Nitrite and nitrate represent the final products of nitric oxide (NO) oxidation pathways, and their hematic concentrations are frequently assessed as an index of systemic NO production. However, their intake with food can influence their levels. Nitrite and nitrate could have a role by producing NO, because nitrite can release NO after reaction with deoxyhemoglobin and dietary nitrate can be reduced substantially to nitrite by commensal bacteria in the oral cavity. Different methods have been applied for nitrite/nitrate detection, with the most commonly used being the spectrophotometric assay based on the Griess reagent. However, a reference methodology for these determinations is still missing and many possible interferences have been reported. This chapter assesses how different experimental conditions can influence the results when detecting nitrite and nitrate in human plasma by the Griess assay and provides a simple method characterized by high reproducibility and minimized interferences by plasma constituents.

Effect of ethanol addition on the measurement of nitrite by the Griess reagent in the presence of plasma. Aliquots (100 ml) of nitrite-free plasma were added with 1 ml of 5 mM sodium nitrite and 5 ml of 300 mM NEM. After 2 min, samples were diluted with 400 ml of water/ethanol solutions: (a) water, (b) 25% ethanol, (c) 50% ethanol , and (d) 62.5% ethanol; 500 ml of the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric acid) was then added. After 30 min, samples were deproteinized byTCA addition and analyzed by a spectrophotometer in the 650-to 480-nm range; in the graph, a typical experiment of four replicates is shown.
Nitrite detection by the Griess reagent prepared at different pH values in the presence of NADPH. Sodium nitrite solutions containing 80 mM NADPH (final concentration) were freshly prepared, reacted with the Griess solutions (1:1 ratio) for 30 min, and then analyzed by a spectrophotometer in the 650-to 480-nm range; the height of the peak at 545 nm is reported.The value of nitrite in the abscissa is referred to its final concentration (after 1:1 dilution with the reagent). The number of replicates is four.
Nitrite detection by the Griess reagent prepared at different pH values. Sodium nitrite solutions in MilliQ water were freshly prepared, reacted with the Griess solutions (1:1 ratio) for 30 min, and then analyzed by a spectrophotometer in the 650-to 480-nm range; the height of the peak at 545 nm is reported. The value of nitrite in the abscissa is referred to its final concentration (after 1:1 dilution with the reagent). The number of replicates is three.
Calibration curve. One hundred-microliter aliquots of plasma samples (previously passed through PD10 columns and brought to the initial protein concentration by ultrafiltration) were added with 1 ml of 0.5 to 8 mM sodium nitrate solutions,1 ml of 12.5 mM NADPH,1 ml of 0.5 mM FAD, and 4 ml of nitrate reductase (5 U/ml dissolved in 50 mM phosphate buffer, pH 7.5). After 90 min, samples were treated with 5 ml of 300 mM NEM and, after 2 min, were diluted with 400 ml of a 62.5% ethanol solution and 1:1 reacted with the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric acid). After 30 min, samples were deproteinized by TCA addition and analyzed by a spectrophotometer in the 650-to 480-nm range.The height of peaks recorded at 545 nm is reported.The number of replicates is four.The value of nitrate in the abscissa is referred to as the final concentration of added nitrate to plasma.The calibration curve (closed circles ) was compared with the values reported in Fig. 23.10 (open circles) after subtraction of the basal NOx concentration.

Calibration curve. One hundred-microliter aliquots of plasma samples (previously passed through PD10 columns and brought to the initial protein concentration by ultrafiltration) were added with 1 ml of 0.5 to 8 mM sodium nitrate solutions,1 ml of 12.5 mM NADPH,1 ml of 0.5 mM FAD, and 4 ml of nitrate reductase (5 U/ml dissolved in 50 mM phosphate buffer, pH 7.5). After 90 min, samples were treated with 5 ml of 300 mM NEM and, after 2 min, were diluted with 400 ml of a 62.5% ethanol solution and 1:1 reacted with the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric acid). After 30 min, samples were deproteinized by TCA addition and analyzed by a spectrophotometer in the 650-to 480-nm range.The height of peaks recorded at 545 nm is reported.The number of replicates is four.The value of nitrate in the abscissa is referred to as the final concentration of added nitrate to plasma.The calibration curve (closed circles ) was compared with the values reported in Fig. 23.10 (open circles) after subtraction of the basal NOx concentration.

…

Analysis of the recovery of the added nitrite. Aliquots (100 ml) of nitritefree plasma were added with 1 ml of 1 to 4 mM sodium nitrite solutions, 1 ml of 10 mM NADPH, and 5 ml of 300 mM NEM. After 2 min, samples were diluted with 400 ml of a 62.5% ethanol solution and added with 500 ml of the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric acid). After 30 min, samples were deproteinized by TCA addition and analyzed by a spectrophotometer in the 650-to 480-nm range.The height of peaks recorded at 545 nm is reported (black bars), together with those obtained in similar experiments in which plasma was omitted (gray bars).The number of replicates is four.

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CHAPTER TWENTY-THREE

Nitrite and Nitrate Measurement by

Griess Reagent in Human Plasma:

Evaluation of Interferences and

Standardization

Daniela Giustarini,* Ranieri Rossi,* Aldo Milzani,

†

and

Isabella Dalle-Donne

†

Contents

1. Introduction 362

2. Experimental Procedures 364

2.1. Materials 364

2.2. Blood collection and plasma separation 364

2.3. Evaluation of the effect of pH and NADPH on the

measurement of nitrite 364

2.4 . Evaluation of the effect of plasma dilution 365

2.5. The effect of ethanol addition 365

2.6. Nitrate reductase activity 365

2.7. Recovery of added nitrite 365

2.8. Effect of incubation time with nitrate reductase 366

2.9. Measurement of NOx in plasma samples 366

3. Results 366

4. Discussion 373

Acknowledgment 378

References 378

Abstract

Nitrite and nitrate represent the final products of nitric oxide (NO) oxidation

pathways, and their hematic concentrations are frequently assessed as an

index of systemic NO production. However, their intake with food can influence

their levels. Nitrite and nitrate could have a role by producing NO, because

nitrite can release NO after reaction with deoxyhemoglobin and dietary nitrate

can be reduced substantially to nitrite by commensal bacteria in the oral cavity.

Methods in Enzymology, Volume 440 # 2008 Elsevier Inc.

ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)00823-3 All rights reserved.

*Department of Evolutionary Biology, University of Siena, Siena, Italy

{

Department of Biology, University of Milan, Milan, Italy

361

Different methods have been applied for nitrite/nitrate detection, with the most

commonly used being the spectrophotometric assay based on the Griess

reagent. However, a reference methodology for these determinations is still

missing and many possible interferences have been reported. This chapter

assesses how different experimental conditions can influence the results

when detecting nitrite and nitrate in human plasma by the Griess assay and

provides a simple method characterized by high reproducibility and minimized

interferences by plasma constituents.

1. Introduction

Nitric oxide (NO) mediates many physiological functions and, conse-

quently, it is commonly investigated under many different pathological

conditions. Nevertheless, NO, per se, is difficult to quantify, as a conse-

quence of its short half-life (milliseconds or less, depending on the environ-

ment) in the presence of O

2

and other scavenging molecules, for

example, hemoglobin (Gally et al. , 1990). Therefore, assays that indicate

the presence of NO indirectly are commonly carried out. In particular,

accumulation of the stable degradation products of NO, nitrite (NO

2

–

)and

nitrate (NO

3

–

), S-nitrosothiols, or the increase in cGMP levels (due to NO-

dependent activation of guanylyl cyclase) are measured preferably as an index

of NO production (Forstermann and Ishii, 1996; Giustarini et al., 2003; Tsikas,

2005). Nitrite and nitrate represent the final products of the NO oxidation

pathways and, consequently, their concentration in human body fluids

depends on NO production itself. Other variables can influence the levels of

nitrite/nitrate, such as alimentary intake (van Vliet et al ., 1997).

Plasma nitrite/nitrate content is assessed frequently as an index of NO

production in the whole organism, as plasma is in a dynamic equilibrium

with all organs and interstitial fluids. NO

2

–

and NO

3

–

have always been

considered only as NO end products, without any physiological meaning.

It has been proposed that NO

2

–

may have a role as a signaling molecule by

producing NO, either in the stomach at acidic pH or by reaction with

deoxyhemoglobin, thus inducing hypoxic vasodilation (Gladwin, 2005).

It also seems to act as a modulator of ischemia/reperfusion tissue injury and

infarction (Lundberg and Weitzberg, 2005). Finally, because plasma NO

2

–

sensitively reflects changes in endothelial NOS activity following shear stress in

healthy subjects, but not when endothelial dysfunction occurs, its detection in

human plasma may represent a valid diagnostic tool (Rassaf et al ., 2006).

Analogously, it has been suggested that nitrate of dietary origin can have a

physiological role, as it is reducedsubstantially to nitrite by commensalbacteria

in the oral cavity following an enterosalivary cycle (Lundberg et al ., 2006).

In fact, plasma levels ofnitrite increase significantly after an oral load of sodium

nitrate, corresponding to 300 g of spinach (or other nitrate-rich green leafy

vegetables). This could ensure enough systemic NO levels for being protective

362 Daniela Giustarini et al.

for the cardiovascular system (Lundberg and Govoni, 2004). Furthermore,

dietary NO

3

–

can reduce diastolic blood pressure in healthy subjects (Larsen

et al., 2006). As a consequence, interestregarding NO

2

–

and NO

3

–

is increasing

and the needs of simple, time-sparing, and inexpensive methods to detect them

would be welcome.

A reliable blood measurement of nitrite is difficult because it is unstable,

being oxidized rapidly to nitrate by hemoglobin, with a t

1/2

of about 180 s

(Giustarini et al ., 2004). Once plasma is separated from blood, both nitrite and

nitrate are stable at 20 C for at least 1 year (without hemolysis) (Moshage

et al., 1995). A combined measure of nitrate þ nitrite (NOx) can be performed,

as the procedure is easier than that for nitrite analysis and some critical steps

in sample manipulation needed for nitrite analysis can be avoided. Because

NO

3

–

is largely more abundant than NO

2

–

in body fluids, NOx is almost

synonymous with nitrate in most cases (Tsikas, 2007). Different methods

have been applied for nitrite/nitrate detection, namely colorimetric and fluo-

rometric assays, chemiluminescence, gas chromatography/mass spectrometry,

and capillary electrophoresis (Bryan and Grisham, 2007; Ellis, 1998). The most

commonly used method is the spectrophotometric assay, which is based on

formation of an azo dye by reaction of NO

2

–

with the Griess reagent. Specifi-

cally, NO

2

–

reacts directly with sulfanilamide under acidic conditions and is

then revealed after diazotization with N -(1-naphthyl)ethylenediamine (NED)

(Fig. 23.1). Nitrate can also be measured by this method after its reduction to

nitrite. This can be performed enzymatically (by nitrate reductase) or by

metallic reduction (Cd, Vn) (Ellis, 1998). Notwithstanding their widespread

H2 NO 2S

H2 NO 2 S

H2 NO 2 S

N≡N+

+

N= N

−Ο–N=O

NNH 2

NH2

NH2

H

N

H

+

Figure 23 .1 Schematic diagram representing the Griess reaction principle. Under

acidic conditions, nitrite reacts with sulfanilamide to produce a diazonium ion, which is

then coupled to NED to produce a chromophoric azo product, which strongly absorbs

at 545 nm.

NOx Measurement by Griess Reagent 363

application, a reference methodology for these determinations is still lacking

and many different variants of the Griess reaction are available. Significant

differences can be observed for the type of reagents used to prepare the Griess

solution and their relative ratio, sample manipulation during the preanalytical

phase, protein removal, and reduction of NO

3

–

to NO

2

–

(Tsikas, 2007).

This chapter assesses how different experimental conditions can influ-

ence the results when detecting NOx in human plasma by Griess reagents

and provides an improved method, characterized by high reproducibility

and minimized (and quantified) interferences by plasma constituents.

2. Experimental Procedures

2.1. Materials

Nitrate reductase from Aspergillus , FAD, NADPH, sulfanilamide, N-ethyl-

maleimide (NEM), N -(1-naphthyl)ethylenediamine, and all other reagents

of highest purity available are from Sigma-Aldrich Chemie GmbH. PD10

gel-filtration columns are from Amersham, and ultrafiltration cartridges

(MW 10,000 cutoff ) are from Supelco. Before use, cartridges are washed

extensively with abundant MilliQ water.

2.2. Blood collection and plasma separation

Human blood is obtained from healthy volunteers after informed consensus

by venipuncture, using K

3

EDTA as an anticoagulant. Blood is collected and

centrifuged rapidly at 15,000g for 10 s to separate plasma. This rapid plasma

separation has been shown not to provoke any visible red blood cell

hemolysis.

2.3. Evaluation of the effect of pH and NADPH on the

measurement of nitrite

Griess solutions at different pHs are prepared by adding 1% (w/v) sulfanil-

amide and 0.1% (w/v) NED to different acid solutions: (a) pH 3.3: 2.5% (v/v)

acetic acid brought to the indicated pH with 5 M NaOH; (b) pH 2.5: 2.5%

(v/v) acetic acid; (c) pH 1.46: 2% (v/v) phosphoric acid; (d) pH 1.16: 6% (v/v)

phosphoric acid; (e) pH 0.75: 10% (w/v) trichloroacetic acid (TCA); and (f )

pH 0.6: 0.25 M HCl.

Sodium nitrite solutions in MilliQ water are freshly prepared and reacted

with the different Griess solutions (1:1 ratio) for 30 min in the dark. Samples

are then analyzed in the 650- to 480-nm range by a Jasco V550 spectropho-

tometer. Spectra are recorded against a blank prepared in the same condi-

tions but with a Griess reagent where NED is omitted. The same

364 Daniela Giustarini et al.

experiment is also performed by reacting the different Griess solutions under

the same conditions (a to f) with sodium nitrite solutions containing 80 m M

NADPH [stock solution 10 mM in 0.5% (w/v) sodium bicarbonate].

The dose dependence of NADPH interference on nitrite detection is

assessed by preparing 10 mM sodium nitrite solutions (in MilliQ water) in

the presence of 0 to 100 mM NADPH. These solutions are incubated with

the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric acid) and

analyzed as indicated earlier.

2. 4. Evaluation of the effect of plasma dilution

Nitrite-free plasma is obtained after the storage of human blood at 37 C for

1 h by a 10-s centrifugation at 15,000g . Plasma is diluted up to 80% with

MilliQ, and 500 m l of each dilution is added with 10 m l of 500 mM sodium

nitrite and 5 m l of 300 mM NEM; after 2 min, 500 m l Griess solution (1%

sulfanilamide, 0.1% NED, 6% phosphoric acid) is added. After a 30-min

incubation in the dark, samples are deproteinized by the addition of 3%

(w/v, final concentration) TCA and centrifuged for 2 min at 10,000g, and

supernatants are analyzed by a spectrophotometer as described earlier.

2.5. The effect of ethanol addition

Aliquots (100 m l) of nitrite-free plasma are added with 1 m lof5m Msodium

nitrite and 5 m l of 300 mM NEM. After 2 min, samples are diluted with

400 m l of water/ethanol solutions (0–62.5% ethanol), 1:1 reacted with the

Griess solution (1% sulfanilamide, 0.1% NED, 6% phosphoric acid), TCA

treated, and measured as described earlier.

2.6. Nitrate reductase activity

Nitrate reductase is dissolved in 0.05 M phosphate buffer, pH 7.5, and

assayed by mixing in a cuvette 1 ml of 0.05 M phosphate buffer, pH 7.5,

containing 1 mM sodium nitrate, 0.2 mM NADPH, 0.005 mM FAD, and

3m l of the enzyme-containing solution. NADPH oxidation is evaluated at a

340-nm wavelength. Aliquots of the enzyme-containing solution are then

stored at 4, 20, or 80 C, and enzyme activity is measured with time.

2.7. Recovery of added nitrite

Aliquots (100 m l) of nitrite-free plasma are added with 1 m lof1to4m M

sodium nitrite solutions, 1 m l of 12.5 mM NADPH, and 5 m l of 300 mM

NEM. After 2 min, samples are diluted with 400 m l of a 62.5% ethanol

solution. All samples are then 1:1 reacted with the Griess solution

NOx Measurement by Griess Reagent 365

(1% sulfanilamide, 0.1% NED, 6% phosphoric acid), TCA treated, and

measured as described previously.

2.8. Effect of incubation time with nitrate reductase

Plasma aliquots (0.8 ml) are treated with 8 m lof1to4m Msodium nitrate

solutions. Samples are then added with 8 m l of 12.5 mM NADPH, 4 m lof

1m MFAD, and 32 m l of nitrate reductase (5 U/ml dissolved in 50 mM

phosphate buffer, pH 7.5). At specified times, a 0.1-ml sample is added with

5m l of 300 mM NEM. After 2 min, samples are diluted with 400 m lofa

62.5% ethanol solution, 1:1 reacted with the Griess solution (1% sulfanil-

amide, 0.1% NED, 6% phosphoric acid), TCA treated, and measured as

described earlier.

2.9. Measurement of NOx in plasma samples

One hundred microliters of plasma is added with 1 m l of 12.5 mMNADPH,

1m l of 0.5 mM FAD, and 4 m l of nitrate reductase (5 U/ml dissolved in

50 mM phosphate buffer, pH 7.5); after a 90-min incubation, samples are

added with 5 m l of 300 mM NEM. After 2 min, samples are diluted with

400 m l of a 62.5% ethanol solution, 1:1 reacted with the Griess reagent

(1% sulfanilamide, 0.1% NED, 6% phosphoric acid), TCA treated, and

measured as described previously. For experiments where nitrate is added,

100 m l of plasma is added with 1 m l of 0.5 to 8 mM sodium nitrate. Nitrite/

nitrate-free plasma is obtained by gel filtration on PD10 columns equili-

brated with 50 mM phosphate buffer, pH 7.5. After gel filtration, the initial

protein concentration is restored by ultrafiltration on Supelco Centrisart

(10 kDa cutoff) cartridges. Protein concentration is measured by the

Bradford assay.

3. Results

Colorimetric methods based on the Griess reaction fundamentally

detect NO

2

–

that, under acidic conditions, reacts with sulfanilamide and

NED (Fig. 23.1) to produce an azo compound, which strongly absorbs in

the visible region with a peak around 545 nm. To obtain the acidic pH

necessary for the reaction to proceed, different acids (HCl, acetic acid, or

phosphoric acid) at different final concentrations (and, consequently, at

different final pH values) have been used indifferently (Granger et al.,

1996; Tsikas, 2007). However, little is known about the influence of various

acids on the yield of the reaction.

366 Daniela Giustarini et al.

This chapter evaluated the reaction of NO

2

–

with sulfanilamide and

NED under different pH values. Incubation of sodium nitrite solutions (2–

30 mM ) with the Griess reagent (1% sulfanilamide, 0.1% NED) prepared

with HCl, phosphoric acid, acetic acid, or TCA in the 0.6 to 3.3 pH range

gave different results and indicated that lower pH values resulted in a lower

final absorbance (Fig. 23.2). These differences were not due to an insuffi-

cient incubation time to complete the reactions, as measurements repeated

after a further 30 min did not show any significant increase in absorbance

(data not shown). Data from Fig. 23.2 thus suggest that higher pH values

(2.5–3), resulting in a higher response, are preferred. However, nitrite is not

usually measured by the Griess reaction in water or in buffers, but in

biological samples, which may contain several molecules possibly interfer-

ing with the assay. Among these molecules, NADPH, used to reduce nitrate

to nitrite, is one of the most important and studied (Verdon et al ., 1995).

Therefore, we repeated the experiment shown in Fig. 23.2 in the presence

of 80 mM NADPH. Figure 23.3 shows that NADPH interference with

NO

2

–

detection is higher at higher pH values. Data from Figs. 23.2 and

23.3 suggest that the Griess reagent prepared using 6% phosphoric acid

could be the most suitable solution to measure nitrite in complex biological

samples and in the presence of significant amounts of NADPH. Because

NADPH significantly decreases the slope of the curve even when a 6%

0

0

100

200

300

400

Acetic acid pH 3.3

Acetic acid pH 2.5

Phosphoric acid pH 1.46

Phosphoric acid pH 1.16

TCA pH 0.75

HCl pH 0.60

500

600

700

800

2

Absorbance x 1000

468

Nitrite (mM )

10 12 14 16

Figure 23 .2 Nitrite detection by the Griess reagent prepared at different pH values.

Sodium nitrite solutions in MilliQ water were freshly prepared, reacted with the Griess

solutions (1:1 ratio) for 30 mi n, and the n analyzed by a spect rophotometer in the 650- to

480-nm range; the height of the peak at 545 nm is reported. The value of nitrite in the

abscissa is referred to its final concentration (after 1:1 dilution with the reagent). The

number of replicates is th ree.

NOx Measurement by Griess Reagent 367

solution of phosphoric acid is used (Figs. 23.2 and 23.3), we assessed the dose

dependence of NADPH interference on nitrite detection. Solutions contain-

ing the same amount of sodium nitrite and 0 to 100 mM NADPH were

analyzed for the NO

2

–

content with the Griess reagent prepared with 6%

phosphoric acid (Fig. 23.4). The interference of NADPH in these conditions

was minimal at concentrations below 25 m M . Furthermore, the possible effect

of plasma on nitrite colorimetric measurement was analyzed by adding a NO

2

–

standard concentration to human plasma diluted to different final percentages

with MilliQ. Samples were then assayed with the Griess reagent. Figure 23.5

indicates that plasma constituents decreased the 545-nm peak; in particular, in

the presence of 100% plasma, the height of the peak was less than half of that

obtained in the absence of plasma (i.e., 100% MilliQ).

In the experiments carried out with plasma (Fig. 23.5), proteins were

removed before spectra analysis by TCA precipitation: TCA was added to

samples after a 30-min reaction with the Griess reagent. The use of TCA is

necessary, as sample acidification with the Griess reagent alone does not allow

its deproteinization; consequently, the sample turbidity is excessively high.

After protein precipitation, we noticed that the pellet had an intense purple

color. This was likely a consequence of the coprecipitation of the diazo

compound with the proteins. This phenomenon could be minimized by

adding an alcoholic solution to the final reaction mixture. Plasma samples

0

0

100

200

300

Absorbance x 1000

400

500

2468

Nitrite (m M)

10 12 14 16

Acetic acid pH 3.3

Acetic acid pH 2.5

Phosphoric acid pH 1.46

Phosphoric acid pH 1.16

TCA pH 0.75

HCl pH 0.60

Figure 23. 3 Nitrite detection by the Griess reagent prepared at different pH values in

the presence of NADPH. Sodium nitrite solutions containing 80 mM NADPH (final

concentration) were freshly prepared, reacted with the Griess solutions (1:1 ratio) for

30 min, and then analyzed by a spectrophotometer in the 650- to 480-nm range;

the height of the peak at 545 nm is reported. The value of nitrite in the abscissa is refer red

to its final concentration (after 1:1dilution with the reagent).The number of replicates

is four.

368 Daniela Giustarini et al.

0

40

50

60

70

Absorbance at 545 nm (%)

80

90

100

10 20 30 40

NADPH (mM )

50 60 70 80 90 100

Figure 23 .4 Dose dependence of NADPH interference on nitrite detection. Solutions

of 10 mM sodium nitrite and NADPH at different final concentrations in MilliQ water

were reacted with the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric

acid) for 30 min a ndthen analyzed by a spectrophotometer in the 650- to 480-nm range.

In the graph, the percentage of the absorbance of peaks measured at 545 nm in the

presence of NADPH with respect to those recorded with nitrite alone is reported.

The number of replicates is four.

0

40

50

60

70

Absorbance at 545 nm (%)

80

100

90

10 20 30 40

Plasma (%)

50 60 70 1009080

Figure 23 .5 Evaluationof the effectofplasmaon the measurementofnitritebytheGriess

reaction. Nitrite-free plasmawas diluted from0 to 80% with MilliQ.Five hundredmicroli-

ters of each sample was then added with 10 m lof500mM sodium nitrite and 5 ml of 300 mM

NEM; after 2 min, 500 m l of the Griess reagent (1% sulfanilamide, 0.1% NED, 6%

phosphoric acid) was added. After 30 min, samples were deproteinized by the addition of

TCA and analyzed by a spectrophotometer in the 650- to 480-nm range. In the graph, the

percentage of the absorbance of peaks measured at 545 nm in the presence of plasma with

respectto those recorded with nitritealone is reported.The numberof replicatesis four.

NOx Measurement by Griess Reagent 369

containing a fixed amount of nitrite were added with different ratios of ethyl

alcohol/water and then allowed to react with the Griess reagent (Fig. 23.6).

The heightof the recorded peaks indicates thatthe addition of ethyl alcohol at a

62.5% final concentration to human plasma helps the efficiency of the reaction,

probably diminishing the unspecific binding of the azo dye to albumin.

The experiments shown in Figs. 23.2 to 23.6 as a whole suggest that use

of the Griess reagent containing 6% phosphoric acid, NADPH below

30 mM , four- to fivefold diluted plasma, and 62.5% ethyl alcohol could be

the best condition to minimize both NADPH and plasma constituent

interferences. We tested the efficacy of these conditions by applying them

to detect known amounts of nitrite added both to plasma and to buffer only

(Fig. 23.7). At the three concentrations of NaNO

2

tested (namely, 10, 20,

and 40 m M ), spectra recorded in the presence of plasma were only about

12% lower than those obtained in buffer. Similar values were measured

when plasma from different healthy donors was used, as evidenced by the

low standard deviation reported in Fig. 23.7, indicating a reproducible

event. In addition, the intersample coefficient of variation and the intraday

variation on measurements carried out on the same sample were as low as

2%, suggesting a high reproducibility.

500 520

d

c

b

a

540 560 580 600 620 640

0.00

0.05

0.10

0.15

Absorbance

0.20

Wavelen

th (nm)

Figure 23 .6 Effect of ethanol addition on the measurement of nitrite by the Griess

reagent in the presence of plasma. Aliquots (100 m l) of nitrite-free plasma were added

with 1 m lof5m Msodium nitrite and 5 m l of 300 m MNEM. After 2 m in, samples were

diluted with 400 ml of water/ethanol solutions: (a) water, (b) 25% ethanol, (c) 50% etha-

nol, a nd ( d) 62.5% etha nol;500 m l of the Griess reagent (1% sulfan ilamide, 0.1% NED,

6% phosphoric acid) was then add ed. After 30 min, samples were de proteiniz ed byTCA

addition and analyzed by a spectrophotometer in the 650- to 480 -nm range; in the

graph, a typical ex periment of four replicates is shown.

370 Daniela Giustarini et al.

With the experiments shown in Figs. 23.2 through 23.7, we have

defined the best conditions to minimize interferences and variability due

to plasma constituents during NO

2

–

detection. However, nitrate, and not

nitrite, is the main component of NOx in plasma, as well as in other

biological fluids (Tsikas, 2007). Therefore, it is also crucial to assess the

method used to reduce nitrate to nitrite. Either chemical or enzymatic

reduction can be performed, with the latter being applied most frequently

(Bryan and Grisham, 2007). We have verified the capability of the enzyme

nitrate reductase from Aspergillus niger to reduce nitrate to nitrite under our

conditions. First of all, we have measured the enzyme activity after resolu-

bilization of the lyophilized enzyme and followed it with time after storage

under various conditions (Fig. 23.8). It is noteworthy that, once resolubi-

lized, nitrate reductase decreases its activity rapidly, in particular after cycles

of freezing and thawing; thus, its storage at 70 C, after reconstitution in

aliquots, to limit freezing and thawing cycles and checking its activity before

use are recommended actions. Known concentrations of nitrate were added

to human plasma, and 125 mM NADPH, 5 mM FAD, and 0.2 U/ml of

nitrate reductase were used for its reduction. Figure 23.9 indicates that 40 to

50 min was necessary to reduce all nitrate present in the samples. From these

experiments, we concluded that 90 min was the time that surely allowed the

enzyme to reduce all nitrite under our conditions. This was confirmed by

10

0

20

60

40

100

80

Absorbance x 1000

140

120

180

160

20 40

Nitrite (m M)

Figure 23 .7 Analysis of the recovery of the added nitrite. Aliquots (100 m l) of nitrite-

free plasma were added with 1 m lof1to4m Msodium n itrite s olutions, 1 m lof10m M

NADP H, and 5 m lof300 m MNEM. After 2 min, samples were diluted with 400 m lofa

62.5% ethanol solution and added with 500 m l of the Griess reagent (1% sulfanilamide,

0.1% NED, 6% phosphoric acid). After 30 min, samples were deproteinized by TCA

addition and analyzed by a spectrophotometer in the 650- to 480-nm range.The height

of peaks recorded at 545 nm is reported (black bars), together with those obtained in

simil ar experiments in which plasm a was omitted (g ray ba rs).The number of replicates

is four.

NOx Measurement by Griess Reagent 371

the fact that when nitrite instead of nitrate was added to the samples

(Fig. 23.9, stars), the same absorbance values were obtained.

Finally, we tested the capability of our standardized procedure (as detailed

earlier) to measure nitrate in human plasma samples spiked with know

amounts of NO

3

–

. Figure 23.10 shows the values of absorbance obtained

following the addition of known amounts of nitrate to four different plasma

samples. The perfect linearity obtained for each sample and the similar slope

of the curves suggest high reproducibility.

Usually, calibration curves for NOx measurements are carried out in

water or buffer, as also recommended by manufacturers of kits for NOx

analysis. However, from an analytical point of view, it is more correct to

carry out calibration curves in a matrix as similar to that where NOx will be

measured. Thus, to perform calibration curves, human plasma was passed

through PD10 columns to remove nitrate and nitrite, and samples were

then ultrafiltrated to restore the initial protein concentration. Data are

reported in Fig. 23.11 (closed circles), where a comparison with mean

values obtained from data of Fig. 23.10 after subtraction of the value

measured without the addition of nitrate (basal value) for each sample is

also shown (Fig. 23.11, open circles). A perfect linearity was obtained,

together with high reproducibility of the method, and the slope obtained

with plasma passed through PD10 columns was only slightly lower than that

obtained with plasma samples spiked with nitrate, thus suggesting little

interference by low molecular weight compounds.

0

0

1

2

3

Activity (U/ml)

4

5

1234

Weeks

56

+4⬚C

Freezing thawing

−20⬚C

−70⬚C

78

Figure 23 .8 Variation of nitrate reductase activity with time and under different stor-

age conditions. Lyophilized enzyme was dissolved in 50 mM phosphate buffer, pH 7.5,

and stored at 4, 20, or 70 C divided into aliquots. Enzyme activity was measured

weekly; some samples (freezing/ thawing) were maintained at 20 C but frozen/

thawed once every 3 days.

372 Daniela Giustarini et al.

Once our experimental conditions were verified as a valid procedure to

detect nitrite and nitrate in plasma, we finally measured the NOx concen-

tration in 15 fasting, healthy subjects; the mean plasmatic value was 27.3

8.3 mM (12–45 mM ).

4. Discussion

The most commonly applied method to measure nitrite and nitrate is

based on the Griess reaction, first described by Johann Peter Griess in 1879.

The original Griess reaction has been modified since and, instead of the

original reagents, that is, sulfanilic acid and a -naphthylamine (the so-called

original Griess reagents), sulfanilamide and NED were introduced, follow-

ing the observation that NED offers several advances over a-naphthylamine

in terms of reproducibility, sensitivity, and pH independence of the color

10

Absorbance x 1000

50

100

150

200

250

Incubation time (min)

20 30 40 50 60 70 80 90

Figure 23 .9 Effect of incubation time with nitrate reductase. Plasma aliquots (0.8 ml)

were t reated with 8 m lof1to4m Msodium nitrate solutions to obtain the following

final concentration of added nitrite: 0 mM (closed ci rcles), 10 mM (open circles), 20 m M

(closed triangles), and 40 mM (open triangles). All samples were then added with 8 m lof

12.5 mM NADPH, 4 m lof1m MFA D, a n d 3 2 m l of nitrate reductase (5 U/ml dissolved in

50 mM phosphate buffer, pH 7.5). At the specified times, a 0.1-ml sample was added with

5m lof300 m MNEM. After 2 min, samples were diluted with 400 m l of a 62.5% ethanol

solution and then 1:1 reacted with the Griess reagent (1% sulfanilamide, 0.1% NED,

6% phosphoric acid). After 30 min, samples were deproteinized by TCA addition and

analyzed by a spectrophotometer in the 650- to 480-nm range. The height of peaks

recorded at 545 nm is reported. Stars indicate height of the peaks measured when

the same amounts of nitrite instead of nitrate were added to the sample under the same

conditions.The numbe r of replicates is four.

NOx Measurement by Griess Reagent 373

(Tsikas, 2007). Several colorimetric methods, essentially based on a batch

reaction with Griess reagents with various modifications, for detection of

NOx (namely, nitrite and nitrate) in biological fluids, such as plasma and

urine, have been introduced and described (Tsikas, 2005, 2007). Specifi-

cally, the diazotization reactions are specific for nitrite, but these analyses

also allow the nitrate measurement after its reduction to nitrite. Given the

high content in protein and the relative lower concentration of NOx in

plasma with respect to urine, analytical methods developed to measure

NO

2

–

and NO

3

–

in plasma have to deal with a major number of possible

interferences (Tsikas, 2007). Basically, two crucial points in NOx measure-

ments are evident: (i) quantitative nitrite detection by the Griess reaction in

different biological samples and (ii) efficiency of reduction of nitrate to

nitrite. As a general rule, the more complex nitrite-containing sample, the

higher the probability of interferences. Potential, mechanism-based inter-

ferences may become significant at any step of the diazotization reaction;

in addition, recovery of the azo dye generated by the reaction of nitrite with

sulfanilamide and NED depends on many factors, such as pH, temperature,

0

0

100

200

300

400

500

Absorbance x 1000

Nitrate added (mM )

10 20 30 40 50 60 70 80 90

Figure 23 . 10 Measurement of NOx in plasma samples. One hundred-microliter ali-

quots of plasma samples were added with 1 m lof12.5m MNADPH,1 m lof0.5m MFA D,

and 4 m l of nitrate reductase (5 U/ml dissolved in 50 mM phosphate buffer, pH 7.5). After

90 min, samples were treated with 5 m lof300m MNEM. After 2 min, samples were

diluted with 400 ml of a 62.5% ethanol solution and 1:1 reacted with the Griess reagent

(1% sulfanilamide, 0.1% NED, 6% phosphoric acid). After 30 min, samples were depro-

teinized by TCA addition and analyzed by a spectrophotometer in the 650- to 480-nm

range. Aliquots (100 m l) of plasma samples were added with 1 m lof0.5to8m Msodium

nitrate solutions and treated as described in the text. The height of peaks recorded at

545 nm is reported.The number of replicates is four.The value of nitrate in the abscissa is

referred to as the concentration of n itrate added to plasma.

374 Daniela Giustarini et al.

and relative concentration of the reagents, only to name a few (Fox, 1979).

Interference in the assay by proteins and NADPH is generally recognized,

and most of the previously described methods include protein separation

by ultrafiltration (Miranda et al ., 2001) and NADPH oxidation to NADP

þ

by incubation with NADPH consuming mixtures (e.g., lactate dehydroge-

nase þ piruvate) (Wang et al. , 1997). Alternatively, a NADPH regenerating

system is included, that is, glucose-6-phosphate dehydrogenase þ glucose-

6-phosphate, which allows the use of NADPH at lower concentrations

(Verdon et al ., 1995).

Nevertheless, there is a general belief that Griess assays are reliable for

accurate, quantitative measurement of NOx in biological samples. In par-

ticular, the commercial availability of ''ready-to-use'' kits is extremely

tempting, and NO researchers are making increase use thereof. However,

it is worth mentioning that commercial availability does not automatically

guarantee the accuracy and robustness of the method.

0

0

Absorbance x 1000

50

100

150

200

250

300

350

Nitrate added (mM )

10 20 30 40 50 60 70 80 90

Figure 23 . 11 Calibration curve. One hundred-microliter aliquots of plasma samples

(previously passed through PD10 columns and brought to the initial protein concentra-

tion by ultrafiltration) were add ed with 1 m lof0.5to8 m Msodium nitrate solutions,1 ml

of 12.5 mM NA DPH , 1 m lof0.5m MFA D, a n d 4 ml of nitrate reductase (5 U/ml dissolved

in 50 mM phosphate buffer, pH 7.5). After 90 min, samples were treated with 5 m lof

300 mM NEM and, after 2 min, were diluted with 400 m l of a 62.5% ethanol solution

and 1:1 reacted with the Griess reagent (1% sulfanilamide, 0.1% NED, 6% phosphoric

acid). After 30 min, samples were deproteinized by TCA addition and analyzed by a

spectrophotometer in the 650- to 480-nm range.The height of peaks recorded at 545 nm

is reported.The number of replicates is four.The value of nitrate in the abscissa is referred

to as the final conce ntration of added nitrate to plasma.The calib rationc urve (closed cir-

cles) was compared with the values reported in Fig. 23.10 (open circles) after subtraction

of the basa l NOx concentration.

NOx Measurement by Griess Reagent 375

Over the last years, dozens of papers have included the measurement of

NOx in biological fluids, particularly in plasma/serum, with values ranging

from 4 to 108 mM (Tsikas, 2007). Increased/decreased values for NOx, with

respect to healthy controls, were found in various physiopathological con-

ditions, for example, Crohn's disease, stenosis, diabetes, and renal failure

(Adachi et al., 1998; Ishibashi et al. , 2000; Maejima et al. , 2001; Oudkerk

Pool et al. , 1995).

This chapter evaluated the main interferences to NOx measurements in

plasma and provided an inexpensive, easy, and time-sparing standardized

procedure for plasma NOx measurement. It is noteworthy that the same

methodology could likely be applied to also measure NOx in other

biological samples. All the reported measurements were carried out record-

ing the whole 650- to 480-nm spectra and not a single point(s); moreover,

spectra were recorded against a blank constituted by the sample to be

analyzed and the same reagents, where only NED was omitted.

First, we evaluated the effect of different pH values on the Griess

reaction. The pH influence was described extensively by Fox (1979), who

reported that many factors may influence the diazotization reaction, among

these pH, with a maximal pigment formation in the 2.5 to 3.5 pH range.

Commonly used Griess reagents can be prepared by using different acids,

namely phosphoric acid, acetic acid, and HCl, and, frequently, the final

concentrations are different too (Granger et al ., 1996; Tsikas, 2007). We

confirmed that pH can influence the yield of nitrite titration significantly,

with higher performance at higher pH values (Fig. 23.2). It was also evident

that NADPH interference is maximized at 3 to 4 pH values (Fig. 23.3).

From our data it is clear that the Griess solution prepared with 6% phos-

phoric acid (pH 1.16) is the best condition, allowing both a good yield of

the reaction and low NADPH interference. We also identified at what

concentration both NADPH and plasma interference could be tolerated

(Figs. 23.4 and 23.5). In addition, we showed that ethyl alcohol addition to

the mixture greatly reduces the coprecipitation of the azo dye with plas-

matic proteins (Fig. 23.6), which must be removed before measurements

because they can produce turbidity and absorb at 540 nm, particularly if the

sample is slightly hemolyzed. Combining all the information obtained from

experiments shown in Figs. 23.2 to 23.7, we concluded that sample dilu-

tion, ethyl alcohol addition to plasma samples, and use of a Griess solution

prepared with a higher phosphoric acid content (6% instead of 2%) mini-

mized all the possible interferences satisfactorily. Nitrite detection under

these conditions, both in buffered solutions and in plasma samples (added

with the same amounts of nitrite), gave similar results, thus confirming our

findings (Fig. 23.7).

Once verified that added nitrite was measured by our method quantita-

tively, we analyzed the other crucial step in NOx measurement, that is,

nitrate reduction by nitrate reductase. Enzymatic reduction is carried out

376 Daniela Giustarini et al.

more commonly than chemical reduction, whose major drawback is that

some metals lead to incomplete reduction or carry the reduction further

than nitrite (Cortas and Wakid, 1990), and various conditions have been

reported (Tsikas, 2007). Most commercial kits also include reduction of

nitrate by nitrate reductase from Aspergillus . However, little is reported

about the stability with time of this enzyme. In some kits (e.g., from

Cayman Chemical Company), it is reported that the enzyme can be frozen

and thawed once after its reconstitution with buffer. Information on nitrate

reductase stability was also reported by Ricart-Jane

´and colleagues (2002),

who evidenced how stability of the bacterial nitrate reductase is limited and,

after storage at both 4 and 20 C, is lost rapidly. We pointed out that

nitrate reductase is not stable after its reconstitution with buffer (Fig. 23.8).

In particular, cycles of freezing and thawing should be avoided and mea-

surement of its activity is highly recommended before use. The reason for

such instability, to the authors' knowledge, is unknown. Experiments with

nitrate added to plasma (Fig. 23.9) led us to conclude that, under our

conditions, the reduction of nitrate is complete within 45 to 60 min and

that nitrate added is recovered quantitatively (Figs. 23.10 and 23.11).

Moreover, since only a slightly different slope was obtained after nitrate

addition both to human plasma and to the same sample after gel filtration,

we suggest that calibration curves can be carried out by the addition of

nitrate to plasma after G25 gel filtration. The mean nitrate concentration

measured (27.3 8.3 m M ) in healthy humans is consistent with many other

previous findings (Tsikas, 2007).

The method for NOx measurements in plasma/serum described here is

based on the following points.

i. Accurate measurement of spectra in the 480- to 650-nm range against

the same samples reacted with a modified Griess solution, where NED

was omitted. This procedure allowed us to minimize, and eventually

evaluate, deviations of recorded spectra due to turbidity or colored

protein presence. This procedure allowed us to also detect spectra

with peaks at 545 nm as low as 0.02 OD, with the limit of detection

in the final mixture being close to 0.5 m M.

ii. Sample dilution: a fivefold dilution can be carried out, considering the

method detection limit and plasma NOx levels. This allowed us to

eliminate both ultrafiltration, which is critical, as the material contained

in some cartridges could release significant amounts of nitrate and nitrite

(Everett et al ., 1995), and procedures for NADPH interference mini-

mization (e.g., elimination by lactate dehydrogenase), which are time-

consuming and expensive.

iii. Use of a 6% phosphoric acid solution to prepare the Griess reagent

minimizes pH variation due to a protein-rich sample addition.

NOx Measurement by Griess Reagent 377

iv. Addition of ethanol to the solution used to dilute samples largely

decreases the unspecific binding of the azo compound to plasmatic

proteins.

The method was found to be reproducible with intersample coefficient

of variance and intraday variation on measurements carried out on the same

sample as low as 2%, with high linearity in the range of 5 to 80 mM NOx.

In view of the large number of applications that the Griess reaction has

found in clinical and experimental studies, it is crucial to apply easy-to-

perform and virtually low artifact-prone methods. Investigators from vari-

ous disciplines are the users of these analytical methods, and there is no

doubt that assays based on the Griess reaction are the most frequently

applied analytical methods for the analysis of nitrite and nitrate in the NO

area of research. However, as stated by Tsikas (2007), we agree that accurate

quantitative determination of NOx in plasma (or serum) is still a challenging

analytical task. Many of the common methodological problems may arise

from preanalytical factors, particularly from the shortcoming in the assay

related to the Griess reaction itself. In addition, it must be emphasized that

calibration curves are usually performed with nitrite instead of nitrate (i.e.,

the main component of NOx) and, moreover, in water or buffer, which are

very different from plasma. The most critical point in the measurement of

NOx in biological samples by batch Griess assays seems to be the unknown

recovery rate (Tsikas, 1997).

This chapter reported on a new Griess assay able to measure NOx in

plasma samples with high precision and accuracy and also quantified the

recovery rate of both nitrate and nitrite. Because there are no time-

consuming and expensive steps (e.g., ultrafiltration), it appears suitable for

use in future clinical studies performed to analyze further the role of nitrite/

nitrate as potential reservoirs of NO activity and the link between NOx

levels and disease conditions.

ACKNOWLEDGMENT

This work was supported by FIRST 2006 (Fondo Interno Ricerca Scientifica e Tecnologica)

University of Milan.

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380 Daniela Giustarini et al.

... Nitrate can be measured with the same method after its stoichiometric reduction to nitrite [15][16][17]. This reaction can be accomplished with several inorganic reagents and with a specific natural enzyme that uses NAD(P)H as the reducing co-factor [18]. In this case, both nitrite (if any present) and nitrate are measured in the sample, and speciation can be achieved by separately measuring nitrite without and following the reduction of sample nitrate [15][16][17]19]. ...

... The Griess method has been adapted to the measurement of nitrite/nitrate in biological samples, such as plasma/plasma, tissue homogenates, and urine [11,18]. Preanalytical sample preparation, especially the elimination of proteins, which interfere with the assay, is necessary for obtaining robust results. ...

... The use of Cadmium granules in packed columns for the Jones method is widely employed, especially in automatic water analyzers [18]. However, the method employs a carcinogenic reagent, for which a risk assessment procedure should be performed, and use is not authorized when alternatives exist. ...

The metabolism of nitric oxide plays an increasingly interesting role in the physiological response of the human body to extreme environmental conditions, such as underwater, in an extremely cold climate, and at low oxygen concentrations. Field studies need the development of analytical methods to measure nitrite and nitrate in plasma and red blood cells with high requirements of accuracy, precision, and sensitivity. An optimized spectrophotometric Griess method for nitrite–nitrate affords sensitivity in the low millimolar range and precision within ±2 μM for both nitrite and nitrate, requiring 100 μL of scarcely available plasma sample or less than 50 μL of red blood cells. A scheduled time-efficient procedure affords measurement of as many as 80 blood samples, with combined nitrite and nitrate measurement in plasma and red blood cells. Performance and usefulness were tested in pilot studies that use blood fractions deriving from subjects who dwelt in an Antarctica scientific station and on breath-holding and scuba divers who performed training at sea and in a land-based deep pool facility. The method demonstrated adequate to measure low basal concentrations of nitrite and high production of nitrate as a consequence of water column pressure-triggered vasodilatation in deep-water divers.

... Ten grams of processed meat were chopped using a blender (Moulinex, France) for 30 s and then added 100 mL phosphate buffer (Na 2 HPO 4 : NaH 2 PO 4 ; pH = 2; 30 ± 1 °C). The acidic conditions stimulate nitrite reacts with sulfanilamide to produce a diazonium ion, which is then coupled to NEDA to produce an azo product (Giustarini et al. 2008), while phosphate buffer for nitrite extraction easily and keeps the nitrite stable in solution. The mixture was sonicated for 15 min, 35 ± 1 °C, and then left to settle for 10 min. ...

Sodium nitrite is a common practice additive in meat products for enhancing color, flavor, and microbial quality; however, several studies link its post-procession to the formation of carcinogenic N-nitrosamines. The aim of this study was to fabricate and validate a portable, rapid, and accurate microfluidic polydimethylsiloxane (PDMS) analytical device to determine the nitrite concentration in meat products. The colorimetric determination of nitrite using PDMS is based on Griess reaction. The proposed PDMS method was validated and the uncertainty evaluated according to Eurachem guidelines. Results demonstrated that PDMS device's detection and quantification limits for nitrite were 0.1 and 0.4 mg L−1, respectively. A complete correlation (R2 = 0.99) and RSD value was 1.3–2.1% (inter-day precision) and 1.5–2.4% (intra-day precision). The PDMS method is highly robust, accurate 98.8%, and uncertainty measurement 3.5%. No significant differences between PDMS device and spectrophotometry were found during the determination of nitrite in examined processed meat products. The overall analysis of the PDMS device could be completed within 10 min. The PDMS-based device has potential applications for rapid detection and routinely screen the nitrite concentration of meat products and guarantee food safety.

... In brief, after adding 100 µL supernatant to the Griess reagent, samples were transferred to a 96-well flat-bottomed microplate, and the absorbance was read at 520 nm using a microplate reader (EL800, BioTek Instrument, Winooski, VT, USA). Final values were calculated from standard calibration plots (Kleinbongard et al., 2002;Giustarini et al., 2008;Baghcheghi et al., 2018b). Each standard and sample was measured in duplicate. ...

... In this method, the degree of DPPH radical discoloration is often used as an index of the antioxidant capacity of the tested samples. In our previous work [9], we observed that GEO has a higher power to diminish the dark violet color of DPPH radical to yellow diphenylpicrylhydrazine radical in comparison to CEO and that GEO was found to be rich in monoterpenoid and citronellol and geraniol were the major component of the oil and they also have been previously identified as a potential antioxidant [26,27]. The DPPH radical scavenging capacity of extracted EOs was previously reported by us as 85:51 ± 0:020% (for GEO) and 78:06 ± 0:04% (for CEO) at 250 μg/ml [9]. ...

The present study deals with the evaluation of the age-defying potential of topical cream formulations bearing Geranium essential oil/Calendula essential oil-entrapped ethanolic lipid vesicles (ELVs). Two types of cream formulations were prepared, viz., conventional and ELVs spiked o/w creams. Essential oil- (EO-) loaded ELVs were characterized by vesicle size, polydispersity index, encapsulation efficiency, and scanning electron microscopy. The cream formulations were evaluated for homogeneity, spreadability, viscosity, pH, in vitro antioxidant capacity, sun protection factor, and in vitro collagenase and elastase inhibition capacity. Confocal laser scanning microscopy (CLSM) was performed to ascertain skin permeation of conventional and vesicular cream. The results of in vitro antioxidant studies showed that GEO-/CEO-loaded vesicular creams have notable antioxidant capacity when compared to nonvesicular creams. GEO- or CEO-loaded vesicular creams exhibited the highest SPF value 10.26 and 18.54, respectively. Both the EO-based vesicular creams showed in vitro collagenase and elastase enzyme inhibition capacity. CLSM images clearly depicted that vesicular cream deep into the skin layers. From the research findings, the age-defying potential and photoprotective effects of GEO and CEO were confirmed. It can be concluded that ELVs are able to preserve the efficiency of EOs and have the potential to combat skin aging.

... The concentration of nitrites (NO 2 − ) was determined using the Griess reaction method [53][54][55] . Griess reagent was obtained by dissolving 1% wt/v sulphanilamide, 0.1% wt/v N-(1-naphtyl) ethylenediamine and 5% wt/v phosphoric acid in Milli-Q water. ...

Atmospheric pressure plasma jets have been shown to impact several cancer cell lines, both in vitro and in vivo. These effects are based on the biochemistry of the reactive oxygen and nitrogen species generated by plasmas in physiological liquids, referred to as plasma-conditioned liquids. Plasma-conditioned media are efficient in the generation of reactive species, inducing selective cancer cell death. However, the concentration of reactive species generated by plasma in the cell culture media of different cell types can be highly variable, complicating the ability to draw precise conclusions due to the differential sensitivity of different cells to reactive species. Here, we compared the effects of direct and indirect plasma treatment on non-malignant bone cells (hOBs and hMSCs) and bone cancer cells (SaOs-2s and MG63s) by treating the cells directly or exposing them to previously treated cell culture medium. Biological effects were correlated with the concentrations of reactive species generated in the liquid. A linear increase in reactive species in the cell culture medium was observed with increased plasma treatment time independent of the volume treated. Values up to 700 µM for H2O2 and 140 µM of NO2− were attained in 2 mL after 15 min of plasma treatment in AdvDMEM cell culture media. Selectivity towards bone cancer cells was observed after both direct and indirect plasma treatments, leading to a decrease in bone cancer cell viability at 72 h to 30% for the longest plasma treatment times while maintaining the survival of non-malignant cells. Therefore, plasma-conditioned media may represent the basis for a potentially novel non-invasive technique for bone cancer therapy.

... Another example, the Griess reaction assay-based methods are widely used in nitrate and nitrite determination. It has been reported that the high concentration of hydrogen ion increases the diazotization rate and lower the coupling rate, since the hydrogen ion act first as a reactant and then as a product in principle color reaction [46]. Hence, the best reaction time is determined by the acidities in the whole reaction system. ...

Soil health monitoring is crucial to maintain a sustainable agricultural yield and food security. Therefore, the efficiency and accuracy of monitoring methods are of paramount importance. Colourimetric detection relies on a color change given by interaction or reaction between the analyte and a chemical reagent. The colourimetric method must be robust, low-cost, and straightforward and is ideally the best option for the in situ test. Commercial colourimetric test kits often rely on visual inspection with the naked eye to estimate the target level, but the interferences, such as background light intensity and soil color, limit their accuracy and therefore their applicability. As such, these commercial test kits can only provide an estimation of a concentration range instead of quantified results for each analyte. With the use a smartphone, some of these issues can be overcome through normalization and digitization of colourimetric test results. The smartphone is a good starting point to link new technologies, such as artificial intelligence (AI), unmanned devices, and human-machine interface (HMI), to traditional analysis in both agricultural and environmental protection. Furthermore, if combined with the implementation of the chemistry in a microfluidic device, it could provide a cost-effective "lab-on-chip" system for in-field colourimetric application. In this chapter, we focus on the selection of chemical reagents for colourimetric detection, and discuss the possible interferences in both the colourimetric reaction and the digital data processing using smartphones, and then link to the recent progress of microfluidic devices used for colourimetric soil nutrients testing.

... Control groups were treated with or without LPS in the absence of sample. The supernatant (100 µL) was harvested and NO production was determined using the Griess assay [26]. Briefly, cells were treated with each compound at the same above concentrations. ...

  • ChoEen Kim
  • DucDat Le
  • Mina Lee

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest pain, arthritis, rheumatism, and sexually transmitted diseases. To identify natural products having efficacy against inflammatory bowel disease (IBD), we identified a new, 16-hydroxy-4β-carboxy-O-β-D-glucopyranosyl-19-nor-totarol (4) together with three known diterpenoids from P. macrophyllus. Furthermore, all the extracts, fractions, and isolates 1-4 were investigated for their anti-inflammatory effects by assessing the expression on nitric oxide (NO) production and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 and HT-29 cells. Among them, nagilactone B (2) exhibited a potent anti-inflammatory effect against NO production on RAW 264.7 cells; therefore, nagilactone B was further assessed for anti-inflammatory activity. Western blot analysis revealed that nagilactone B significantly decreased the expression of LPS-stimulated protein, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and phosphorylated extracellular regulated kinase (pERK)1/2. In addition, nagilactone B downregulated tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels in LPS-induced macrophages and colonic epithelial cells. To our best knowledge, this is the first report on the inhibitory effect of nagilactone B (pure state) and rakanmakilactone G against NO production in LPS-stimulated RAW 264.7 cells. Thus, diterpenoids isolated from P. macrophyllus could be employed as potential therapeutic phytochemicals for IBD.

Aim Male reproductive toxicity is becoming of growing significance due to clinical chemotherapy usage. Methotrexate (MTX) is an anti-folate used on a large scale for different tumors and autoimmune conditions. Despite its wide clinical use, MTX is associated with severe testicular intoxication. The exact underlying mechanism is unclear. Methods Our study was conducted to explore the pathogenesis mechanism of MTX-induced testicular damage and the potential testicular protective effects of apocynin (APO) on testicular injury induced by single i.p. MTX (20 mg/kg). APO was administered orally (100 mg/kg) for ten days. Results As compared to rats given MTX alone, co-administration of MTX with APO demonstrated multiple beneficial effects evidenced by a marked increase in testosterone, FSH, and LH and significantly restored testes histopathological alterations. Mechanistically, APO restored antioxidant status through up-regulation of Nrf2, cytoglobin, PPAR-γ, SIRT1, AKT, and p-AKT, while effectively lowering Keap-1. Moreover, APO significantly attenuated inflammation by down-regulating NF-κB-p65, iNOS, and TLR4 expressions confirmed by in-silico evidence. Additionally, network pharmacology analysis, a bioinformatics approach, was used to decipher various cellular processes' molecular mechanisms. Significance The current investigation proves the beneficial effects of APO in MTX-associated testicular damage through activation of cytoglobin, Keap-1/Nrf2/AKT, PPAR-γ, SIRT1, and suppressing of TLR4/NF-κB-p65 signal. Our data collectively encourage extending the investigation to the clinical setting to explore APO effects in MTX-treated patients.

  • Sasi Bhushan Gottimukkala
  • Giridhar Sethuraman
  • Srinivasan Kitchanan
  • Surajit Pathak

Background & objectives: Phototherapy (PT) has become the standard of care for treating neonatal jaundice. This study was aimed to find out if intermittent PT (IPT) results in comparable rate of fall of bilirubin level to continuous PT (CPT) and results in lesser side effects and better acceptance. Methods: In this non-inferiority trial, 174 neonates ≥35 wk gestation and >2000 g with jaundice requiring PT were randomized to receive either IPT (one hour on and two hours off) or CPT (with minimum interruptions for feeding) after device stratification [light-emitting diode (LED) or compact fluorescent light (CFL)]. Bilirubin was checked 12th hourly, and calcium, vitamin D and nitric oxide (NO) levels were analyzed along with the clinical side effects and nursing and maternal satisfaction scores (CTRI Registration No. CTRI/2018/01/011072). Results: The rate of fall of bilirubin was similar in both the CPT and IPT groups [0.16 (0.10, 0.22) vs. 0.13 (0.09, 0.20) mg/dl/h, P=0.22]. The median difference with 95 per cent confidence interval of 0.03 (0.03, 0.03) mg/dl was also within the pre-defined inferiority limits. There was no significant change in the duration of PT and side effects such as fall in calcium levels, rise in vitamin D and NO levels or the clinical side effects. Maternal satisfaction favoured the IPT group, but the nurses opined that IPT was difficult to implement. Subgroup analysis for PT devices used showed that efficacy of both CFL and LED devices was equivalent. Interpretation & conclusions: IPT was non-inferior to CPT in reducing bilirubin levels in ≥35 wk neonates, irrespective of device used, and also mothers reported better satisfaction with IPT. Although IPT appears promising, CPT does not increase clinical and biochemical side effects compared to IPT.

  • Afolabi Clement Akinmoladun Afolabi Clement Akinmoladun
  • Morenikejimi Bello
  • Emmanuel Oluwafemi Ibukun

Context The effect of Eucalyptus globulus in diabetic cardiac dysfunction and the possible mechanisms involved have not been explored. Objective To evaluate the effect of ethanol leaf extract of E. globulus (NEE) on the cardiac function of fructose/streptozotocin-induced diabetic rats. Materials and methods Type-2 diabetes was induced in rats with 10% fructose feeding for 14 days and an intraperitoneal administration of 40 mg/kg streptozotocin. Diabetic animals were treated with NEE (100–400 mg/kg) or 5 mg/kg glibenclamide orally for 21 days. Biochemical assays, histopathological examination and analyses of PCSK9, Rho kinase and Cardiac troponin expression were performed. Results The untreated diabetic group showed decreased expression of the genes, oxidative stress, dyslipidemia, increased activities of creatine kinase MB and lactate dehydrogenase, reduced nitric oxide level, and depletion of cardiomyocytes, which were reversed in NEE treated groups. Conclusions Eucalyptus globulus ameliorated diabetic cardiac dysfunction through increased PCSK9, Rho kinase and Cardiac troponin expression.

Several observations suggest that the Ca2(+)-dependent postsynaptic release of nitric oxide (NO) may be important in the formation and function of the vertebrate nervous system. We explore here the hypothesis that the release of NO and its subsequent diffusion may be critically related to three aspects of nervous system function: (i) synaptic plasticity and long-term potentiation in certain regions of the adult nervous system, (ii) the control of cerebral blood flow in such regions, and (iii) the establishment and activity-dependent refinement of axonal projections during the later stages of development. In this paper, we detail and analyze the basic assumptions underlying this NO hypothesis and describe a computer simulation of a minimal version of the hypothesis. In the simulation, a 3-dimensional volume of neuropil is presented with patterned afferent input; NO is produced, diffuses, and is destroyed; and synaptic strengths are determined by a set of synaptic rules based on the correlation of synaptic depolarization and NO levels. According to the hypothesis, voltage-dependent postsynaptic release of this rapidly diffusing substance links the activities of neurons in a local volume of tissue, regardless of whether the neurons are directly connected by synapses. This property is demonstrated in the simulation, and it is this property that is exploited in the hypothesis to account for certain aspects of long-term potentiation and activity-dependent sharpening of axonal arbors.

  • M Oudkerk Pool
  • G Bouma
  • J J Visser
  • Amado Salvador Peña Amado Salvador Peña

Nitric oxide is an important mediator in inflammatory and autoimmune-mediated tissue destruction and may be of pathophysiologic importance in inflammatory bowel disease. We studied whether serum levels of nitrate, the stable end-product of nitric oxide, are increased in active Crohn's disease or ulcerative colitis, in comparison with quiescent disease and healthy controls. The setting was the gastroenterology unit of the Free University Hospital, Amsterdam. In 146 patients--75 with ulcerative colitis and 71 with Crohn's disease--and 33 controls serum nitrate was measured by the Griess reaction after enzymatic conversion of nitrate to nitrite with nitrate reductase. Median serum nitrate concentrations did not differ statistically significantly between ulcerative colitis (median, 34.2 mumol/l; range, 15.6-229.4 mumol/l), Crohn's disease (median 32.3 mumol; range 13.2-143.2 mumol/l), and healthy controls (median, 28.7 mumol/l; range, 13.0-108.4 mumol/l). However, when active ulcerative colitis patients (median, 44 mumol/l; range, 29.1-229.4 mumol/l were compared with inactive ulcerative colitis patients (median, 31.2 mumol/l; range, 15.6-59.7 mumol/l), a significant difference in nitrate concentration was found (p < 0.0001). A significant positive correlation was found between serum nitrate levels in ulcerative colitis and erythrocyte sedimentation rate (ESR) (r = 0.30, p - 0.01), leucocyte count (r = 0.27, p = 0.02), and thrombocyte count (r = 0.24, p = 0.04). Comparing active Crohn's disease patients (median, 37.5 mumol/l; range, 13.2-143.2 mumol/l) with inactive Crohn's disease patients (median, 31.3 mumol/l; range, 14.5-92.3 mumol/l) also showed a significant difference in serum nitrate concentration (p < 0.009). Serum nitrate levels correlated with the ESR (r = 0.26, p = 0.028) and serum albumin (r = 0.38, p = 0.004) as well. Nitric oxide production is increased in both active ulcerative colitis and Crohn's disease and may be implicated in the pathogenesis of inflammatory bowel disease.

Plasma nitrite and nitrate determinations are increasingly being used in clinical chemistry as markers for the activity of nitric oxide synthase and the production of nitric oxide radicals. However, a systematic evaluation of the determination of nitrite and nitrate in plasma has not been performed. In this study the recovery and stability of nitrite and nitrate in whole blood and in plasma, the relation between nitrite and nitrate concentrations in plasma, and possible sources of artifacts were investigated. The main conclusions are: (a) Recovery of nitrite and nitrate from plasma is near-quantitative (87%) and reproducible; (b) nitrite and nitrate are stable in (frozen) plasma for at least 1 year; (c) nitrite in whole blood is very rapidly (> 95% in 1 h) oxidized to nitrate, and therefore plasma nitrite determination alone is meaningless; (d) the ranges of nitrite and nitrate concentrations in plasma samples of 26 healthy persons are 1.3-13 mumol/L (mean 4.2 mumol/L) and 4.0-45.3 mumol/L (mean 19.7 mumol/L), respectively; (e) plasma nitrite and nitrate concentrations were not correlated (nitrite as % of total nitrite + nitrate varied from 3.9% to 88% in plasma samples); and (f) plasma samples should be deproteinized, and background controls for each sample should be included in the assay, to avoid measuring artifactually high nitrite and nitrate concentrations in plasma.

  • Jay B. Fox

The kind and position of substituants on the aniline and naphthylamine reagents used in the Griess method of nitrite analysis were assayed, and the factors that control the rate, amount, and stability of the pigment formed from the reaction were defined. This work has shown that the Hammett relationship may be used to determine in part the utility of the aniline derivatives under the unique conditions of nitrite analysis. However, specific reagent combinations result in poor pigment production because of multiple reactions, multiple products, incomplete conversion, and pigment instability. Impediments to complete conversion of nitrite to diazo pigment include multiple nitrous acid reactions with both the aniline and naphthylamine derivatives and instability of reaction intermediates. Other factors critical to pigment production, including pH, temperature, and concentration of reagents, both relative and absolute, were quantitated. Added reductants usually result in lessened pigment production, except with certain reagent combinations where the same or higher concentrations are produced. Criteria are given for establishing the utility of reagent combinations to be used for nitrite analysis.

  • Junko Adachi
  • Sumiharu Morita
  • Hisafumi Yasuda
  • Y Tatsuno

We investigated the nitric oxide profile in the plasma of ten healthy controls and 29 patients hurt by the Kobe Earthquake. The levels of nitrite (NO2-) and nitrate (NO3-) were measured simultaneously by high-performance liquid chromatography (HPLC) with UV detection using the Griess reaction after the reduction of nitrate to nitrite. Arginine consumed in food or diet-derived nitrite had no effect on the plasma nitrite and nitrate contents of the healthy volunteers. Plasma nitrate was elevated in the crush syndrome patients; whereas neither nitrite nor nitrate was increased in patients with normal renal function. This finding suggests increased nitric oxide synthesis, decreased excretion of nitric oxide in the crush syndrome or both.

  • Najwa K. Cortas
  • Nabll W Wakid

Nitrate in serum and urine was assayed by a modification of the cadmium-reduction method; the nitrite produced was determined by diazotization of sulfanilamide and coupling to naphthylethylene diamine. After samples were deproteinized with Somogyi reagent, the nitrate was reduced by Cu-coated Cd in glycine buffer at pH 9.7 (2.5 to 3 g of Cd granules for a 4-mL reaction mixture). The reduction followed pseudo-first-order reaction kinetics, a convenient time interval for assay being 75 to 90 min. Maximum reduction (85%) occurred at about 2 h. Detection limits in urine or serum were 2 to 250 mumol/L. This method does not require the reaction to go to completion, does not require expensive reagents or equipment, and can assay several samples simultaneously. Repeated assays of two serum pools gave CVs of 9.0% and 4.7% for nitrate concentrations of 31.4 and 80.2 mumol/L, respectively (n = 20 each). The mean concentration of nitrate was 1704.0 +/- 1294 (SD) mumol/L (n = 21) in untimed normal urine, 81.8 +/- 50.1 mumol/L in serum of 38 renal dialysis patients, and 51.2 +/- 26.4 mumol/L in serum of 38 controls.

The analysis of nitric oxide-derived nitrite and nitrate ions in biological fluids represents a proven strategy for determining nitric oxide participation in a diverse range of physiological and pathophysiological processes in vivo. In this article we describe a versatile method for the simultaneous measurement of NO2- and NO3- anions in both plasma and isolated tumour models based on anion-exchange chromatography with spectrophotometric detection (214 nm). This method compares well with the capillary electrophoresis technique, exhibiting an equivalent sensitivity for NO2-/NO3- anions and short run-times, i.e. not greater than 4 min. Comparisons are also made with two alternative but less satisfactory methods which employ ion-exchange or reversed-phase ion-pair chromatography with conductimetric as well as spectrophotometric detection. Technical problems associated with each method, particularly those arising from nitrate contamination, have been addressed.

  • C.P. Verdon
  • B.A. Burton
  • Ronald L Prior Ronald L Prior

An assay for the simultaneous measurement of nitrite and nitrate, products of nitric oxide metabolism, is described. Others have reported pretreating sample by using nitrate reductase (NR) and NADPH to reduce endogenous NO3- before assaying the resultant NO2- using the Griess reaction. However, we found that the NADP+ formed during pretreatment interfered with the Griess reaction when NADPH was used at concentrations necessary to drive the NR reaction. For instance, 500 microM NADP+ in 100 microM NaNO3- (without NR) causes a 90% interference with the formation of Griess reaction product. To limit interference, we modified the method by decreasing the NADPH concentration to 1 microM. NADPH was regenerated by coupling the NR reaction with that catalyzed by glucose-6-phosphate dehydrogenase (GD). Using this method, NaNO3- standard curves were linear up to 100 microM and coincided with control curves obtained using NaNO2- incubated in parallel. Addition of urine up to a strength of 20% did not interfere with the assay. Comparison with an alternative assay based on cadmium reduction resulted in the following linear regression: [Cd method] = 0.915*[NR-GD method] + 0.37, r2 = 0.997. Coupling GD to NR to recycle NADPH allows this cofactor to be used at a low concentration so that interference with the Griess reaction is negligible.

Assay methods based on the Griess reaction are frequently used to measure nitrite and nitrate in urine, plasma, and other biological fluids. With minor exceptions, careful attention has not been paid in extending the Griess assay from aqueous solutions to biological fluids, In the present study, parallel measurements of nitrite and nitrate were performed in urine, plasma, and aqueous solutions with a published batch assay based on the Griess reaction and with gas chromatography-mass spectrometry (GC-MS). We report here further interferences by free reduced thiols, proteins, and other plasma constituents in the Griess assay but not in GC-MS. The best correlation (r2 = 0.985) between the Griess assay and GC-MS was observed for aqueous solutions in the absence of thiols. Unlike GC-MS, the Griess assay was not applicable to whole human plasma and urine samples. For the measurement of nitrate in diluted human urine samples, reduction by cadmium was performed both under acidic (pH 2 or 5) and alkaline (pH 8.8) conditions. The mean recovery rate of nitrate from urine samples was quantitative in the GC-MS but amounted to only 30-80% in the Griess assay. Measurement of nitrate in human urine samples (n = 33) resulted in an excellent correlation between two GC-MS techniques (r2 = 0.979) but only in a poor correlation (r2 < 0.64) between the Griess assay and GC-MS. Unlike GC-MS, the batch Griess assay is associated with many problems in measuring nitrate in biological fluids.