Breath Tests in Medicine Since antiquity, it has been known that the breath contains clues to many diseases. Researchers are developing a new generation of breath tests as an alternative to invasive diagnostic procedures by Michael Phillips

F

or many people, the idea of breath testing conjures up a vision of a

as alcohol or methyl mercaptan (the fra­

Carbon dioxide is relatively easy to

grant compound in garlic), will diffuse

detect, because it makes up approxi­

roadside interrogation by a grim­

across the alveolar membrane from

mately 5 percent of the breath. Unfortu­

faced police officer. A motorist sus­

the compartment with the higher vapor

nately, most other volatile compounds

pected of being intoxicated must blow

pressure to the lower, from the air into

in the breath are present in much lower

into a hand-held instrument that, after

the blood, or vice versa.

concentrations, in parts per million or less. Their detection had to wait until

a few seconds, indicates the amount of

The detection of volatile organic com­

alcohol in the blood. Despite this unflat­

pounds in the breath has a long histo­

the mid-19th century, when colorimet­

tering association with inebriation and

ry. Since the time of Hippocrates, phys­ icians have known that the aroma of human breath can provide clues to di­ agnosis. The astute clinician is alert for the sweet, fruity odor of acetone in pa­ tients with uncontrolled diabetes, the musty, fishy reek of advanced liver dis­ ease, the urinelike smell that accom­ panies failing kidneys and the putrid stench of a lung abscess.

ric assays were introduced. In such an

arrest, breath testing is blossoming into an exciting area of medical technolo­ gy. Physicians have begun using these tests to diagnose an increasingly wide variety of diseases without the hazards or discomforts of invasive procedures. Furthermore, breath tests are provid­ ing important new insights into the un­ derstanding of basic biochemical func­

analysis, an organic compound inter­ acts with reagents in a solution to pro­ duce a change in color. One of the earliest to exploit color­ imetric analysis was A. Nebelthau, a physician at the Marburg PolycliniC in Germany. He constructed a device to analyze the breath of patients suffering from diabetes mellitus. When uncon­ trolled, this condition causes the blood

tions of the body.

which have made it possible to identi­

B

ical tool could not have pro­

quantities of acetone as a major me­

fy a rapidly growing number of com­

gressed beyond educated sniffing. This

tabolite. When Nebelthau bubbled a pa­ tient's breath through the alkaline io­

The recent upsurge in interest stems from advances in analytic technology,

ut without objective chemical

glucose to rise to excessively high lev­

analysis, breath testing as a med­

els; as a result, the body generates large

pounds in the breath. Clinical studies

problem attracted the attention of sci­

with these improved assays have shown

entists more than 200 years ago. An­

dine trap of his apparatus, he observed

that the presence of abnormal chemi­

toine Laurent Lavoisier, celebrated as

a rapid and intense change in color.

cals in the breath can aid in the early

the father of the "chemical revolution"

The change demonstrated that unusual

diagnosis of many diseases.

and renowned for his discovery of the

amounts of acetone were being excret­

The breath contains valuable informa­ tion because only a slender barrier sep­

role of oxygen in combustion, was also

ed through the lungs.

a pioneer in breath testing.

arates the air in the alveoli of the lung

In 1 7 84 he and Pierre Simon Laplace,

In 1874 the British physician Francis E. Anstie applied colorimetric analysis

from the blood in the capillaries. This

the French mathematical physicist, ana­

to study the fate of alcohol in the hu­

barrier is called the pulmonary alveolar

lyzed the breath of a guinea pig. They

man body. His objective was to resolve

membrane. Like water flowing down a hill, a volatile organic compound, such

found that the animal consumed oxygen and expired carbon dioxide. This finding

the controversy raging between physi­

was the first direct evidence that food

down alcohol as it does food, and tem­

undergoes combustion in the body. In

perance activists, who thought the body

this discovery lies both the foundation

excreted it unchanged as if it were a

MICHAEL PHILLIPS is associate direc­ tor of medicine at St. Vincent's Medical Center of Richmond in Staten Island and clinical professor of medicine at New York Medical College in Valhalla. He re­ ceived his degree from the University of

of modem biochemistry and the expres­

foreign substance. Anstie's breath trap

sion "to be a guinea pig," as Lavoisier

contained a solution of chromic acid,

confirmed the finding by using himself

which changed from red-brown to green

as a subject.

in the presence of ethyl alcohol. In an

Lavoisier's apparatus included an in­

Western Australia and has had appoint­

genious innovation that researchers to­

ments at the University of Connecticut and Georgetown University. He writes

day still use in various guises: the breath

that his main interests are in the clinical pharmacology of alcohol abuse and in "the less popular exhalations of the hu­ man body-breath and sweat-for the neglected windows they open onto meta­ bolic processes."

74

SCIENTIFIC AMERICAN

ologists, who believed the body broke

trap. This device accumulates and con­ centrates components of the breath. La­ voisier's trap was a chemical solution through which he bubbled a large vol­ ume of breath. The carbon dioxide in the breath reacted with the solution to form a visible precipitate.

July 1992 © 1992 SCIENTIFIC AMERICAN, INC

BREAlH-COLLECTION DEVICE in the au­ thor's laboratory provides the patient with air purified by activated carbon (in containers at the left). Exhaled breath passes through a drying agent (in con­ tainer at the right) and is trapped for later analysis. The nose clip ensures the patient breathes through the tubing.

elegant series of experiments, he dem­

disease reveals itself when an abnor­

small intestine, such as bacterial over­

onstrated that the amount of alcohol ex­

mal quantity of the breakdown prod­

creted via the breath and other routes

ucts appears in the breath.

growth, can also be diagnosed by breath testing. This condition may result from

fell far short of the amount consumed.

For example, physicians use this ap­

any cause of stagnation in the small

Therefore, most of the alcohol must

proach to diagnose an intestinal disor­ der known as malabsorption syndrome.

from a surgical procedure or damage

have been metabolized. (He is also re­

intestine, including scarring resulting

membered as the originator of Anstie's

This condition causes severe and chron­

to the nerves that control the propul­

limit: more than two alcoholic drinks a

ic diarrhea because the diseased intes­

sion rate of food through the gut.

day may be injurious to one's health, a

tine cannot absorb food efficiently. One

Breath testing is also used to identify

concept strongly supported by modern

consequence of the syndrome is that

damage to the pancreas. Jay A. Perman

findings.)

most of the sugar in food arrives at

of the Johns Hopkins University School

Current hand-held breath analyzers

the colon intact, where bacteria break

of Medicine studied children with cystic

for alcohol are electronic: the donor

it down. The bacterial action releases

fibrosis, an often fatal condition that

blows into a plastic tube, and a spring­

hydrogen, which is absorbed into the

severely harms the pancreas and lungs.

loaded piston withdraws approximate­

blood and excreted through the lungs.

He gave them a dose of rice starch, a

ly one cubic centimeter of breath, which

As a test, a physician gives the patient

complex carbohydrate. Children with

is oxidized in a fuel cell. The resulting

an oral dose of xylose, a five-carbon

electric current is proportional to the

sugar normally absorbed completely by

impaired pancreatic function could not secrete sufficient amylase to digest the

concentration of alcohol in the breath.

the small intestine. The appearance of

meal in the small intestine. Instead the

After a few seconds, the breath analyz­

large amounts of hydrogen in the breath

bacteria in the colon completed the di­

er calculates and then displays digitally

within the next few hours confirms the

gestive task, producing hydrogen, which

the blood alcohol concentration.

diagnosis of malabsorption syndrome.

was detectable in the breath.

onstrate less common kinds of malab­

Although the presence of hydrogen can help diagnose several disorders of

it is easy to detect a volatile or­

sorption, in which the intestine fails to

the intestine, other markers are also

ganic compound in the breath

absorb a speCific carbohydrate. For in­

useful. For example, radioactive carbon

if the substance has been consumed

stance, a patient who has a defiCiency of

14 is used to identify diseases of the

in large quantities beforehand. Conse­

lactase in the small bowel will produce

pancreas. The carbon 14 is combined

quently, many breath tests require a patient to consume a dose of a specif­

hydrogen after a dose of lactose but not after a dose of a different sugar, such as

tabolized by pancreatic enzymes. The

ic precursor to a volatile chemical. The

xylose or glucose. Other disorders of the

amount of radioactive carbon dioxide

This test can also be refined to dem­

A

testing for alcohol demonstrates,

© 1992 SCIENTIFIC AMERICAN, INC

with other compounds that are me­

doscopy in the detection of

ter

infections.

Helicobacter

Helicobac­ possesses

an enzyme called urease, which is not present in humans. If an infected pa­ tient consumes a dose of urea labeled with carbon 14, the urease in

bacter will

Helico­

break down the urea in the

stomach. One of the metabolites will be 14C02, which is absorbed by the blood and excreted in the breath. A similar test is also proving useful in uncovering damage to the liver. Con­ ditions such as cirrhosis and hepatitis often go unnoticed in their early stag­ es. By the time bilirubin, a major me­ tabolite of hemoglobin, builds up in the lAVOISIER'S LABORATORY to study human breath may be the earliest recorded. In

blood to cause the characteristic jaun­

this woodcut, a subject breathes oxygen through a tube, so that the French chemist

dice, a patient may have lost more than

can study respiration. Madame Lavoisier, seated at the right, records the data.

50 percent of the cells in the liver. Breath testing can measure the im­ pairment of speCific metabolic path­ ways before jaundice sets in. Specifi­

(14C02) that subsequently appears in

labeling may be especially useful in

the breath indicates how well the pan­

diagnosing two of the most common

cally, it can test the N-demethylation

creas is functioning. For instance, a dis­

stomach disorders seen in general med­

pathway in the hepatic microsomes,

eased pancreas may not secrete suffi­

ical practice: peptic ulcer disease and

which is affected early by any kind of

cient lipase to break down dietary fats.

chronic gastritis. The conditions may

liver damage. The demethylation pro­

A physician can identify the disorder

result from

infec­

cess in a healthy liver releases substan­

if a reduced amount of 14C02 appears

tions, which appear to be quite com­

tial amounts of carbon dioxide. To de­

in the breath after the patient has con­

mon. The infections are probably under­

tect liver injury, a phYSician can give

sumed a dose of a radiolabeled triglyc­

diagnosed because confirmation usually

a patient a dose of a tracer compound,

eride, a low-molecular-weight fat.

requires endoscopy, in which the phys­

such as aminopyrine, phenacetin or ga­ lactose, labeled with radioactive carbon

Helicobacter pylori

Although breath tests for disorders

ician passes an instrument into the

of the small intestine and pancreas are

stomach to collect a sample of the mu­

14. Any impairment of N-demethyla­

the primary ones now used by the med­

cosal lining.

tion capacity will reveal itself through a

ical community, recent studies have

Barry

J

Marshall of the University of

marked reduction in the rate at which 14C02 appears in the breath.

shown that the technique holds promise

Virginia Health Sciences Center has

in recognizing other conditions. Breath

shown that breath tests are a highly re­

There are other markers of liver dis­

tests employing radioactive carbon 14

liable noninvasive alternative to en-

ease. For example, the liver normally

Breath Tests in Gastroenterology

HYDROGEN IN BREATH SUGAR

14C-LABELED UREA

,.....--- 14C-LABELED BILE SALT

14C021N BREATH BACTERIA BREAK DOWN SUGAR

STOMACH Peptic ulcers and chronic gastritis may result from infections of

Helicobacter pylori. The bacteria will break down a dose of 14C­ labeled urea into 14C02, which is absorbed by the blood and will appear in the breath.

76

SCIENTIFIC AMERICAN

SMALL INTESTINE

In malabsorption syndrome, the small intestine is unable to absorb a sugar, such as xylose, lactose, sucrose, glucose, fructose or lactose. Hydrogen will appear in the breath. A dose of a bile salt labeled with carbon 14 will reveal bacterial overgrowth.

July 1992 © 1992 SCIENTIFIC AMERICAN, INC

breaks down climethyl sulfide, a volatile

suIts in a greater buildup of acetalde­

metabolite of the amino acid methio­

hyde in the blood, causing flushing and

nine. But if the liver's functions are im­

other unpleasant symptoms.

paired, a greater quantity of dimethyl sulfide "spills over" into the breath.

tube chilled by dry ice. He then assayed the condensate by gas chromatography and mass spectroscopy. He observed ap­

Breath testing is already proving use­

proximately 2 50 different compounds.

ful in monitoring the exposure of in­

This surprisingly large number indi­

Diagnosis of disease is only one realm

dustrial workers to potentially hazard­

cated that the composition of human

in which breath testing can be useful.

ous solvents and petrochemicals. Sev­

breath was more complex than had

Drug mOnitoring is a neglected but po­

eral of these toxic compounds can be

been previously suspected. Other lab­

tentially fruitful field, because most pre­

readily measured in human breath. Sen­

oratories have confirmed his findings,

scribed and illicit drugs have low mo­

sitive breath tests developed by Edo D.

and researchers have now isolated near­

lecular weights. Their vapor pressures at

Pellizzari, now at the Research Triangle

ly 400 volatile organic compounds in normal human breath.

body temperature may be sufficiently

Institute in North Carolina, and his col­

high so that they or their metabolites

leagues at the Environmental Protection

Why have scientists not routinely as­

are propelled into the alveolar air in

Agency have shown that toxic chemi­

sayed these compounds, if the technol­ ogy has been available for more than

measurable quantities. Indeed, breath

cals can pollute air far from the factory

tests for alcohol are so widely employed

floor. It is now clear that indoor air in

20 years? Let me attempt to answer

that it is curious that testing for other

the workplace, office and home may

this question by inviting the reader to

drugs is still comparatively unexplored.

be polluted with small amounts of sev­

accompany me on an imaginary visit to

Metabolites of marijuana can be readily

eral compounds, some of them carcin­

my laboratory at Bayley Seton Hospital

measured in the breath for several days

ogens. In one study Pellizzari and his

in Staten Island. As you enter, I point

after use.

colleagues found that workers in hard­

out the instruments we use to analyze

Breath testing can also illuminate dif­

ware stores had ingested volatile com­

volatile compounds in breath: thermal

ferences in metabolism. It had long been

pounds that may have originated from

desorbers, gas chromatographs and a

carpet solvents and plastics.

mass spectrometer, their functions all

observed that Asians react to the con­ sumption of alcohol in a different man­ ner from most whites. Many Asians ex­ perience flushing after a drink, as well

�

orchestrated by microcomputers.

l the breath tests described so

But, you wonder, how does one ac­

far detect either a volatile or­

tually collect breath samples in the

as more severe reactions, such as palpi­

ganic compound that had been

first place? I now display our collect­

tations, nausea and malaise. Breath tests

previously consumed (such as alcohol)

ing apparatus, which is something of

have confirmed that concentrations of

or the metabolites of a precursor. Re­

an anticlimax. It is a handcrafted and

acetaldehyde, a volatile metabolite of al­

searchers have attempted to advance

somewhat bizarre-appearing device on

cohol, are considerably higher in Asians

one step further by detecting the vola­

a wheeled cart. Several meters of plas­

than in whites after consumption of an

tile compounds present in the breath

tic tubing wind in seemingly disparate

alcoholic beverage. Further research has

under normal circumstances. In 1971 li­

directions; a plywood container conceals a pump and even more tubing. The

shown that more than 80 percent of

nus Pauling described an elegantly sim­

Asians generally have relatively low lev­

ple method for microanalysis of normal

unit contrasts oddly with the polished

els of aldehyde dehydrogenase in the

breath. He first passed breath through a

elegance of the analytic instruments.

liver microsomes. A dose of alcohol re-

cold trap consisting of a stainless-steel

COMPLEX CARBOHYDRATE

14C-LABELED LIPID

This clash of technological styles is

14C-LABELED COMPOUND

1

HYDROGEN IN BREATH

METHIONINE

�

DIMETHYL SULFIDE

IMPAIRED N- DEMETHYLATION PATHWAY

BACTERIA BREAK DOWN CARBOHYDRATE

REDUCED 14C02 IN BREATH

LIVER

PANCREAS

A lipid labeled with carbon 14 will be broken down by lipase, so any deficiency will result in a below normal amount of 14(02 in the breath. Insufficient production of amylase, which breaks down carbohydrates, will be revealed by the presence of hydrogen.

Impaired functioning of the N-demethylation pathway can be detected by giving aminopyrine, phenacetin or galactose labeled with carbon 14. Sulfur metabolism can be gauged by methionine, which is broken down by the body into dimethyl sulfide.

SCIENTIFIC AMERICAN

© 1992 SCIENTIFIC AMERICAN, INC

July

1992

77

often encountered in laboratories where

contaminate the chemically clean trap.

breath compounds are studied. The ana­

For meaningful results, the appara­

lytic instruments are purchased "off the shelf," while the collection technology

tus must be able to sample breath that

freezes the water and carbon dioxide

has originated deep in the lungs, be­

in the breath and may rapidly become

consists of the unstandardized gadgetry

cause human breath is not a homoge­

plugged by ice.

created by individual tinkerers. Despite

neous gas. The first 1 50 milliliters of ev­

Adsorptive trapping has become the

two centuries of experience gained since

ery expiration consists of "dead space"

most convenient and most widely used method today. It captures volatile com­

nitrogen, which is at -196 degrees Cel­ sius. Unfortunately, a cold trap also

Lavoisier's time, there is still no gener­

air from the upper airways, where no

al agreement on the best way to trap a

gas exchange has occurred (a healthy

pounds by binding them to agents such

sample of concentrated breath.

individual exhales half a liter or more

as activated carbon and adsorptive res­

Much of the debate is caused by sev­

with each breath). The trap should also

ins. Recent advances in microprocessor­

eral difficulties in designing breath-col­

capture only the volatile compounds of

controlled thermal desorbers, which au­

lection equipment. First and foremost,

interest and allow the remainder, in­

tomatically "bake off " and concentrate

a patient should have no trouble exhal­

cluding nitrogen and oxygen (which to­

trapped compounds, have made the

ing into the device. Blowing through

gether constitute more than 90 percent

technique even more convenient.

a liquid trap can be hard work, which

of the breath), to pass unimpeded.

probably explains why Lavoisier's sub­

Several investigators have developed highly sensitive assays using adsorp­

G

jects stripped to the waist before using the instrument. Even Pauling's device, which included a tube five feet long and

enerally speaking, there are three

tive traps. They include Pellizzari, M.

types of breath traps: chemical,

Sydney Gordon of Battelle, Jack O'Neill

cryogenic and adsorptive. None

of the IIT Research Institute in Chicago

0.2 inch in diameter, could not have

is perfect. Chemical trapping usually

and E. S. Kovaleva and R. Liedeman of

been especially easy to blow through. In

uses traditional "wet chemistry": breath

the All-Union Mental Health Research Center in Moscow.

addition, mouthpiece parts must be re­

is bubbled through a reagent solution

placeable to avoid any risk of transmit­

that captures a specific compound, such

The apparatus that my colleague

ting infections from patient to patient.

as ethanol or acetone. The method is

Joel Greenberg and I have made il­

Contamination of the sample is also

simple and direct, and the trapped de­

lustrates how adsorptive trapping can

a major consideration. Even the cleanest

rivative, often colored, can be measured

overcome most of the technical diffi­

room is polluted with volatile chemicals

easily. The disadvantages are poor sen­

culties

from such sources as ventilation units

sitivity and the physical effort required

our device the donor inhales purified

and solvents in carpeting adhesive.

of the donor.

[see illustration on this page]. In

air and breathes out into an exhaust

The trace compounds are measurable

In cryogenic trapping, the volatile

tube. One-way valves ensure that there

as background "noise." In addition,

compounds are captured by freezing.

is no reflux of breath into the reservoir

the moisture in breath will condense

The breath travels through a tube im­

of purified air. There is virtually no re­

into droplets on the tubing, which may

mersed in such cooling fluids as liquid

sistance to breathing because the tubing is wide (nearly three centimeters in di­ ameter), and the one-way valves require very little pressure to open and close. Even severely ill patients can donate a

Collecting Human Breath

T

sample without much discomfort.

his device, in the author's laboratory, is mounted on a cart, which can be wheeled to a patient's bedside

[see illustration on page 75].

Room

air is pumped through the activated carbon, supplying the volunteer

with chemically clean air. Exhaled alveolar breath, drawn through the water trap to remove moisture, passes through the stainless-steel breath trap, which captures the volatile organic compounds. The one-way valves prevent backflow. The respirometer measures the amount of each exhalation .

ONE-WAY VALVE +--- W4�

VENT

� /

tion yields 10 liters of alveolar breath. The breath is dessicated in water traps and drawn through a stainless-steel

ganic compounds. Later, a thermal de­

- �

. -

the exhaust tube. Five minutes of collec­

sorptive resin capture the volatile or­

/ j \.

--

dead-space air. Only alveolar breath from deep in the lungs is extracted from

tube, where activated carbon and an ad­

MOUTHPIECE

. t ��i��

No sample is collected from the first part of the breath, which contains the

,�

sorber automatically strips the volatile compounds from the trap and concen­

WATER

V TRAP

trates them more than 200,000 times,

;

for separation by gas chromatography and assay by mass spectrometry. The main drawback of adsorptive trapping is that it may have a greater

ACTIVATED CARBON

affinity for some compounds than for

WATER TRAP

others. The gas chromatograph is also a source of variation. It separates samples

ROOM AIR PUMP

PUMP/FLOW METER

�

�

BREATH TRAP

I

in a long, narrow tube (called the sep­

TAP I

r ::::=, :: 0 ···· ..

-

-,

VENT

arating column), whose chemical com­ position influences the number of de­ tectable compounds. Thus, our method

BYPASS TUBE

yields only about 50 to 60 compounds. This comparatively small number has proved more than sufficient for re­ search purposes.

78

SCIENTIFIC AMERlCAN

July 1992 © 1992 SCIENTIFIC AMERICAN, INC

At our current state of knowledge, microanalysis of volatile compounds in the breath has raised more questions than it has answered. The first and most pressing question is, from where do the compounds come? The origin of most is still unknown. Some may be environmental pollutants. For instance, we found carbon disulfide in the breath of all our human volunteers. The com­ pound might be a breakdown product of sulfur-containing amino acids. On the other hand, we have also found car­ bon disulfide in air samples collected in and around New York City. The ori­ gin of the compound is not a trivial

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