale: Cystic fibrosis (CF) patients are known to produce cyanide (CN^-) although challenges exist in determinations of total levels, the precise bioactive levels, and specificity of its production by CF microflora, especially P. aeruginosa. Our objective was to measure total CN^- levels in CF sputa by a simple and novel technique in P. aeruginosa positive and negative adult patients, to review respiratory tract (RT) mechanisms for the production and degradation of CN^-, and to interrogate sputa for post-translational protein modification by CN^- metabolites. Methods: Sputa CN^- concentrations were determined by using a commercially available CN^- electrode, measuring levels before and after addition of cobinamide, a compound with extremely high affinity for CN^-. Detection of protein carbamoylation was measured by Western blot. Measurements and main results: The commercial CN^- electrode was found to overestimate CN^- levels in CF sputum in a highly variable manner; cobinamide addition rectified this analytical issue. Although P. aeruginosa positive patients tended to have higher total CN^- values, no significant differences in CN^- levels were found between positive and negative sputa. The inflammatory oxidant hypochlorous acid (HOCl) was shown to rapidly decompose CN^-, forming cyanogen chloride (CNCl) and the carbamoylating species cyanate (NCO^-). Carbamoylated proteins were found in CF sputa, analogous to reported findings in asthma. Conclusions: Our studies indicate that CN^- is a transient species in the inflamed CF airway due to multiple biosynthetic and metabolic processes. Stable metabolites of CN^-, such as cyanate, or carbamoylated proteins, may be suitable biomarkers of overall CN^- production in CF airways.

Full text of DOI:10.1016/j.freeradbiomed.2018.09.007

Author’s Accepted Manuscript
Quantitative Assessment of Cyanide in Cystic
Fibrosis Sputum and its Oxidative Catabolism by
Hypochlorous Acid
Jason P. Eiserich, Sean P. Ott, Tamara Kadir, Brian
M. Morrissey, Keri A. Hayakawa, Michele A. La
Merrill, Carroll E. Cross
www.elsevier.com
PII:
S0891-5849(18)31406-0
https://doi.org/10.1016/j.freeradbiomed.2018.09.007
FRB13909
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Free Radical Biology and Medicine
Cite this article as: Jason P. Eiserich, Sean P. Ott, Tamara Kadir, Brian M.
Morrissey, Keri A. Hayakawa, Michele A. La Merrill and Carroll E. Cross,
Quantitative Assessment of Cyanide in Cystic Fibrosis Sputum and its Oxidative
Catabolism by Hypochlorous Acid, Free Radical Biology and Medicine,
https://doi.org/10.1016/j.freeradbiomed.2018.09.007
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Free Rad Bio Med
Submitted 08/15/2018
Quantitative Assessment of Cyanide in Cystic Fibrosis Sputum and its Oxidative
Catabolism by Hypochlorous Acid
Jason P. Eisericha,b,1, Sean P. Otta, Tamara Kadira, Brian M. Morrisseya, Keri A. Hayakawaa,
Michele A. La Merrillc and Carroll E. Crossa,b,*
aDepartment of Internal Medicine, Division of Pulmonary/Critical Care and Sleep Medicine,
University of California, Davis, CA 95616
bDepartment of Physiology and Membrane Biology, University of California, Davis, CA 95616
cDepartment of Environmental Toxicology, University of California, Davis, CA 95616
*Corresponding Author: Carroll E. Cross, M.D. Phone: +1-916-734-3564, Fax: +1-916-734-
7924, cecross@ucdavis.edu
ABSTRACT
Rationale:
Cystic fibrosis (CF) patients are known to produce cyanide (CN-) although challenges exist in
determinations of total levels, the precise bioactive levels, and specificity of its production by CF
microflora, especially P. aeruginosa. Our objective was to measure total CN- levels in CF sputa
by a simple and novel technique in P. aeruginosa positive and negative adult patients, to review
respiratory tract (RT) mechanisms for the production and degradation of CN-, and to interrogate
sputa for post-transcriptal protein modification by CN- metabolites.
Methods:
1 Current affiliation: Senior Environmental Scientist (Supervisory), California Environmental Protection
Agency, Department of Pesticide Regulation, Sacramento, CA, 98517.
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Sputa CN- concentrations were determined by using a commercially available CN-
electrode, measuring levels before and after addition of cobinamide, a compound with extremely
high affinity for CN-. Detection of protein carbamoylation was measured by Western blot.
Measurements and Main Results:
The commercial CN- electrode was found to overestimate CN- levels in CF sputum in a highly
variable manner; cobinamide addition rectified this analytical issue. Although P. aeruginosa
positive patients tended to have higher total CN- values, no significant differences in CN- levels
were found between positive and negative sputa. The inflammatory oxidant hypochlorous acid
(HOCl) was shown to rapidly decompose CN-, forming cyanogen chloride (CNCl) and the
carbamoylating species cyanate (NCO-). Carbamoylated proteins were found in CF sputa,
analogous to reported findings in asthma.
Conclusions:
Our studies indicate that CN- is a transient species in the inflamed CF airway due to multiple
biosynthetic and metabolic processes. Stable metabolites of CN-, such as cyanate, or
carbamoylated proteins, may be suitable biomarkers of overall CN- production in CF airways.
PICTORIAL ABSTRACT
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Key words:
cystic fibrosis, cyanide, ion-specific electrode, cyanogen chloride, cyanate, P. aeruginosa,
neutrophils, myeloperoxidase, hypochlorous acid, carbamylated protein
Abbreviations:
Cyanide (CN-), Cystic Fibrosis (CF), carbamoylating species cyanate (NCO-), hypochlorous acid
(HOCl), cyanogen chloride (CNCl), respiratory tract (RT), Pseudomonas aeruginosa (P.
aeruginosa)
Introduction
Pseudomonas aeruginosa (P. aeruginosa) is the most persistent respiratory tract (RT)
pathogen, eventually dominating cultures obtained from over 80% of adult Cystic Fibrosis (CF)
patients [1]. P. aeruginosa releases numerous metabolites and virulence factors that likely
contribute to RT deterioration in the lungs of CF patients [2]. These species include hydrogen
cyanide/cyanide (CN-), which binds with high affinity to multiple metalloproteins (i.e. iron and
copper) to varying degrees and particularly inhibits cytochrome c oxidase in mammalian cell
mitochondria [3]. Of particular interest, Bacillus pyocyaneus (later determined to be what is
today regarded as P. aeruginosa), was found to produce CN- as early as 1913 [4]. The heavily
infected inflammatory hypoxic conditions of the CF RT represent a fertile milieu for cyanogenic
pathogens. CN- is presumably produced to gain a survival advantage against the competing
growth of CN--sensitive microorganisms and/or possibly aggravating metabolic conditions of the
host RT) [2, 5, 6]. Biosynthesis of CN- is carried out by cyanogenic bacteria using an enzyme
known as hydrogen cyanide synthase, using the amino acid glycine, abundant in CF sputa
(range 0.27 to 1.73mM) as a substrate [2, 7]. The reaction proceeds as shown in Scheme 1,
with an unstable imino acetic acid intermediate, with ultimately the stoichiometric liberation of
HCN and CO2.
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Scheme 1: Enzymatic production of hydrogen cyanide (HCN) by the Pseudomonas
HCN synthase enzyme system
CN- levels of up to 200 µM have been reported in the sputa of CF patients, whereas
much lower to nil amounts have been measured in non-CF patients with bronchiectasis and in
CF patients not culturing P. aeruginosa [2, 6-10]. While the commercial CN- electrode provides
a convenient method for quantifying CN-, the complex nature of CF sputum presents significant
challenges including non-specificity. Moreover, sputa also have pigments and other substances
that interfere with spectrophotometric determination of CN- complexed with cobinamide, a
compound with very high affinity for CN-. Cobinamide itself has been used as a method for
measuring cyanide and as a therapeutic agent for CN- poisoning [11, 12].
In the present study CN- concentration was quantified in the sputa of adult CF patients
infected with P. aeruginosa and/or non - P. aeruginosa bacterial strains using a combination of
an ion-specific electrode system and cobinamide to correct for non-specific background
interference. The refined method is simple, convenient and accurate and would be particularly
useful in the clinical setting for routine analyses.
Methods
Subjects:
Twelve adult CF-outpatients attending the UC Davis Adult CF Center clinic were recruited for
this study. The study was approved by the Office of Human Research/Institutional Review
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Board (IRB) of the University of California, Davis. All subjects provided informed consent. All
subjects recruited were confirmed CF patients based on the published diagnosis criteria given
by the CF Foundation. Subjects were asked to void their mouths of saliva prior to producing a
spontaneous sputum sample. Sputum samples were expectorated into sterile containers and
immediately frozen on dry ice and subsequently stored at -80°C for analyses.
Sputum processing:
The -80°C frozen sputum samples were processed within two weeks of collection. The samples
were homogenized with ice cold PBS (10 mM phosphate, pH 7.4, containing 154 mM NaCl; 1:1
w/v) using a dounce homogenizer. Homogenized samples were then incubated with DNase
(0.2 mg/mL) for 10 min at room temperature and made alkaline with NaOH to prevent escape of
HCN by volatilization.
Cobinamide synthesis:
Hydroxoaquocobinamide (cobinamide) was synthesized using a modified method by Armitage
et al. [13]. Briefly, 20 mg hydroxocobalamin was incubated with 150 µL concentrated HCl for 8
min at 65 °C. The reaction was stopped by placing the mixture on ice. The pH was increased
to 5 by the slow addition of 6 M NaOH. Cobinamide was isolated using a PrepSep C18 solid
phase extraction column. The column was rinsed with double deionized water to remove
excess salt and then 10% acetone to remove unreacted hydroxocobalamin. Cobinamide was
eluted from the column with 20% acetone. The cobinamide solution was then concentrated
under N2 gas. Cobinamide was quantified by converting it to diaquocobinamide with 0.1 M HCl
and absorbance read with a spectrophotometer at 348 nm. Diaquocobinamide has an
extinction coefficient of ε348 = 2.8 x 104 M-1 · cm-1[14]. Purity of cobinamide synthesis was
assessed by thin layer chromatography. Reverse-phase silica plates were developed in
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isopropanol:ammonium hydroxide:H2O (7:1:2) and 2 mM CN-. Purity of cobinamide product was
also assessed by converting it to dihydroxocobinamide in 0.1 M NaOH and reading the
absorbance at 344 nm and 356 nm. If the ratio of A344/A356 is between 1.05 and 1.11 the
cobinamide sample is considered pure [15].
Cyanide measurement in sputum:
CN- concentrations were measured with a micro-cyanide ion-selective electrode (ISE) (Lazar
Research Laboratories, Los Angeles, CA). All data were collected using a PowerLab/16SP
recording unit and PowerLab Chart v3.6.5 data acquisition software package (ADInstruments
Inc., Colorado Springs, CO). Sputum samples were brought to a concentration of 0.1 M NaOH
by a 1:2 dilution in a 96-well plate to trap CN- in its non-volatile ionic form (CN-) and to bring the
sample within optimal ISE working conditions. To determine non-specific electrode responses
cobinamide (100 µM final concentration) was added to scavenge any CN- in the sputum sample.
The mixture was incubated 5 min at room temperature and re-measured with the electrode. A
second addition of the cobinamide solution was added to confirm that no additional signal
change occurred. The resultant non-specific response was then subtracted from the sample’s
original measurement to obtain the corrected CN- concentration in the sputum sample. A linear
standard CN- curve (0 – 250 µM) was made utilizing this method of cobinamide addition. The
lower limit of CN- detection of the ISE with cobinamide background correction was 2.5 µM.
Intra-assay variabilities were determined by making HCN/CN- measurements on 3
aliquots of the same sample. Inter-assay varabilities were determined by HCN/ CN-
measurements on aliquots of the same sample on 3 different days. Several samples were
spiked with known concentrations of HCN/ CN- to estimate recoveries of HCN/ CN- when added
to sputa. To validate and assess the precision and accuracy of our method, we determined the
intra-assay and inter-assay variability. This new method has an intra-assay variability of 7.2%,
and an inter-assay variability is 10.2%. To further define recovery yields of CN- by this method
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in sputum, we spiked samples with known concentrations of CN- and achieved a recovery of
109.5% 6.8%.
Reactions of CN- with HOCl:
CN- (100mM) was reacted with various concentrations of HOCl (0-200mM) in phosphate buffer
(50 mM), pH 7.4) at room temperature using a vortex mixer. After 15 min of incubation, an
aliquot of the reaction mixture was made alkaline for determination of CN- concentration using
the CN- electrode (described above), and a second aliquot was used for analysis of CNCl as
described below. Stock solutions of both HOCl (pH 12) and CN- (pH 7.4) were freshly prepared
prior to each experiment. The concentration of HOCl (as the deprotonated form, OCl-) was
determined spectrophotometrically at 290 nm using an extinction coefficient of 362 M-1cm-1.
Analysis of cyanogen chloride (CNCl):
CNCl was detected using the pyridine-1,3-dimethyl barbituric acid reagent as previously
described [16,17]. CNCl reacts with pyridine to form glutacon dialdehyde, that then reacts with
1,3-dimethyl barbituric acid and condenses to form a violet-colored polymethine complex.
Coloring reagent (100 µl) was added to the HOCl/CN- reaction mixture (100 µl) and was
incubated for 15 min at room temperature. The absorbance of the resulting violet-colored
solution was measured at 587 nm. The concentration of CNCl was calculated using the
extinction coefficient of 1.03 x 105 M-1cm-1.
Western blot analyses for carbamoylated proteins.
Aliquots of sputum samples containing 20 µg of total protein were separated by SDS-PAGE on
12% gels under reducing conditions. Bovine serum albumin (BSA, 10 mg/ml) was reacted with
sodium cyanate (NCO-, 1 mM) for 12 hrs at room temperature in PBS (pH 7.4) to serve as a
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positive control for carbamoylated proteins. Native BSA or carbamoylated BSA (20 µg) was
separated by SDS-PAGE as noted above. Proteins were transferred to nitrocellulose
membranes and incubated overnight in PBS containing 0.05% Tween-20 and 5% non-fat dry
milk. Membranes were incubated with anti-carbamyllysine (CBL) antibody (rabbit polyclonal
antibody against carbamoylated Keyhole Limpet hemocyanin; Academy Biomedical Company,
Inc.; Catalog #CBL30S-R1a) at a dilution of 1:5,000. A horseradish peroxidase-conjugated goat
anti-rabbit IgG was used as the secondary antibody (diluted 1:3,000), followed by detection
using enhanced chemiluminescence.
Statistical analyses.
Data were analyzed in general linear models (PROC GLM, SAS version 9.4). Sputum cyanide
was modeled as a function of Pseudomonas status, with lsmeans statement to generate least
square means, standard error, and p-values for their differences. Lung function (FEV1) was
modeled as a function of either apparent or corrected cyanide concentration. Data are
presented using Graphpad Prism.
Results
Demographics:
Demographic data of the 12 subjects are summarized in Table 1. All sputum samples collected
cultured positive for common CF microbes. Eight subjects cultured one or more strains of P.
aeruginosa.
Effect of cobinamide on the detection of CN-:
The CN- electrode is known to respond non-specifically in RT secretions [8,9]. We first sought to
use a simple model buffer system to establish whether cobinamide can be used to bind CN- and
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prevent its detection from the CN- electrode. The structure of cobinamide is shown in Fig 1A,
and illustrates the cobolt atom in the center of the molecule. Cobinamide is capable of forming
complexes with two molecules of CN- sequentially with very high affinity, as illustrated in Fig 1C
[18]. As shown in Fig 1B, a linear standard curve is obtained with increasing amounts of CN- (5
- 80 µM in 0.1 M NaOH) in the absence of cobinamide. With the addition of cobinamide
(25mM), the electrode does not detect CN- until concentrations exceed 50 µM. This confirms
that cobinamide can bind 2 CN- ions and the cobinamide – CN- complex does not produce a
reading from the electrode. When the stoichiometry exceeds 2:1, a linear electrode response is
observed (Fig 1B).
Measurement of CN- in sputum:
When measuring the sputum samples without the addition of cobinamide, we observed that
there was an average CN- concentration of 104.4 10.4 µM (Mean SEM) (Table 2). To
correct for non-specific interference of sputum components with the electrode, we repeated the
measurements for each sputum sample in the presence of cobinamide. The electrode signal
that was observed in the presence of cobinamide was considered non-specific detection due to
inference. This value (‘non-specific background’) was then subtracted from the initially obtained
measurement (‘apparent’) to provide a ‘corrected’ concentration value of CN-. When using this
approach, the average CN- concentration was significantly lower at 37.0
6.8 µM (Mean
SEM, p-value ≤ 0.0001, paired t-test), a value nearly 3 times lower than the uncorrected
(apparent) CN- concentration average. The degree of sputum background interference also
varied extremely widely between sputum samples (Table 2 and Fig. 2), indicating that the
degree of non-specific interference is vastly different with sputum taken from individual patients.
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Cyanide concentration in sputum obtained from P. aeruginosa positive and negative
patients:
Sputum from 8 patients cultured positive for P. aeruginosa and 4 subjects cultured negative for
P. aeruginosa, but were positive for other microbes (Table 1). As shown in Fig 3, both P.
aeruginosa positive and P. aeruginosa negative patients displayed significant levels of CN-, both
utilizing uncorrected electrode measurements (Fig 3A), as well as cobinamide corrected CN-
levels (Fig 3B). There were a general trend indicating decreased levels of CN- in the P.
aeruginosa negative sputum samples. Values of CN- in the P. aeruginosa negative patients
were 23.1 11.1 µM whereas P. aeruginosa positive patients were 43.9 7.8 µM (Fig 3B).
Correlation of FEV-1 (% predicted) with sputum CN- concentrations:
In an attempt to determine whether our measured CN- levels could reflect the severity of lung
injury in CF patients, we plotted FEV-1 (% predicted) versus CN- concentrations using both
apparent and corrected measurement methods. As shown in Fig 4, when apparent (A) or
corrected (B) concentrations of CN- in sputa are plotted against FEV-1 (% predicted) it is clear
that there is no statistically significant correlation.
Reaction of HOCl with CN-: As myeloperidase (MPO)-mediated HOCl generation is known to
be high in CF [19] we asked whether HOCl could alter the stability of CN-. As shown in Fig 5,
we found that HOCl reacts with and decreases CN- in a near 1:1 stoichiometry. In parallel with
the loss of CN-, we observed the formation of cyanogen chloride (CNCl). However,
quantitatively, we could only attribute approximately 35-40% of the lost CN- to CNCl. When the
ratio of HOCl:CN- exceeded 1:1 stoichiometry, CNCl was completely consumed. The further
reaction of CNCl with HOCl has been shown to result in the formation of cyanate (NCO-) [20].
These data clearly indicate that in the presence of HOCl, CN- is an unstable species and may
be only transiently present in the highly inflamed CF RT, and thus may not be a biomarker that
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adequately reflects the overall degree of RT bacterial CN- production or lung function (i.e. FEV -
1, Fig 4).
Analysis of carbamoylated proteins in CF sputum:
We next determined whether carbamoylated proteins could be detected in CF sputum. As
shown in Fig 6B, BSA incubated with NCO-, but not unreacted native BSA, showed
immunoreactivity in Western blots against antibodies that recognize carbamoylated proteins.
When sputum proteins from adult CF patients were subjected to Western blot analysis using the
same anti-carbamyl lysine antibodies, immunoreactive bands from 15-200 kD were detected
(Fig 6C). Interestingly, CF sputum from Pseudomonas-positive patients showed more robust
staining for carbamoylated proteins compared to a Pseudomonas-negative patient. Whereas a
comprehensive quantitative assessment of carbamoylated protein in CF patient sputum was
beyond the scope of this investigation, the data provide a proof of concept and illustrate the
potential for carbamoylated proteins to serve as biomarkers of NCO--dependent protein
modification in the airway of CF patients.
Discussion
The present data confirms the findings of many others that RT secretions contains
considerable levels of CN- (14.1-98.1 mM), that a combined CN- electrode/cobinamide
methodology can conveniently be used as a simplified methodology for sputum total CN-
determinations and that the presence of CN- is not specific for the presence of Pseudomonas.
The rapid reactions of the CN- with the myeloperoxidase derived oxidant HOCl indicate that CN-
is not likely to be stable species in the CF airway and is likely to be involved in the production of
NCO-, a compound capable of generating carbaml-lysine, homocitruline). Findings are
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discussed from the perspective of technological advances and challenges, issues related to RT
CN- metabolism and chemical reactions as well as some pathobiological perspectives.
Technological advances and specificity of CN- presence in infected CF patients:
The present methodology makes use of a commercially available cyanide ion-selective
electrode and cobinamide, a substrate with a very high affinity for CN-. Our data demonstrate
that caution should be exercised in assuming that CN- is the only substance detected by the
electrode in the complex matrix of CF sputum. The method presented here utilizes cobinamide
to correct for the non-specificity of the CN- electrode.
Limitations of the currently described method include that the non-CN- species detected by the
electrode remain uncharacterized. Uncertainties also exist regarding the magnitude of the CN-
species remaining sequestered in non-solubilized portions of RT secretions after processing.
Common ions at concentrations found in CF RT secretions (thiocyanate, nitrate and nitrite) were
not detected by the CN- electrode (data not shown). It is also likely that some limited amount of
HCN escapes as a volatile gas prior to alkalization of the collected RT secretions. HCN has a
pKa of 9.3 and is detected in low concentrations in the collected breath of control non-CF
subjects (≈ 5 ppb) [21] and in the breath of CF patients in higher concentrations (≈ 13.5 ppb)
[22].
Is CN- a specific marker of P. aeruginosa?
There are considerable discordant data in the literature related to the specificity of RT CF CN-
levels to reflect the presence of P. aeruginosa. Interestingly, one study reports that exhaled
breath HCN levels in P. aeruginosa positive CF patients (~ 7.95 ppb) and in P. aeruginosa
negative patients (~ 6.95 ppb) are not dramatically different [23]. Recently Staphylococcus
aureus, a prominent CF pathogen, has also been shown to produce CN- in both S. aureus
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cultures and breath from S. aureus infected CF patients, albeit in lower quantities than CF
patients infected with P. aeruginosa [24]. Whereas a previous study [8] concluded that CN- was
detected only in P. aeruginosa positive patients, our present study and those of others [9,24]
reveal that CN- is detected in CF patients who culture either positive or negative for the bacteria
(Fig 3) and thus is not a specific marker for P. aeruginosa.
Mechanisms of airway CN- formation:
There are multiple potential mechanisms (summarized in Fig 7) that may explain why P.
aeruginosa negative patients still have significant quantities of CN- in sputum and exhaled
breath: 1) There are multiple other microorganisms that are cyanogenic and could contribute in
the absence of P. aeruginosa [25]. For instance, in addition to S. aureus, Burkholderia cepacia
appears to be cyanogenic under biofilm colonial growth conditions [26] although there are
controversial data [27]. 2) The microbiome colonizing the CF lung is highly diverse [28] and they
are only partially culturable in routine clinical assays; thus it could be reasonable to speculate
that microbes previously not recognized as being cyanogenic may be participants in
biosynthesis of CN- in the CF airway. 3) There are multiple potential mechanisms for
microorganism-independent formation of CN- in the CF airway. It has been demonstrated that
neutrophil-derived myeloperoxidase (MPO), which is highly abundant in the CF airway of all
adult CF patients (even in P. aeruginosa negative patients) [19], is capable of producing CN-. In
fact, the reaction is analogous to some degree with that of the bacterial HNC synthase. MPO-
derived hypochlorous acid (HOCl) is capable of reacting with glycine to form the N-
dichloroglycine, which spontaneously rearranges to liberate HCN and CO2 [29]; the same
products formed by the HCN synthase enzyme of cyanogenic bacteria.
Analogous reactions have also been demonstrated with the amino acid serine
undergoing reaction with HOCl [17]. Additionally, HOCl and/or N-chloramines can react with
thiocyanate (SCN-) [30, 31] or uric acid [32] to form CN-. Both of these substrates are present in
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high concentrations in CF sputum [33-35] and hence may be additional chemically-mediated
pathways for the synthesis of CN- in the inflamed CF airway. The contribution of MPO/HOCl to
CN- formation in the CF airway remains to be fully characterized and is worthy of further
investigation. As shown in Fig 7B there are multiple potential biological and chemical potential
pathways for the synthesis of CN- in the CF RT.
Mechanisms of airway CN- metabolism:
Similar to the multiple mechanisms of CN- synthesis in the CF airway, a number of both
enzymatic and chemical pathways for CN- metabolism/catabolism potentially exist, as
simplistically illustrated in Fig 7A (and in mechanistic detail in Fig 7B). The major CN-
metabolizing enzyme in most living organisms, rhodanese (thiosulfate cyanide sulfur
transferase), is present in vertebrate lung mucosal tissues [36]. Rhodanese enzymatically
converts CN- into thiocyanate (SCN-), which itself has been demonstrated to be present in CF
airway fluids [33] and has the potential to alter a number of biological pathways including
antimicrobial activity and modulating reactive oxidant pathways [37]. It is worth noting that there
is considerable functional polymorphism of this major human CN- detoxification enzyme. This
should be taken into consideration when interpreting CN- levels in RT secretions [38]. In
addition, selected microbial species, including P. aeruginosa, express enzyme systems capable
of metabolizing/detoxifying CN- and utilizing CN- for biosynthetic pathways [2, 39].
In addition to enzymatic pathways of CN- metabolism, there also exist a number of
potential chemical mechanisms for the catabolism of CN-. First, CN- reacts with MPO-derived
HOCl at near-diffusion limited rates (k=109 M-1s-1) [40] to produce CNCl as an intermediate,
which is then further converted by HOCl to cyanate (NCO-) [41]. Moreover, CN- reacts with
peroxidase-derived hypothiocyanous acid (HOSCN) to form thiocyanate (SCN-) and NCO- via
intermediate formation of dicyanosulfide (NCSCN) [42]. The formation of NCO- by these two
independent pathways can lead to carbamoylation of proteins that could potentially be useful as
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biomarkers of CN- and could also impact protein structure and function [43, 44]. One final
pathway of CN- “metabolism” is the high affinity sequestration of CN- by metalloproteins and
metal complexes such as metal-containing siderophores [45].
Carbamoylation reactions in CF airways:
As illustrated in Fig 7B, metabolism of CN-/HCN in the presence of the abundant MPO/HOCl in
inflamed CF airways can be expected to yield cyanate (NCO-), a potent carbamoylating species
[20,46]. In fact, our data in Fig 6 revealing the presence of carbamoylated proteins in CF
sputum serves as a proof-of-concept for this pathway in the CF airway. Carbamoylated proteins
are increasingly being identified from sites of chronic inflammatory processes, most notably in
rheumatoid arthritis [47,48], asthma [44], kidney disease [4,46-51], atherosclerosis [43,49] and
aging [50]. It is likely that protein carbamoylation results from the chemical and enzymatic
metabolism of both CN- and SCN- (Fig 7B). The posttranslational carbamoylation of proteins is
likely to not only serve as a marker for CN-/SCN-, but is also likely to alter the structure and
function of proteins and potentially exert biological pro-inflammatory effects [44,46,51].
Biological implications of CN- in the CF airway:
It is important to address potential hypotheses regarding the consequences of the increased
CN- produced by cyanogenic microbes (largely but not exclusively P. aeruginosa) in CF airways
[2,8,9,18,52-54]. The fact that CN- is produced only under hypoxic conditions suggests that the
CN- synthase is likely to be highly active in the adjunct mucus and host mucosal cell region of
the microbial biofilm [2,55]. Like the case for the strong P. aeruginosa virulence factor pyocyanin
[56], the CN- can be expected to have multiple pathobiologic effects including those on
respiration, metabolism and mucociliary clearance [57].
CN- would be expected to augment the already hypoxic microenvironment of the biofilm
thus having synergistic effects on mitochondrial reactive oxygen species activations of hypoxia-
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induced transcription factor (HlF-1) and presumably impacting transcription nuclear factor
erythroid 2-like2 (Nrf-2). Of interest, both HlF-1 activation and Nrf-2 activation have been
reported to be protective against CN- toxicity in select model systems [58,59]. Further insights
will be needed to more fully understand the effects of airway CN- concentrations of the
magnitude being generated in CF airways on juxtapositioned cellular actors of signaling
transduction networks.
A major understudied issue in the CF-related CN- community relates to the levels of
bioavailable CN- in the complex matrix of CF secretions. Our study and those of others do not
address this important consideration. For instance, it is not currently known how much of the
CN- in the CF airway is free vs. bound to metalloproteins. Thus, it is difficult to estimate the
precise bioavailable CN- levels impacting the biology of the extracellular and cellular host milieu
in the proximity of the CN- producing biofilms [60]. Depending on such factors as binding affinity,
equilibrium kinetics and pH, sputa CN- sequestrations are likely to depend on a wide spectrum
of species including metalloproteins and metal complexes such as siderophores which are
capable of binding with CN- [45]. The presence of measurable amounts of HCN in breath
validates the presence of sizable amounts of free CN- in CF secretions [52-54].
Conclusions:
Bacterial cyanogenesis in the CF RT is likely to have broad-ranging implications related to not
only the CF RT microbiome, but also to CF RT pathophysiology [2, 6]. Simplified and more
accurate assay methods, such as the one presented here, and including newer clinic adapted
breath analysis methodologies [2], should facilitate more in-depth studies of the significance of
CN- production and metabolism in the CF RT. The ratio of free bioavailable CN- to sequestrated
metal bound CN- represents an important remaining challenge. Finally, therapeutic intervention
strategies that decreases airway levels of CN- (such as cobalamin/cobinamide or small
16

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Submitted 08/15/2018
molecular inhibitors of bacterial HCN syntheses) should allow for clinical elucidation of the
impact that CN- plays in CF pathobiology.
Author contributions:
Conception and design: J.P.E., C.E.C.; Provided patient materials: B.M.M., C.E.C.; data
analysis and interpretation: J.P.E., M.A.L.M., C.E.C.; manuscript preparation: J.P.E., S.P.O.,
M.A.L.M., C.E.C.; final revision and approval to be published as well as agreement to be
accountable for all aspects of the work: J.P.E., S.P.O., T.K., B.M.M., K.A.H., M.A.L.M., C.E.C.
Acknowledgements
The authors gratefully acknowledge the UC Davis adult CF patients contributing to this
study, Maya Juarez, the Clinical Coordinator that managed the IRB processes, and Chue Xiong
for manuscript preparation. Supported by N.I.H. grant R21-HL092506 (J.P.E and C.E.C.) and by
the CF Foundation in support of the UC Davis Adult CF Program (B.M.M. and C.E.C)
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Table 1. Demographics of adult cystic fibrosis patients. A: Aspergillus, Bcc: Burkholderia
cepecia complex, MRSA: Methicillin – resistant Staphylococcus aureus, PA: Pseudomonas
aeruginosa, SA: Staphylococcus aureus. Averages stated as average SD.
Subject
Age
(years)
25
Sex
FEV1
Culture
(% predicted)
1
M
F
28
91
PA, Bcc
SA, A
MRSA
PA
2
51
3
23
M
M
M
M
M
F
20
4
43
47
5
27
60
PA
6
40
55
PA, SA
Bcc, SA
PA, MRSA
PA, MRSA
SA
7
44
18
8
44
35
9
10
26
F
30
20
F
58
11
34
M
M
65
PA
12
26
35
PA
Average
34 10
45 22
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Table 2. Cyanide concentration detected in CF sputa. CN- was determined using an ISE.
Apparent CN- was the concentration of CN- in sputum samples measured before cobinamide
addition. Cobinamide was added to sputum samples to determine the background interference
to calculate the corrected CN- concentration. Values listed are the average SEM.
Subject
Apparent CN-
(µM)
Corrected CN-
(µM)
% Difference
1
133.5
74.8
21.1
632.7
411.0
2
18.2
3
68.8
44.7
153.9
4
49.7
30.0
165.7
5
105.7
179.1
80.6
32.3
327.2
6
98.1
182.6
7
14.1
571.6
8
112.4
113.0
78.3
25.9
434.0
9
10
44.4
254.5
15.7
498.7
11
147.1
109.2
104.4 10.5
58.8
250.2
12
41.0
266.3
Average
37.0 6.8
345.7 46.8
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Figure 1. Cobinamide prevents the detection of CN- by an Ion-Selective Electrode (ISE).
(A) Structure of dihydroxocobinamide (B) Simplified structure of cobinamide indicating the
location of cyanide binding and illustrating cobinamide binds CN- with a 2:1 stoichiometry. (C)
Standard curves of CN- detection with the ISE in the absence (- Cobinamide) and presence (+
Cobinamide). ISE response (mV) to cyanide (5 - 80 µM) with (+ Cobinamide) and without (-
Cobinamide) 25 mM cobinamide in 0.1 M NaOH. In the presence of cobinamide, the ISE only
detects CN- once the concentration exceeds the 2:1 binding stoichiometry, and then a linear
detection curve is obtained. Thus, cobinamide is an effective method of removing cyanide from
ISE detection.
Figure 2. Utilization of cobinamide to correct for non-specific measurements of the CN-
electrode in CF sputum. Homogenized sputum samples from CF patients (1:1 w/v PBS, 10
mM phosphate, 154 mM NaCl, pH 7.4) were brought to a concentration of 0.1 M NaOH. Using
an ISE electrode apparent CN- was the concentration of CN- in sputum samples measured
before cobinamide addition. Cobinamide was added to sputum samples (100 µM final
concentration) to determine the background interference and calculate the corrected CN- levels.
Lines indicate individual CF patient sputum samples.
Figure 3. Cyanide concentration in sputum between P. aeruginosa-positive and P.
aeruginosa-negative CF patients. Shown are data obtained directly from the CN- electrode
measurements (A) and data utilizing the corrected method presented herein (B). Neither sets of
data are statistically significant (apparent measurement, p = 0.049; corrected measurement, p =
0.158). However, P. aeruginosa negative patients showed a trend of lower CN- concentrations
by both methods.
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Figure 4. Cyanide (CN-) concentration in sputum does not correlate with lung function
(FEV1) in CF patients. Assessment of CN- concentration using either (A) apparent CN-
concentrations or (B) corrected CN- concentrations does not reveal a statistically significant
correlation between CN- concentration and lung function as assessed by FEV1 (% predicted).
Statistical p-values and R2-values are provided in each figure panel.
Figure 5. Hypochlorous acid (HOCl)-dependent oxidative consumption of CN-. Reactions
of HOCl (0-200 µM) with CN- (100 µM) were conducted in phosphate-buffered solutions (50 mM,
pH 7.4) at room temperature. Quantification of CN- and cyanogen chloride (CNCl) were
performed 10 min after initiation of the reaction as described in the Materials and Methods
section. CN- was stoichiometrically consumed by HOCl to form CNCl, maximally at a 1:1 ratio.
As the concentration of HOCl was increased beyond the 1:1 ratio with CN-, CNCl concentrations
were decreased and non-existent at a ratio of 2:1 (HOCl/CN-), suggesting the formation of other
chemical species, most likely cyanate (NCO-).
Figure 6. Detection of carbamoylated proteins in CF sputum. (A) Schematic illustrating that
cyanate (NCO-) reacts with protein lysine residues to form carbamyl-lysine (homocitrulline). (B)
Western blot indicating that bovine serum albumin (BSA) reacted with cyanate (NCO-), but not
native BSA, is recognized by an antibody against protein carbamyl-lysine. (C) Western blot
illustrating that proteins in CF sputum harbor carbamyl-lysine post-translational modifications.
CF sputum from Pseudomonas-positive and Pseudomonas-negative patients are noted by (+)
and (-), respectively. Western blots are representative of multiple experiments.
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Figure 7. Summary of the various potential biochemical pathways for the synthesis and
metabolism of CN- in the CF airway. (A) General scheme illustrating synthesis and
metabolism of CN-. Multiple chemical/biochemical pathways are potentially involved in the
synthesis and metabolism/fate of CN- in the CF airway and which illustrate its’ dynamic and
transient nature. (B) Detailed mechanistic scheme illustrating pathways involved in the synthesis
and metabolism of CN-. Pathways of CN- synthesis: (1) Oxidative biosynthesis of CN- by
cyanogenic bacteria via the HCN Synthase enzyme, utilizing the amino acid glycine as a
substrate. [2] (2) HOCl-dependent chlorination of glycine. [16,29] (3) HOCl-dependent
chlorination of serine. [17] (4) HOCl- and N-chloramine-dependent oxidation of thiocyanate
(SCN-). [30,31] (5) HOCl-dependent chlorination/oxidation of uric acid. [30] (6) Metabolism of
CN- to thiocyanate (SCN-) by both host and microbial enzymatic systems (ie. rhodanese,
thiosulfate sulfurtransferase). [25,36-39] (7) Microbial utilization of CN- as carbon and nitrogen
source for biosynthesis. [39] (8-10) HOCl-dependent conversion of CN- into CNCl and NCO-,
and its utilization in protein carbamoylation reactions. [41,43] (11) Reactions hypothiocyanous
acid (HOSCN) with CN- to form NCO-, and its utilization in protein carbamoylation reactions.
[42,43] (12) Sequestration of CN- by high-affinity interactions with metaloproteins and/or metal
complexes. [45]
HIGHLIGHTS
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Ion-Specific electrode and cobinamide used to quantify CN- in CF sputum
Sputum CN- levels do not correlate with infection or lung function
Hypochlorous acid rapidly degrades CN- into cyanogen chloride and cyanate
CF respiratory tract biosynthetic and catabolic pathways of CN- reviewed
Carbomoylated proteins are present in CF sputum.
28

Figure 1
Eiserich et al.
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