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| Proc Natl Acad Sci U S A. 2001 April 10; 98(8): 4617–4621. doi: 10.1073/pnas.071057598. | PMCID: PMC31883 |
Copyright © 2001, The National Academy of Sciences Medical Sciences Correlation of breath ammonia with blood urea nitrogen and
creatinine during hemodialysis L. R. Narasimhan, * William Goodman, † and C. Kumar N. Patel *‡*Department of Physics and Astronomy, University of California, Los
Angeles, CA 90095; and †Department of Nephrology, School
of Medicine, University of California, Los Angeles, CA 90095 Accepted February 5, 2001. |
Abstract We have spectroscopically determined breath ammonia levels in seven
patients with end-stage renal disease while they were undergoing
hemodialysis at the University of California, Los Angeles, dialysis
center. We correlated these measurements against simultaneously taken
blood samples that were analyzed for blood urea nitrogen (BUN) and
creatinine, which are the accepted standards indicating the level of
nitrogenous waste loading in a patient's bloodstream. Initial levels
of breath ammonia, i.e., at the beginning of dialysis, are between
1,500 ppb and 2,000 ppb (parts per billion). These levels drop very
sharply in the first 15–30 min as the dialysis proceeds. We found the
reduction in breath ammonia concentration to be relatively slow from
this point on to the end of dialysis treatment, at which point the
levels tapered off at 150 to 200 ppb. For each breath ammonia
measurement, taken at 15–30 min intervals during the dialysis, we also
sampled the patient's blood for BUN and creatinine. The breath ammonia
data were available in real time, whereas the BUN and creatinine data
were available generally 24 h later from the laboratory. We found
a good correlation between breath ammonia concentration and BUN and
creatinine. For one of the patients, the correlation gave an
R2 of 0.95 for breath ammonia and BUN
correlation and an R2 of 0.83 for breath
ammonia and creatinine correlation. These preliminary data
indicate the possibility of using the real-time breath ammonia
measurements for determining efficacy and endpoint of hemodialysis. Keywords: end-stage renal disease, breath analysis |
Expired human breath
has been analyzed extensively by mass spectrometric techniques for the
existence of a variety of trace amounts of volatile organic compounds
and several small inorganic molecules such as ammonia, nitric oxide,
carbon disulfide, and carbon dioxide ( 1). Of these, several gases
exhaled in human breath, e.g., ammonia, nitric oxide, aldehydes, and
ketones, have been linked to kidney and liver malfunction, asthma,
diabetes, cancer, and ulcers ( 2– 4). Others, such as carbon disulfide,
ethane, butane, and pentane have been linked to neurological disorders,
including schizophrenia ( 5, 6). A few nascent technologies promise the
ability to detect some of these compounds at the required
parts-per-million (ppm) or parts-per-billion (ppb) concentration levels
while in the presence of other interfering species. Recently, a
chemiluminescence detector is being deployed for quantifying nitric
oxide in human breath ( 7– 9). Of the above afflictions, end-stage renal
failure forces over 197,000 patients who require hemodialysis in the
United States to undergo lengthy, three-times-per-week, often painful,
in-clinic treatment (paid by Medicare) to compensate for the loss of
their kidney functions. Another 28,000 patients undergo peritoneal- or
hemodialysis in their homes. Improper, insufficient, and/or
delayed treatment leads quickly to secondary organ failures and a rapid
death. Our study indicates that a breath ammonia measurement may be
capable of providing patients requiring kidney dialysis and their
physicians a fast, painless, and cost-effective in situ
monitor that measures the progress of dialysis in real time, and which
could potentially improve the quality of renal care. We have assessed
the quantitative measure of ammonia exhaled in human breath as an
instantaneous, noninvasive, and low-cost alternative to blood tests in
evaluating the effectiveness of kidney dialysis. We report results of
deploying a laser-based breath ammonia sensor (100 ppb
ammonia-detection sensitivity) into the University of California, Los
Angeles, kidney dialysis center to correlate the reduction in the
accepted blood markers—creatinine (≈14 mg/dl to ≈5
mg/dl) and blood urea nitrogen (BUN) (≈90 mg/dl to
≈30 mg/dl)—with a reduction in the breath ammonia
concentration from ≈2,000 ppb to ≈200 ppb. We have observed a
monotonic reduction in breath ammonia as the dialysis proceeds and we
present quantitative correlation between breath ammonia and BUN and
creatinine measured in blood samples. Once refined and established, the
use of the breath ammonia sensor can serve (i) as an
endpoint detector for dialysis treatment, (ii) to measure
painlessly the rate of buildup of waste products in blood after
treatment, and (iii) as a means for physicians to rapidly
tailor the dialysis regimen to the changing needs of their patients. Kidney dialysis adequacy is determined presently through the use of a
dimensionless parameter called the urea reduction ratio (URR) that
compares the pre- and postdialysis levels of BUN as determined through
laboratory analyses of blood samples taken at the beginning and at the
end of dialysis treatment:
A URR of at least 65% is the current standard of care in the US.
According to the National Institute of Diabetes, Digestive, and Kidney
Diseases ( 10, 11), the “… URR is normally measured only once
every 12 to 14 treatments (i.e., once a month). URR is often averaged
over several months. And therefore, it may vary considerably from
treatment to treatment.” Thus, with the establishment of breath
ammonia as the real-time quantitative indicator of BUN, it could become
a reliable real-time surrogate of the URR for patients requiring
hemodialysis during each of their three-times-per-week dialysis
sessions. Note that URR, by itself, measures the removal of BUN as the
result of a dialysis session but says nothing about the absolute level
of BUN that may be acceptable for assuring long-term health of the
patient. § |
Experimental Methods Spectroscopic Measurements of Ammonia. Absorption and emission of light by molecules has long been used as a
means of identifying which molecules are present in a mixture
(qualitative analysis) and in what concentration (quantitative
analysis). Most molecules with two or more atoms show distinct
absorptions in the infrared region of the spectrum, generally defined
as light with a wavelength between 1 μm and 15 μm (1 μm =
10−6 m). These features can be extremely sharp
for molecules that are in the gas phase and at low pressure, enabling
both the qualitative and quantitative assays with very high selectivity
of species through their so-called “fingerprint” absorptions. Laser spectroscopy has been used extensively for detection of a large
number of industrially produced pollutant gases such as NO,
NO 2, NH 3,
SO 2, and CH 4. Many of these
gases are found in large concentrations at their sources, e.g., nitric
oxide at the tailpipe of an automobile ( 12), and at very low
concentrations in ambient atmosphere and stratosphere ( 13). The
concentration of an unknown sample is determined by characterizing the
optical absorptivity of a sample of known concentration. A number of
techniques have been developed to obtain the necessary spectroscopic
parameters for a wide variety of molecular gases. These methods include
conventional measurements of light throughput, calorimetry, cavity-ring
down spectroscopy (refs. 14 and 15 and references cited in ref. 15),
and thermal distortion spectroscopy (ref. 16 and references cited
therein). Of these, calorimetric techniques have been shown to be
widely applicable for ultra low-absorption measurements, leading to
sub-ppb detection of many gaseous components ( 17). There is increasing evidence that the chemical composition of expired
breath can be an accurate, timely, and painless indicator of the health
of an individual ( 1). The exhaled gases can be used as surrogates for
inferring the makeup of blood and the functioning of vital organs. Here
we describe our early results on the application of optoacoustic
spectroscopy for the detection of minor constituents of human breath of
patients undergoing hemodialysis (see ref. 18 for a preliminary
report). The experimental scheme is shown in Fig.
1. It consists of a tunable laser and an
optoacoustic cell. The patient's breath is conducted to the
optoacoustic cell and is illuminated by the chopped laser beam. Any
light absorbed by the gases in breath is converted into heat, thereby
generating an acoustic signal that is detected by sensitive
microphones. The optoacoustic signal is processed to yield an absolute
absorption coefficient and therefore yields the concentration of the
absorbing species of molecules. This technique has been applied to the
measurement of a variety of gases ( 19). Detection of a specific gas,
even in the presence of other absorbing species, is possible because of
its distinct and characteristic fingerprint absorption. Thus, tuning
the laser frequency permits us to discriminate among different gases of
interest that may be present simultaneously in the optoacoustic cell.
Even when continuous frequency tuning of the laser is not possible,
there are a number of laser systems in the
infrared—CO 2, CO, HF, DF, etc.—that possess
discrete laser transitions, and it is possible to tune in to any one of
these lines.
 | Figure 1 Schematic of the sensitive optoacoustic (OA) measurement system. |
Ammonia Detection. We use a sealed-off, radiofrequency excited CO 2
laser whose operating wavelength can be line switched from R40 of the
9-μm band to P50 of the 10-μm band (by using an intracavity
grating), giving us laser operation on more than 120 discrete
frequencies. These transitions are separated by 1–2
cm −1 and the laser frequency, therefore, is not
continuously tunable. Nonetheless, we take advantage of pressure
broadening of the absorbing species by choosing the gas pressure
appropriately (see ref. 18 for additional details). In particular, we
operate the optoacoustic cell at nearly atmospheric pressure. At this
pressure, ammonia presents a large absorption at a particular
CO 2 laser wavelength and is transparent at
another nearby laser line. We divert a small portion of the laser beam
into another optoacoustic cell, which contains a reference mixture of
ammonia in air, and normalize the breath measurements to this
calibrated mixture. At the selected wavelength of the
CO 2 laser, there are two additional components in
human breath that could interfere with NH 3
absorption. These are saturated water vapor at human body temperature
(37°C) and CO 2 (≈4% by volume). We have
found that water vapor interference is negligible at the wavelength
chosen for NH 3 absorption measurements ( 20).
Independent measurements of breath on another laser line show that the
CO 2 level is relatively constant. Hence, the
background absorption signal caused by breath CO 2
and H 2O is assumed to be relatively constant, and
this amount is subtracted as a constant offset for all measurements.
Patients requiring dialysis do dehydrate during treatment. We examined
the effect of moisture content through a “synthetic patient”
protocol as part of the system calibration procedure ( vide
infra). These data indicate that moisture content is a
second-order effect in the measured signal, and hence we believe the
assumption of a constant (and small) water-vapor contribution to be
valid. This scheme allowed us to measure NH 3
levels as low as 100 ppb in human breath by using a 3-second data
integration time. The breath ammonia measurements involve the patient breathing into a
lightweight disposable mouthpiece (or a face mask) and hose that
conveys the breath to the instrument. The instrument continuously
analyzes the sample and displays an absolute measure of the ammonia
concentration in ppb. An accurate measurement can be obtained in well
under a minute, the time required for the breath sample to reach the
measurement chamber. |
Clinical Background Kidney Failure and Malfunction. Kidney failure can be a result of diseases such as diabetes,
glomerulonephritis, certain viral infections, and/or direct
trauma to the organ. Nephrons, the filtering agents that remove
nitrogen-bearing wastes from the blood, are damaged either partially or
fully during kidney failure. Renal disease is perforce signaled by a
rise in the nitrogen-bearing compounds in the patient's blood stream,
with serious consequences to other organs and to the patient's
lifespan. Two of the important compounds are BUN
[CO(NH2)2] and creatinine
(2-amino-1,5-dihydro-1-methyl-4H-imidazol-4-one). Patients
with end-stage renal disease (ESRD) must have their blood filtered
through reverse osmosis every other day for several hours. In the U.S.,
dialysis times range from 2 to 5 h. Standard practice during a
dialysis session involves withdrawing 3–5 ml of blood immediately
before and immediately after treatment, and then sending the samples
for analysis with typically a 1-day turnaround time. The decrease in
concentration of BUN is used to compute the URR, as defined earlier. Under normal circumstances, the predetermined period of hemodialysis
functions reasonably well but it does not account for the patient's
change in lifestyle or any change of diet. There is, however,
substantial agreement among nephrologists that the present methods of
determining dialysis times and sufficiency are too empirical. The blood
workups do provide useful long-term information about anemia and other
conditions but they are not a source of timely information on the
progress during any particular session. Dialysis is a chemical
titration that presently has no effective real-time endpoint detector. Nitrogenous Wastes and Ammonia. In a healthy individual, ammonia and ammonium ions are converted to
urea in the liver through the enzymatically moderated and energetically
expensive linked urea and citric acid cycles identified by Krebs and
Henseleit ( 21). The urea is then transported through the bloodstream to
be excreted into urine by the kidneys. The reversibility of the process
requires an equilibrium concentration of ammonia related to the BUN
loading of the blood. As small molecules, ammonia and ammonium ions can
penetrate the blood–lung barrier, mix, loft, and appear in exhaled
breath. Given a reliable correlation between breath ammonia and blood
markers, we can use breath ammonia concentration as an instantaneous
tracer of nitrogen-bearing wastes in the human body and provide
( i) an important real-time indicator of the efficacy of the
dialysis treatment and ( ii) a reliable and real-time
endpoint detector of the level of BUN in the blood of the patient with
ESRD to determine an acceptable termination of the dialysis session.
The before and after measurements also provide URRs for comparison with
the accepted standards. |
Results and Discussion Synthetic Patient. Fig. 2 shows an in vitro
ammonia concentration measurement as detected by the instrument on
a synthetic patient. We dilute known concentrations of ammonia in air
(10 or 20 ppm, calibration gases certified by GCMS and Fourier
transform infrared spectroscopy) with a healthy person's breath
(containing water vapor, oxygen, and CO 2) in a
gas manifold connected to the gas analyzer. Stepwise and random
dilutions confirm the high linearity of the system from 15,000 ppb to
200 ppb. We have verified this linear relationship on over 100
measurements of seven healthy individuals who were part of the
development team. From our data it seems, at this time, that the slope
of this line is likely to be specific to each individual. The ammonia
level at a given dilution is recorded for 2 min at a 1-sec sampling
interval and averaged. The uncertainty (standard deviation) was ±
10% of the reading, independent of the actual reading.
 | Figure 2 Linearity of breath ammonia measurement instrument as determined by
diluting calibrated ammonia with human breath (solid line shows a
least-squares fit to the data; R2 =
0.99). |
Patients with ESRD. By using the instrument described above, we successfully measured the
breath ammonia levels of seven patients undergoing dialysis (in the
University of California, Los Angeles, dialysis center) while taking a
fiduciary blood sample concomitant with each breath measurement. Fig.
3 shows the measured breath ammonia as a
function of dialysis time for patient P9. As expected, and as shown by
others using the selected ion flow tube (SIFT) technique ( 22), we see a
reduction in the ammonia concentration in expired breath of patient P9
as dialysis proceeds. Our measured absolute values of ammonia levels
are somewhat lower than those reported by Davies et al.
( 22), but do show a general agreement in the reduction of breath
ammonia with dialysis time. Again, the error bars on the ammonia
measurements are ±10%, as determined by averaging the output of the
detector over 2 min at a 1-sec sampling interval. We obtained similar
breath ammonia reduction results in six other patients with various
session times. We show data for two more patients, P3 and P8, in Figs.
4 and 5,
respectively. The curves in Figs. 3– 5 are one-parameter exponentials
that are meant as a guide for the eye. The exponential fits assume that
dialysis follows first-order kinetics. The fits in Figs. 2, 6, and 7
are from linear regression.
 | Figure 3 Reduction in breath ammonia vs. dialysis time for patient P9. Note that
the total dialysis time is 2 h and 30 min. (Dashed line through
the data points is meant as a guide for the eye.) |
 | Figure 4 Breath ammonia vs. dialysis time for patient P3. Note that the dialysis
session time is 5 h. (Dashed line through the data points is meant
as a guide for the eye.) |
 | Figure 5 Breath ammonia vs. dialysis time for patient P8. Note that the dialysis
session time is 3 h. (Dashed line through the data points is meant
as a guide for the eye.) |
 | Figure 6 Breath ammonia vs. BUN correlation for patient P9. (Solid line shows a
least-squares fit to the data; R2 =
0.95.) |
 | Figure 7 Breath ammonia vs. creatinine correlation for patient P9. (Solid line
shows a least-squares fit to the data;
R2 = 0.83.) |
The most critical test is the correlation of breath ammonia with
constituents of blood that are used traditionally as the measures of
kidney failure in patients with ESRD. Therefore, we collected data on
BUN and creatinine at the same time as breath ammonia measurements were
carried out. Unlike breath ammonia data that were available
instantaneously, the BUN and creatinine data were received 12–24 h
after the blood samples were sent for analysis. Figs.
6 and 7
show the breath ammonia and BUN and creatinine data for patient P9. We
see an encouraging correlation. The uncertainty in the blood
measurements ( y-axis error bars) reflects the ±10%
measurement accuracy specified by the blood-testing laboratory. The
breath ammonia uncertainties ( x-axis error bars) are
determined as before. We independently verified the ±10% quoted
uncertainty in blood measurements by sending samples from one patient
to two different laboratories. We have confirmed both the
time-dependent decrease of breath ammonia and the correlation with BUN
and creatinine on six other patients. These data that show that
noninvasive breath ammonia measurements can be used as a real-time
surrogate measure of the status of patients with ESRD. Returning to Fig. 3, we direct the reader to the last breath ammonia
data point occurring at t ≈3 h, 21 min, showing an ammonia
concentration of about 420 ppb. This patient completed dialysis at
2 h, 30 min, at which point the measured ammonia concentration in
the patient's breath was about 300 ppb. The bounce back in ammonia
concentration from 300 to 410 ppb (and the corresponding increase in
BUN from 30 to 40 mg/dl) and increase in creatinine from 5.5 to
6.5 mg/dl is one of the important observations in understanding
the dynamics and partitioning of BUN and creatinine between the
bloodstream and body tissue. At present, no quantitative data exist
that indicate how BUN and creatinine accumulate in the blood of the
patients with ESRD between dialysis treatments. This is an exceedingly
important question, as dialysis patients are on restricted diets and
their blood wastes can dramatically increase if they do not follow the
specified regimen. Fig. 8 dramatically illustrates the need
for patient diagnostics during dialysis. We plot BUN levels against
creatinine levels as determined by the blood sampling taken during
breath measurements of four patients. Three patients (diamonds and
large and small squares) show excellent correlation with an average URR
as defined above ≈80%. The fourth patient (triangles) shows a
drastically different slope than the others with a URR of only 65%.
Clearly, this patient's body processes BUN and creatinine in a
markedly different way than the others, and at the end of
his/her treatment, s/he had substantially higher levels
of BUN than the other patients. A breath measurement is of clear value
here. Even assuming an inexpensive chairside real-time blood analyzer,
constant blood draws to establish these slopes are not feasible.
Withdrawing 5 ml every 15 min over 3 h removes 60 ml of blood from
the patient. Over a three-treatments-per-week cycle, this amounts to an
unacceptable 180 ml of blood lost. The questions remain, however, of
why this patient is apparently under-dialyzed with respect to the
others and what the consequences are to his/her long-term
health.
 | Figure 8 BUN vs. creatinine levels as determined by periodic blood sampling of
four patients undergoing dialysis, P2, P3, P5, and P9. One patient
(triangles) shows a slope different from the other three. |
Conclusion: Ammonia Detection in Breath. Quantifying the level of ammonia in exhaled breath serves two vital
functions: (i) It can be used as a surrogate for elevated BUN, which,
along with the creatinine level in a patient's blood, is the accepted
indicator of kidney malfunction. Our preliminary measurements are among
the very few quantitative data sets correlating breath ammonia with BUN
and creatinine. No correlations have been made yet with glomerular
filtration rates, but these will be acquired once the breath ammonia
instrument is deployed in planned clinical studies of individuals
at-risk for kidney failure. (ii) The breath ammonia level can be used for determining
the exact time necessary for the desired degree of dialysis for a
patient with ESRD at every session. The breath ammonia monitor will
provide crucial information about when a dialysis treatment may be
stopped, i.e., detect an endpoint. The ability of such instrumentation
to detect partial kidney failure will depend on quantitative
correlation between breath ammonia and BUN, creatinine, and glomerular
filtration rates. In the long run, breath ammonia measurement could
serve as a broad noninvasive screen for incipient kidney failure, as
well as a monitor of kidney functions in at-risk populations such as
diabetics and hypertensives. |
Acknowledgments We thank Professor Gantam Chaudhuri for technical discussions of
the importance of ammonia in human organ functions and disorders. |
Abbreviations | ESRD | end-stage renal disease | | BUN | blood urea
nitrogen | | URR | urea reduction ratio |
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