Author's response to reviews
Title: Ex-vivo changes in amino acid concentrations from blood stored at room
temperature or on ice : implications for arginine and taurine measurements.
Authors:
Joshua S Davis ([email protected])
Christabelle J Darcy ([email protected])
Kim Piera ([email protected])
Yvette R McNeil ([email protected])
Tonia Woodberry ([email protected])
Nicholas M Anstey ([email protected])
Version: 2 Date: 6 October 2009
Author's response to reviews: see over
Response to Reviewers’ Comments Ex-vivo changes in amino acid concentrations from blood
stored at room temperature or on ice : implications for arginine and taurine measurements.
We thank the reviewers for their thorough examination of our manuscript, and the editors for the
opportunity to address the reviewers’ comments. We have addressed each comment in turn.
Reviewer 1
•All single numbers (e.g. 2, 6, etc) should be written out (two, six, etc);
We have changed from figures to words for numbers describing people or objects, and have left
them as figures for those describing proportions or units of measurement (including hours), as per
Fowler’s Modern English Usage, 3rd Edition, 1996.
•“Venepuncture” is misspelled throughout-should be “venipuncture”
The Oxford English Dictionary lists both “venepuncture” and “venipuncture” as acceptable spellings.
We have left it as “venepuncture” at present, but will defer to the editors for a final judgement on
this matter.
•Page 4, paragraph 2, line 8, recommend changing from: “supplementary feeds”
to: “parenteral nutritional supplementation”;
The amino acids we have mentioned are commonly added to enteral as well as parenteral nutrition;
hence we have changed this sentence to:
“Other amino acids which are commonly added to supplementary nutrition for critically ill patients .
..“
•Page 4, paragraph 2, line 9: missing a period at end of the sentence;
We have added a full-stop.
•Page 4, paragraph 3, line 2,change from: “…arginine at 4 hours…” to:
“…arginine following four hours…” and remove the comma before “but”;
We have altered this sentence as below
Hainque and colleagues studied eight healthy volunteers and found a “significant degradation” of
plasma arginine following 4 hours at room temperature but this was not quantified and no other
time points were reported [13].
•Page 4, paragraph 3, line 4, change from: “….at room temperature and a
10%....” to:
“….at room temperature compared with a 10% decrease after six hours at 4 degrees
C,
….”;
We have changed this sentence as follows
Schaefer et al. studied one volunteer and found a 50% decrease in plasma arginine after 6 hours at
room temperature compared with a 10% decrease after 6 hours at 4 degrees centigrade, with earlier
time points not reported [14]
•Page 4, paragraph 3, line 6, change from: “…which showed an approximate
33% decrease…” to: “…which demonstrated a 33% decrease…..by two hours at
room temperature”;
We have left the word “approximate” here, as the paper by Nuttall does not provide
numerical data, only a graph. We have added the reference to room temperature as
requested.
Nutall and colleagues reported time profile data from one volunteer, which showed an approximate
33% decrease in plasma arginine by 2 hours at room temperature [15].
•Page 4, paragraph 4, line 1, change from: “To determine the impact…..a study
aiming…” to: ““To determine the effect…..a study to estimate..…”
We have changed this sentence as follows:
To determine the impact of delayed processing we undertook a study to estimate the rate of
arginine degradation in human plasma at room temperature and on ice.
Methods:
•Page 5, paragraph 1: This reviewer is not in agreement that the Institutional
Review Board (Ethics Committee) of the authors’ university determined the
project to be a “QA-quality assurance project”, based upon the National
Statement of Ethical Conduct in Human Research (Australia). First, collection of
any body fluids (blood in this case) falls under human research (NOT a QA
activity) and meets the criteria for an expedited review of prospective research,
requiring subject informed consent. As informed consent was indeed obtained
(as noted in paragraph 1, line 4), the authors don’t seem to understand the
Australian regulations for human research. This first paragraph must be
corrected to be consistent with the regulations;
We did not state that the Ethics Committee determined this to be a “QA-quality assurance
project”; rather we stated that “The study was considered by the Chair of the Human Research
Ethics Committee of the Menzies School of Health Research and Northern Territory Department of
Health and Families, and was approved as a quality assurance activity which did not require full
ethical review.” The study was an internal laboratory quality assurance activity, pertaining to
validation of sample processing for our amino acid assays. The study was found by the
Chair of the Human Research Ethics Committee to be consistent with The Australian
National Health and Medical Research document “When does quality assurance in health
care require independent ethical review” (NH&MRC, 2003). We chose to seek written
informed consent, as is appropriate for such a study, even though we acknowledge this is
not mandatory in studies deemed to be QA activities.
•Page 5, paragraph 1: Was hand clenching (fist pumping) done during
venipuncture?
Fist pumping by volunteers was not done during venepuncture.
•Were subjects fasted when specimens were drawn? Please add a statement in
paragraph 1 to clarify.
•Were all subjects normotensive? Please add a statement.
As stated, subjects were fasting at the time of venepuncture. A statement has been added
that they were also normotensive.
Following written informed consent, six healthy normotensive fasting volunteers had venous blood
collected into 12x2mL lithium heparin tubes (Vacutainer, Becton Dickinson, Franklin Lakes, New
Jersey) using a 21 gauge needle and vacutainer system.
•Page 5, paragraph 3, line 3: correct from: “summarized” to: “summarized”;
We have changed the English spelling “summarised” to the American spelling “summarized”.
•Page 5, paragraph 3, last line: please remove the following text: “which we had”
so that it states: “….earlier experiment previously performed, which found a
31.8% ……(unpublished data)”. Also, please change from: (sd 14%) to: “(std dev
= 14%) since the abbreviation used by the authors is not correct.
We have altered this sentence as follows:
The sample size was determined using data from an earlier experiment (unpublished data), which
found that there was 31.8% (std dev=14%) degradation of arginine at room temperature by 2 hours.
Results:
•The subjects used in this study are barely described. Please see under “Tables”
below;
We have added a table 1, with age, gender and ethnicity of study subjects; the first sentence
of results now reads:
Of the six study subjects, half were male, and the median age was 37.5 years, with a range of 19-47
years (table 1). All were healthy, of normal weight and normotensive, and none had cardiovascular
disease or diabetes mellitus.
•It isn’t clear to this reviewer why the data aren’t presented as Mean +/ Std Dev.,
since normative data are always presented in this fashion and would have more
meaning to the reader? In Table 1, data should be presented in this manner;
The number of included datapoints is too small to reliably determine if the data are normally
distributed. Inspection of histograms and boxplots at each time point suggests that they are
right-skewed (see below); thus data are presented as median and IQR, as is appropriate for
non-parametric data.
.55
.5
ADMA
.45
.4
.35
•Also, no data are provided to the reader regarding the normative population
values for the amino acid profiles discussed in this study (per gender, age). It is
important for the reader to read and comprehend how the author’s findings relate
to normative values and whether the author’s standard deviations are similar to
the normative population or exceed those values. Please add this information
either as text in the results or as an additional column in Table 1;
Please see below, under “tables”.
•Why didn’t the authors also log transform the data since these data aren’t
normally distributed?
The data are not transformable, as shown below
identity
0
.1
.2
.3
.4
0
0
0
2
2
2
4
4
4
6
6
6
square
8
cubic
.1
.2
.3
.4
.5
.6
.65
.7
.75
.8
.85
4
3
-.4
-.2
-1.6 -1.5 -1.4 -1.3 -1.2 -1.1
0 .02 .04 .06 .08
1/cubic
.2
.1
0
-1
.8
2
-.6
.3
1.5
1
.5
-1.5
.7
1
-.8
1/square
0
-2
.6
0
-1
inverse
-2.5
.5
1/sqrt
0 .5 1 1.5 2 2.5
.6
.4
log
0 2 4 6 8
Density
sqrt
-7
-6
-5
-4
-3
-2
-20
-15
-10
-5
0
ADMA
Histograms by transformation
Discussion:
•Page 8, paragraph 1, line 1: please change from: “concentrations decrease” to:
“concentration decreases”;
The sentence now reads:
Plasma arginine concentration decreases rapidly . . .
•Page 8, paragraph 1, line 2: please change from: “prevented” to: “greatly
attenuated”;
The sentence now reads:
. . .and this decrease is greatly attenuated by placing the blood on ice.
•Page 8, paragraph 1, line 4: please add a comma after “Thus”;
This has been done.
•Page 8, paragraph 1, line 6: please remove the comma before “but” and re-state
the following text: “….were unable to determine which of these is more
important.” As the authors didn’t try to determine the specific causative
mechanism, they can’t state they “were unable to determine”. They need to state
that they didn’t evaluate the specific mechanisms;
The sentence now reads:
This arginase could come from either lysed RBCs or lysed leucocytes, but we did not evaluate the
source of arginase, and thus cannot determine which of these was more important.
We have left the comma before “but”, as is grammatical preceding a subordinate clause (eg. see
Strunk and White’s “The Elements of Style”, 4th edition, page 5)
•Page 8, paragraph 1, last line: The authors can’t state that their
“…..observations strongly support it”. Rather, it is acceptable to state and please
change to: “…our observations strongly suggest it”;
The sentence now reads:
While we have not proven this hypothesis, our observations strongly suggest it.
•Page 8, paragraph 3, line 1: please change from: “”…arginine which we found in
blood…” to: “arginine found in blood…”;
We have not changed this, as it would change the meaning of the sentence. We are
referring specifically to the rate of degradation which we found in the present study.
•Page 8, paragraph 3, line 4: please change from: “…other papers made it
difficult…” to: “…other papers make it difficult…” and change from: “....it was
linear..” to: “..it is linear..”;
The sentence now reads:
The lack of early time points in other papers make it difficult to estimate the rate of decline and
whether it is linear or exponential
•Page 8, paragraph 3, lines 5-6: please change from: “…reported data from only
a single individual, only in graphical form, and only up to 2.5 hours post
venepuncture....” to: “…reported data, in graphical form, from a single subject up
to 2.5 hours post venipuncture”;
The sentence has been changed as suggested.
•Page 9, paragraph 1, last line: please add a period at the end of the sentence.
A full-stop has been added.
•Page 9, paragraph 2, line 5: please change from: “…results as we have found.”
to: “…results we observed.”
The sentence has been changed as suggested.
•Page 9, paragraph 2, lines 7-8: please change from: “…based on indirect
evidence and may be incorrect.” to: “”..is based on our assumption due to its
common pathway of metabolism.”
We have shortened this sentence for clarity. The suggested modification changes the meaning.
We did not directly measure arginase activity in blood or plasma, and thus our inference that plasma
arginase is primarily responsible for the observed ex-vivo arginine degradation is based on indirect
evidence.
•Page 9, paragraph 3, line 1: please add “potential” after “One” at the beginning
of the sentence AND change from: “purposes” to: “purpose”;
The sentence now reads:
One potential implication of these data is that whole blood stored for the purpose of transfusion is
likely to contain non-physiological concentrations of amino acids . . .
•The authors need to add more specifics to the discussion section specific to the
following issues: (1) the clinical consequences (regarding possible treatment
intervention based upon spurious results) of these amino acids should methods
utilize holding specimens at room temperature for an extended period of time.
Although these amino acids are most often measured in research studies as
opposed to clinical evaluation, some amino acids are clearly measured for
clinical purposes (e.g. inborn errors of metabolism-- one example is
phenylalanine); (2) there is never any discussion regarding how the values seen
in the present study compare with normative values for the population. This
should be added at the beginning of the discussion section.
We have added the following sentence to the discussion:
Furthermore, where plasma amino acids are being measured for clinical applications, our data
emphasise the importance of timely separation and freezing of plasma to avoid potential diagnostic
errors.
And the following sentence to the results section:
The median baseline plasma arginine concentration was 74.9 µmol/L, similar to previously reported
mean plasma arginine concentrations from healthy volunteers, the majority of which are between
60 and 80 µmol/L {Martens-Lobenhoffer,2006 #646}.
Tables:
•The subjects used in this study are barely described. Please add a “Table 1” that
provides information regarding the subjects including: age, gender, ethnicity; any
comorbidities (DM, HTN, etc); if adults use BMI and if children use BMI
percentile; if any skinfolds were used to calculate lean mass, please provide lean
and fat mass measures, and waist:hip ratio (if have these data).
We did not collect weight, BMI or skinfold thickness. A table 1 has been added with age,
gender and ethnicity, and the following sentence has been added at the beginning of the
results section:
Of the six study subjects, half were male, and the median age was 37.5 years, with a range of 19-47
years (table 1). All were healthy, of normal weight and normotensive, and none had cardiovascular
disease or diabetes mellitus.
•In what is currently Table 1 (should become Table 2), please add a column with
the normative population Median and IQR for each amino acid (assuming those
values are normally obtained at room temperature) or change all data to Mean +
Std. Dev. The authors should note under the table if any of the amino acids listed
are normally placed on ice immediately.
There are no widely accepted “normative population” values for arginine and ornithine. There
is wide variation in reported normal values between studies, depending on the methodology
used; in addition, very few studies report the time and temperature conditions of the samples
prior to processing. Finally the important point in this study is the change from baseline in
plasma amino acid concentrations, not the baseline values themselves.
Thus we have not added these data to table 2, but have added a sentence to the results
section stating:
The median baseline plasma arginine concentration was 74.9 µmol/L, similar to previously reported
mean plasma arginine concentrations from healthy volunteers, the majority of which are between
60 and 80 µmol/L {Martens-Lobenhoffer,2006 #646}.
Figures: The figures are really great and very interesting!!
•Figures 1a and 1b: please change X axis label from: “TIme to Plasma
Separation” to: “Elapsed Time Prior to Plasma Separation (hours)”;
•Figures 2 and 3: please change X axis label from: “TIme to Plasma Separation”
to: “Elapsed Time Prior to Plasma Separation (hours)”.
The figures have been changed as requested
Reviewer 2
Minor essential revisions:
1. In the section “Arginine and ornithine time profiles…” it would be interesting to
do the parameter fitting not only for arginine, but also for ornithine. By
comparison of the parameters, it could be elucidated whether arginine is
exclusively converted to ornithine, or whether there is another catabolic pathway
at work (from visual inspection of Fig. 2, it seems to me that this is not the case).
We have fitted a non-linear curve of the ornithine time profile, but the data do not fit this model as
well as the arginine data, making it unreliable. Whilst we agree that elucidating the mechanism of
arginine degradation would be very interesting, it was not the primary aim of the study, and is
beyond the scope of this paper.
As a result of this fact, we have changed a sentence in the first paragraph of the discussion:
Thus, it is likely that arginase is the primary mechanism of arginine degradation in ex-vivo blood
samples.
2. The references are not always selected accurately:
Ref. [1] is clearly outdated: The article does not describe the formation of NO, but
of nitrite, proposing a pathway which includes no NOS.
Ref. [2] focuses only on cultured cells and does not mention endothelial function.
References [1] and [2] have been replaced by broader, more recent articles, and the
endothelial function statement has been separately referenced.
Ref. [4] reports divergent data with no clear trend to decreased arginine levels: in
sepsis, they found a significant decrease in arginine, but in sepsis plus trauma
and in trauma only they found a non-significant increase in arginine.
Ochoa
Ref [4] (Ochoa, 1991), actually reported the opposite to what the reviewer states: no change
in plasma arginine in sepsis compared with controls, but a significant decrease in trauma
patients (with or without sepsis). It was cited to support the statement regarding decreased
plasma arginine in trauma patients.
To clarify this, the references have been separated and expanded as follows:
Arginine, the precursor of nitric oxide (NO) {Boger, 2007 #647}, is important for endothelial {Ganz,
2003 #648} and immunological {Bogdan, 2001 #649} function and is acutely decreased in sepsis
{Luiking, 2004 #335; Davis, 2009 #645}, malaria {Yeo, 2007 #260} and trauma {Ochoa, 1991 #377},
and was thus the focus of this study.
Minor revisions:
1. The abbreviations “NOS”, “NO” and “sd” appear in the abbreviations list but
not in the text. However, the abbreviation “IQR” is used and appears not in the
abbreviations list.
Thank you for pointing this out – it has been corrected
2. In Fig. 1, please label the upper and the lower panel clearly with A and B, as in
the figure legend.
The figure has been changed as requested
Yours Sincerely
Dr Josh Davis
On behalf of all the authors.