Structure-activity relationship of novel acridone derivatives as antiproliferative agents

Article abstract of DOI:10.1016/j.bmc.2020.115868

Unlike other DNA topoisomerase II (topo II) inhibitors, our recently identified acridone derivative E17 exerted strong cytotoxic activity by inhibiting topo II without causing topo II degradation and DNA damage, which promoted us to explore more analogues

Full text of DOI:10.1016/j.bmc.2020.115868

J. Dairy Sci. 100:1502–1506
https://doi.org/10.3168/jds.2016-11277
© American Dairy Science Association®, 2017.
Short communication: Analytical method and amount of preservative
added to milk samples may alter milk urea nitrogen measurements
Holley L. Weeks and Alexander N. Hristov1
Department of Animal Science, The Pennsylvania State University, University Park 16802
ABSTRACT
Thus, it is important to maintain consistency in milk
sample preservation and analysis to ensure precision of
MUN results.
Milk urea N (MUN) is used by dairy nutritionists
and producers to monitor dietary protein intake and is
indicative of N utilization in lactating dairy cows. Two
experiments were conducted to explore discrepancies in
MUN results provided by 3 milk processing laborato-
ries using different methods. An additional experiment
was conducted to evaluate the effect of 2-bromo-2-ni-
tropropane-1, 3-diol (bronopol) on MUN analysis. In
experiment 1, 10 replicates of bulk tank milk samples,
collected from the Pennsylvania State University’s
Dairy Center over 5 consecutive days, were sent to 3
milk processing laboratories in Pennsylvania. Aver-
Key words: milk urea nitrogen, bronopol, dairy cow
Short Communication
Indicators for monitoring dietary N adequacy in
dairy cattle include BUN, urinary urea N, and MUN.
Of these, MUN is the most practical analysis because
individual cow or bulk tank milk samples are routinely
collected on dairy farms (Roy et al., 2011). Milk urea
N is used to monitor dietary CP and N utilization in
lactating cows and is linearly related to urinary N ex-
cretion (Broderick and Clayton, 1997; Hof et al., 1997).
High MUN levels can indicate excess CP or RDP in the
diet, which could increase N excretion and ammonia
emission from manure, may have negative effects on re-
production, and also increase feed costs to the producer
(Nousiainen et al., 2004; Burgos et al., 2007; Lean et
al., 2012). It should be pointed out, however, that some
analyses have shown no relationship between individual
cow milk urea concentration and reproduction in dairy
cows on pasture (Trevaskis and Fulkerson, 1999), or
bulk tank MUN and ammonia emissions from manure
(Weeks et al., 2015). Jonker et al. (2002b) concluded
that farms provided with their bulk tank MUN data on
a monthly basis for 6 mo had 0.52 mg/dL lower MUN
than farms who did not received their bulk tank MUN
data over the course of their study. This decrease in
MUN resulted from an 11 g/d per cow decrease in N
intake. Milk analysis laboratories use different analyti-
cal equipment and methods for MUN analysis, which
can result in variability in MUN data (Arunvipas et
al., 2003; Kohn et al., 2004). This can, in turn, cause
confusion on how MUN for a particular farm ranks
relative to optimal MUN values suggested in the litera-
ture (for example, Jonker et al., 2002a; Powell et al.,
2014). Therefore, the objectives of this study were to
(1) investigate consistency among laboratories’ MUN
analytical methods, and (2) investigate differences in
MUN due to milk preservative added.
age MUN differed between laboratory A (14.9
mg/dL; analyzed on MilkoScan 4000; Foss, Hillerød,
Denmark), laboratory B (6.5 0.17 mg/dL; MilkoScan
0.40
FT + 6000), and laboratory C (7.4
0.36 mg/dL;
MilkoScan 6000). In experiment 2, milk samples were
spiked with urea at 0 (7.3 to 15.0 mg/dL, depending
on the laboratory analyzing the samples), 17.2, 34.2,
and 51.5 mg/dL of milk. Two 35-mL samples from
each urea level were sent to the 3 laboratories used in
experiment 1. Average analyzed MUN was greater than
predicted (calculated for each laboratory based on the
control; 0 mg of added urea): for laboratory A (23.2 vs.
21.0 mg/dL), laboratory B (18.0 vs. 13.3 mg/dL), and
laboratory C (20.6 vs. 15.2 mg/dL). In experiment 3,
replicated milk samples were preserved with 0 to 1.35
mg of bronopol/mL of milk and submitted to one milk
processing laboratory that analyzed MUN using 2 dif-
ferent methods. Milk samples with increasing amounts
of bronopol ranged in MUN concentration from 7.7 to
11.9 mg/dL and from 9.0 to 9.3 mg/dL when analyzed
on MilkoScan 4000 or CL 10 (EuroChem, Moscow,
Russia), respectively. In conclusion, measured MUN
concentrations varied due to analytical procedure used
by milk processing laboratories and were affected by
the amount of bronopol used to preserve milk sample,
when milk was analyzed using a mid-infrared analyzer.
Received April 5, 2016.
In experiment 1, bulk tank milk samples were col-
lected from the Pennsylvania State University’s Dairy
Accepted October 23, 2016.
1Corresponding author: anh13@psu.edu
1502

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SHORT COMMUNICATION: MILK UREA NITROGEN
Research and Teaching Center herd. After 5 min of 5 min of continuous agitation in the cooling tank. The
continuous agitation, six 35-mL milk samples were 2 L of milk were divided into four 500-mL samples.
collected at 1400 h daily over 5 d. Two samples from Samples were spiked with urea at 0, 17.2, 34.2, and
each day were shipped overnight to 3 milk processing 51.5 mg urea/dL milk (i.e., added urea levels control, 1,
laboratories (laboratories A, B, and C) to be analyzed 2, and 3, respectively). Two 35-mL samples from each
for MUN, milk fat, and milk true protein (see Table 1). urea level were preserved and sent to laboratories A,
Milk samples were analyzed by mid-infrared spectros- B, and C for MUN analysis according to the methods
copy at laboratory A using MilkoScan 4000 (MS; Foss, listed for experiment 1 (Table 1). Based on the amount
Hillerød, Denmark), at laboratory B using MilkoScan of added urea and the analyzed MUN concentration
FT+ 6000 (Foss), and at laboratory C using MilkoScan of the control milk samples (0 mg of added urea/dL)
6000 (Foss). Samples from 4 of the 5 d submitted to from each laboratory, expected MUN concentrations
laboratory A were also analyzed for MUN using a CL were calculated for each laboratory and each urea level
10 analyzer (CL; EuroChem, Moscow, Russia), accord- (i.e., concentration of MUN in control milk sample plus
ing to procedures described by Luzzana and Giardino amount of added urea for levels 1, 2, and 3). Data in
(1999). The CL 10 method is considered the standard experiment 2 were analyzed using the MIXED proce-
to which other methods are compared due to its accu- dure of SAS. The model contained laboratory, urea
racy (Dairy One Cooperative Inc., Ithaca, NY, personal level, and laboratory × urea level interaction. Similar
communication). Milk samples sent to laboratory A and to experiment 1, when the main effect of laboratory
laboratory B were preserved with 2-bromo-2-nitropro- was significant (P ≤ 0.05), means were separated by
pane-1, 3-diol (bronopol, Janssen Pharmacauticalaan, pairwise t-test (pdiff option of PROC MIXED).
Beerse, Belgium). Milk samples sent to laboratory C
In experiment 3, 48 milk vials were prepared with 16
were shipped refrigerated and without preservative due levels (0 to 1.35 mg/mL) of bronopol (Janssen Phar-
to the inability of laboratory C’s equipment to analyze macauticalaan). Bronopol was added as 15% aqueous
milk preserved with bronopol. Milk MUN data were solution. Each bronopol level was replicated 3 times.
analyzed using the MEANS and MIXED procedures A 2-L bulk tank milk sample was collected from The
of SAS (version 9.4; SAS Institute Inc., 2003). The Pennsylvania State University’s Dairy Research and
MIXED model contained milk sampling day, labora- Teaching Center following the procedure outlined for
tory, and sampling day × laboratory interaction, and experiments 1 and 2. Thirty-five mL of milk was added
data were analyzed as repeated measures assuming to each vial, resulting in final bronopol concentrations
an AR(1) covariance structure. When the main effect of 0, 0.10, 0.19, 0.29, 0.38, 0.47, 0.54, 0.65, 0.72, 0.80,
of laboratory was significant (P ≤ 0.05), means were 0.91, 0.98, 1.07, 1.16, 1.24, and 1.35 mg/mL. All milk
separated by pairwise t-test (pdiff option of PROC vials were sent to laboratory A for MUN analysis us-
MIXED).
ing mid-infrared spectroscopy on a MilkoScan 4000
In experiment 2, a 2-L bulk tank milk sample was col- (Foss) and a CL 10 analyzer. Data in experiment 3
lected from The Pennsylvania State University’s Dairy were analyzed using the MIXED procedure of SAS with
Research and Teaching Center on a single day following bronopol concentration in the model and contrasts to
Table 1. Experimental procedures and sample preparation used in the study1
Laboratory
A—MS
Laboratory
A—CL
Laboratory
B
Laboratory
C
Item
Experiment 1
Equipment used
MilkoScan 4000
CL 10
MilkoScan FT + 6000
MilkoScan 6000
Preservative used (bronopol)
Refrigerated
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Analytical procedure
Infrared analyzer
Wet chemistry
Infrared analyzer
Infrared analyzer
Experiment 22
Added urea (mg/dL of milk)
Control: 0
Not analyzed
Control: 0
Control: 0
Sample 1: 17.5
Sample 2: 34.2
Sample 3: 51.5
Sample 1: 17.5
Sample 2: 34.2
Sample 3: 51.5
Sample 1: 17.5
Sample 2: 34.2
Sample 3: 51.5
Experiment 32
Bronopol added at 0 to 1.35 mg/mL of milk
Analyzed
Analyzed
Not analyzed
Not analyzed
1MS = MilkoScan (Foss, Hillerød, Denmark); CL = CL 10 (EuroChem, Moscow, Russia); bronopol (Janssen Pharmacauticalaan, Beerse,
Belgium).
2Method of analysis, equipment, and sample preparation as in experiment 1.
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WEEKS AND HRISTOV
Table 2. Average MUN, milk fat, and milk true protein concentrations in milk samples analyzed by laboratories A, B, and C (experiment 11)
Laboratory
Laboratory
Laboratory
B
Laboratory
C
Item
A—MS2
A—CL2
SEM
0.32
P-value3
Mean MUN, mg/dL
SD
Minimum
Maximum
CV
Mean milk fat, %
SD
Minimum
Maximum
CV
Mean true protein, %
SD
Minimum
Maximum
CV
14.9a
1.56
12.7
17.3
10.5
3.69a
0.082
3.57
3.79
3.6
14.5a
0.26
14.1
14.9
1.8
6.5c
0.43
6.0
7.4b
0.63
6.6
<0.001
7.2
8.4
6.7
8.5
—
3.55c
0.082
3.45
3.65
2.3
3.58b
0.071
3.49
3.67
2.0
0.003
0.004
<0.001
0.01
—
—
—
3.17b
0.029
3.11
3.20
3.1
—
—
—
—
—
3.18a
0.046
3.10
3.25
1.4
3.16b
0.038
3.10
3.20
1.2
a–cMeans within a row having different letter superscripts differ at P ≤ 0.05.
1Mean values in table are LSM; n = 38 (MUN) and 30 (milk fat and protein), n represents the number of observations used in the statistical
analysis.
2Laboratory A analyzed MUN using 2 methods: MilkoScan 4000 (MS; Foss, Hillerød, Denmark) and CL 10 (CL, EuroChem, Moscow, Russia);
see Table 1 for details. Laboratory B used MilkoScan FT + 6000 and laboratory C used MilkoScan 6000.
3Main effect of laboratory. MUN data: effect of day of milk sampling, P = 0.76; laboratory × day of milk sampling interaction, P = 0.22; milk
fat data: effect of day of milk sampling, P < 0.001; laboratory × day of milk sampling interaction, P = 0.03; milk true protein data: effect of
day of milk sampling, P < 0.001; laboratory × day of milk sampling interaction, P = 0.10.
evaluate linear and quadratic effects of bronopol con- the lowest variation with the CL 10. In the present
centration.
study, average MUN concentrations differed between
The objective of experiment 1 was to set baseline laboratories A (MilkoScan 4000), B (MilkoScan FT+
MUN concentrations and determine differences in MUN 6000), and C (MilkoScan 6000), likely due to variation
analysis among laboratories. Average MUN concentra- in analytical procedure and equipment (Arunvipas et
tion analyzed at laboratory A was greater (P < 0.001) al., 2003; Kohn et al., 2004). If a theoretical benchmark
than laboratories B and C, as shown in Table 2. The for MUN is assumed at 10 to 12 mg/dL (Jonker et al.,
2 methods used by laboratory A, MilkoScan 4000 and 2002a; Powell et al., 2014), the present study indicated
CL 10, returned similar MUN values. Average MUN that MUN results from laboratory A are above the
concentration for laboratory C was also greater (P < benchmark and results from laboratories B and C are
0.001) than laboratory B. There was no effect of day below the benchmark for identical milk samples. Thus,
of milk sampling (P = 0.76) and there was no day of data from experiment 1 indicate that laboratory and
sampling × laboratory interaction (P = 0.22) for MUN. analytical method may cause significant variability in
Variability in MUN data (SD and CV) was larger for bulk tank MUN data.
laboratory A (MS method) than B and C. The low-
The objective of experiment 2 was to quantify the
est variability was associated with the CL method accuracy of each laboratory’s MUN analysis. Average
(laboratory A). As shown in Table 2, the 3 laboratories analyzed MUN and the difference between analyzed and
returned different results for milk fat and true protein predicted MUN are presented in Table 3. The differ-
(P ≤ 0.01) concentrations, although differences were ence between analyzed and expected (calculated based
subtle, particularly for milk protein. Kohn et al. (2004) on MUN concentration in the control milk samples for
concluded that 34% of the variation in bulk tank MUN each laboratory and the amount of added urea) MUN
for Foss (MilkoScan) 4000 was attributed to labora- concentration was largest (P < 0.001) for laboratory
tory. In their study, milk samples analyzed on Foss 4000 C followed by laboratory B, and was least for labo-
resulted in the largest SD ( 2.51 mg/dL) when com- ratory A. The difference was clearly increasing (P <
pared with CL 10. Conversely, the Bentley ( 0.45 mg/ 0.001) for all laboratories with increasing the amount
dL), Foss 6000 ( 0.62 mg/dL), and Skalar ( 0.55 mg/ of added urea, but the ranking among laboratories re-
dL) instruments resulted in smaller SD when compared mained the same. The relationship between amount of
with CL 10 (Kohn et al., 2004). Similarly, Peterson et added urea and the difference between analyzed and
al. (2004) concluded that the highest variation among predicted MUN was linear for all laboratories (R2 =
methods occurred with the Foss 4000 analyzer and 0.82 to 0.99; P ≤ 0.001). Peterson et al. (2004) com-
Journal of Dairy Science Vol. 100 No. 2, 2017

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SHORT COMMUNICATION: MILK UREA NITROGEN
pared recovery of urea N among 5 analytical methods: 0.05 mg/dL and tended to increase linearly (P = 0.06)
Bentley, CL 10, Foss 4000, Foss 6000, and Skalar us- with increasing bronopol concentration. MilkoScan
ing milk samples from 100 individual cows. Recovery 4000 uses mid-infrared spectroscopy, in which a beam
by CL 10 (85.0
2.76%) was lower than for Bentley, of light at specified wavelength for the component be-
Foss 6000, and Skalar, but greater compared with Foss ing measured is passed through milk and the amount
4000 (47.1 9.88%). Furthermore, no differences were of light absorbed is measured (Arunvipas et al., 2003).
detected for Bentley and CL 10 among laboratories; Data for MilkoScan 4000 from experiment 3 suggest
however, results for Foss 4000, Foss 6000, and Skalar that the absence of bronopol in experiment 1, labora-
varied among laboratories. In the present study, all 3 tory C milk samples may have resulted in lower MUN
laboratories overestimated MUN compared with the values than if those samples contained bronopol. In the
expected concentrations, but the least overestimation CL 10 method, the amount of ammonia formed from
was for laboratory A, which used MilkoScan 4000. An urea after hydrolysis with urease is used to calculate
important point in any analysis, including MUN, is MUN (Arunvipas et al., 2003; Kohn et al., 2004). Previ-
calibration of the analytical equipment. All laboratories ous studies indicate greater variability with MilkoScan
have established calibration procedures but as pointed 4000 compared with CL 10 for multiple laboratories
out by Kohn et al. (2004), better calibration methods (Kohn et al., 2004; Peterson et al., 2004). Conversely,
across laboratories may improve the consistency of Arunvipas et al. (2003) reported high reliability and
MUN results. In experiment 2 of the current study, the repeatability for both MilkoScan 4000 and CL 10
range of calibration standards used by the laboratories when analyzed at a single laboratory. In the present
may have not been adequate to cover the range of MUN study, bronopol concentration of 0.54 mg/mL analyzed
in the milk samples with added urea.
on MilkoScan 4000 resulted in about 12% greater (P
The objective of experiment 3 was to determine the < 0.05) MUN than the control (9.6 vs. 8.5 mg/dL,
effect of increasing concentrations of bronopol on MUN respectively). According to one milk processing labora-
as analyzed by laboratory A using 2 MUN methods tory, recommended bronopol concentration is 0.085%,
(MilkoScan 4000 and CL 10). Data from this experi- which is 0.85 mg/mL of milk (Dairy One Cooperative
ment are shown in Figure 1. Concentration of MUN Inc., personal communication). At this concentration,
analyzed on MilkoScan 4000 ranged from 7.7 to 11.9 and based on data from experiment 3 with MilkoScan
0.27 mg/dL and linearly increased (P < 0.001) with 4000 (Figure 1), MUN concentration would likely be
increasing bronopol concentration. Milk urea N concen- overestimated by about 30%. Some have suggested that
trations analyzed on CL 10 ranged from 9.0 to 9.3
bronopol should be used to preserve milk samples at
Table 3. MUN concentrations in milk samples with added urea (experiment 21)
Laboratory
A—MS
Laboratory
Laboratory
C
Item
B
SEM
P-value2
Average analyzed MUN,3 mg/dL
Overall difference,4 mg/dL
Added urea level 1, mg/dL
Analyzed
23.2a
2.2c
18.0c
4.6b
20.6b
5.4a
0.20
0.20
<0.001
<0.001
21.0
19.0
2.2
14.4
11.3
3.1
16.5
13.2
3.3
Predicted
Difference
Added urea level 2, mg/dL
Analyzed
Predicted
Difference
Added urea level 3, mg/dL
Analyzed
0.36
0.55
0.41
0.21
0.02
0.003
25.4
23.0
2.4b
21.5
15.3
6.2a
24.4
17.2
7.2a
31.2
27.0
4.2c
28.6
19.3
9.2b
32.3
21.2
11.2a
Predicted
Difference
a–cMeans within a row having different letter superscripts differ at P ≤ 0.05.
1Difference data are LSM; n = 24 (analyzed MUN) and 6 (added urea levels 1–3), n represents the number of observations used in the statisti-
cal analysis.
2Main effect of laboratory.
3Average MUN across added urea levels. Effect of added urea level, P < 0.001; laboratory × added urea level interaction, P < 0.001.
4Difference between analyzed and expected MUN concentrations (analyzed – expected MUN) across added urea levels. Expected MUN concen-
trations were calculated from amount of urea added and MUN of the control samples from each laboratory. Added urea levels: control = 0, level
1 = 17.2, level 2 = 34.2, and level 3 = 51.5 mg of urea/dL of milk. Differences between analyzed and expected MUN concentrations were close
to zero (10−32 to 9−16 mg/dL) for the control (i.e., 0 mg of added urea) samples.
Journal of Dairy Science Vol. 100 No. 2, 2017

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WEEKS AND HRISTOV
lyzer, MUN concentration may also be affected by the
concentration of bronopol used. Consistently using the
same laboratory and sampling procedure is advisable,
if MUN concentration (bulk tank or individual cows) is
used to monitor protein status of the herd. Thus, es-
tablishing a MUN benchmark relative to the laboratory
and analytical method used may be helpful for on-farm
management purposes.
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Figure1. AverageMUNconcentration( SE)inmilkpreservedwith
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concentrations of 0.01 to 0.02% (Barbano et al., 2010),
or 0.1 to 0.2 mg/mL. At these low concentrations, MUN
analysis is likely not going to be affected by bronopol
(Figure 1). It is noted that MUN analysis by CL 10
was more consistent and was less affected by bronopol
concentration. The CL 10 is widely accepted as the
most accurate measurement of MUN; however, due to
cost and additional labor needed to analyze samples,
it is not practical for commercial use (Arunvipas et
al., 2003). We are not aware of published data on
bronopol interference with MUN analysis. Other milk
components, such as fat, protein, and SCC, do interfere
with the mid-infrared analysis of MUN (Arunvipas et
al., 2003), but the calibration process and computer
algorithm correct for these interferences. Barbano et al.
(2010) studied various milk preservatives and reported
that bronopol-based preservatives interfered with mid-
infrared analyses, particularly for milk protein, com-
pared with K2Cr2O7-preserved milk.
Milk urea N is widely used on farm as an indicator
of dietary CP and RDP concentrations, intake, and N
utilization in dairy cows. Data from the current ex-
periments indicate that reported MUN concentrations
vary between laboratories depending on method and
equipment used. When analyzed by a mid-infrared ana-
Journal of Dairy Science Vol. 100 No. 2, 2017