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Clinical Studies |
Protein Deficiency1
Smell and Taste Center and Department of Otorhinolaryngology: Head and Neck Surgery (R.L.D., A.D.F., D.A.M.), School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; Department of Medicine (M.A.L.), Division of Endocrinology and Metabolism, Johns Hopkins University, Baltimore, Maryland 21205; Department of Medicine (A.M.), College of Medicine, State University of New York Health Science Center, Syracuse, New York 13210
Address correspondence to Richard L. Doty, Ph.D., Director, Smell and Taste Center, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, Pennsylvania 19104.
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Abstract |
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) of
adenylyl cyclase, has been used to support the hypothesis that
Gs plays a major role in human olfactory transduction.
However, only a limited number of olfactory tests have been
administered to such patients, and these patients have other problems
that might cause or contribute to their olfactory dysfunction,
including an unusual constellation of skeletal and developmental
deficits termed Albright hereditary osteodystrophy (AHO). In this
study, we administered tests of odor detection, identification, and
memory to (i) 13 patients with Type Ia PHP; (ii) 8 patients with Type
Ib PHP; (iii) 7 patients with pseudopseudohypoparathyroidism
(PPHP); and (iv) 3 sets of normal controls matched to these groups on
the basis of age, gender, and smoking history. Although we confirm that
PHP Type Ia patients evidence olfactory dysfunction, we also
demonstrate that (i) patients with Type Ib PHP, who have no AHO, no
generalized hormone resistance, and normal Gs activity,
also evidence olfactory dysfunction relative to matched controls; and
(ii) patients with PPHP, who have AHO, no generalized hormone
resistance, and deficient Gs protein activity, have
relatively normal olfactory function. These observations do not support
the hypothesis that the olfactory dysfunction associated with PHP is
the result of generalized Gs protein deficiency and
imply that other mechanisms (e.g. ones associated with PTH
or PTHrP resistance) are responsible for the olfactory deficits of this
disorder. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit of the heterotrimer. The -GTP molecule
rapidly dissociates from the ß
complex and can interact with its
effector until its intrinsic GTPase hydrolyzes it to GDP, thus
promoting reassociation of -GDP with ß. In most cells,
receptors are coupled to activation of one or more forms of adenylyl
cyclase by Gs (2).
It has been suggested that Gs may play an important role in
olfactory transduction, although a genetically distinct G protein,
termed Golf, likely couples olfactory receptors to a unique
form of adenylyl cyclase (3). Support for a role of Gs in
olfactory function comes from reports that Gs-deficient
Type Ia pseudohypoparathyroidism (PHP) patients, who express
generalized hormone resistance, evidence variably decreased olfactory
ability relative to non-Gs deficient Type Ib PHP
patients, whose hormone resistance is specific to parathyroid hormone
(PTH) (7, 8). Although strong positive correlations between intensity
ratings given to odorants by humans and the propensity of such odorants
to stimulate adenylate cyclase activity in an in vitro frog
olfactory ciliary preparation add further credence to the role for
G-proteins in human olfactory transduction (9), such findings do not
shed light on the specific G proteins involved.
While the decreased olfactory function in PHP Type Ia patients seems to provide compelling evidence for a role of Gs in human olfactory processing, few types of olfactory tests have been administered to these unusual patients, and they have other problems that might explain or contribute to their decreased ability to smell. For example, Type Ia PHP patients, unlike Type Ib PHP patients, have Albright hereditary osteodystrophy (AHO), an unusual constellation of skeletal and developmental abnormalities that includes obesity, short stature, brachydactyly, round faces, and subcutaneous ossifications (10).
To better define the apparent association between Gs
protein deficiency and olfactory dysfunction, we administered odor
identification, detection, and memory tests to Type Ia and Type Ib PHP
patients, as well as to patients with pseudopseudohypoparathyroidism
(PPHP) and to normal subjects matched to the patients on the basis of
age, gender, and smoking habits. PPHP is a condition sometimes found in
relatives of patients with PHP Type Ia and shares with Type Ia PHP the
presence of AHO and decreased Gs protein activity.
However, PPHP is unaccompanied by generalized end organ insensitivity
to hormones. Our results confirm that PHP Type Ia patients score lower
on an odor identification test than do patients with PHP Type Ib.
However, we also demonstrate (i) that PHP Type Ib patients, relative to
matched controls, evidence deficits in olfactory function and (ii) that
Gs protein deficient PPHP patients have relatively
normal olfactory function. These observations do not support the
hypothesis that the olfactory dysfunction associated with PHP is the
result of a generalized deficiency of Gs protein.
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Materials and Methods |
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Thirteen patients with Type Ia PHP, 8 patients with Type Ib PHP,
and 7 patients with PPHP served as subjects, along with 3 groups of
normal controls whose members were matched individually to each of the
patients on the basis of age, gender, and smoking history (Table 1
). One Type Ia PHP patient did not receive the odor
detection threshold test, and another did not receive the odor memory
test (see Olfactory Test Procedure section). Most of the
patients were recruited from families with multiple affected members
who were followed by M.A.L. and A.M. at Johns Hopkins University and
the State University of New York, Syracuse, respectively. Two were
patients of Robert Rosenfeld, M.D., of Wyler Children’s Hospital in
Chicago, and one of Andrew Stewart, M.D., of the Veteran’s Affairs
Medical Center, West Haven, Connecticut. Four were patients of Luis
Aparicio, M.D., of Metabolic Associates, Erie, Pennsylvania. All of the
patients provided informed consent to be tested and were tested in
their homes by A.D.F. or D.A.M. The diagnoses of Type Ia PHP, Type Ib
PHP, and PPHP were based on determination of hormone responsiveness,
Gs activity, and the presence or absence of AHO, as
previously described (11, 12). Erythrocyte membrane Gs
was analyzed using either a bioassay to measure activity (12) or by a
quantitative immunoblot technique to determine the relative level of
immunoactive protein (13), as described below. Results for many of the
patients evaluated in this study were reported previously (12, 13, 14, 15).
Demographic information, as well as mean (SD)
Gs erythrocyte levels and scores on the Mini-Mental
State Examination (16), a measure of cognitive function, and the
Picture Identification Test (17), a visual test designed to control for
nonolfactory elements of the odor identification test used in this
study, are presented in Table 1.
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bioactivity
Assays of membrane Gs bioactivity were performed
as previously described (12), based on activation of adenylyl cyclase
in membranes from the cy
clone of the S49 murine lymphoma cell
line, which genetically lacks Gs (18). Membranes from
S49 cyc- cells were prepared as previously described
(19).
Quantitative immunoblot analysis
Membrane proteins (50–100 µg protein) were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the
system of Laemmli (20). In order to optimize resolution among G
proteins, membrane samples were alkylated with N-ethylmaleimide (21)
before electrophoresis through 11% acrylamide gels. The separated
proteins were transferred from the gel to PVDF membranes (Millipore)
using a Transblot chamber (Bio-Rad). We performed immunoblot analysis
using specific polyclonal rabbit antisera that had been raised against
synthetic peptides corresponding to defined regions of the
Gs protein (i.e. antiserum C584 was raised
against a peptide [CTPEPGEDPRVTRAKY] consisting of
residues 325–339 of Gs, and antiserum RM was raised
against the c-terminal decapeptide [RMHLRQYELL] corresponding to
residues 385–394). Both antisera recognize the 45 kDa and 52 kDa forms
of Gs. Each immunoblot was incubated with a saturating
concentration of the appropriate antiserum, and antibody binding was
detected by subsequent incubation with I125-labeled protein
A and autoradiography. The optical density of each band was measured by
two-dimensional scanning densitometry, and regions of the blot that
corresponded to the bands were excised and the amount of radioactivity
was quantitated by -spectrophotometry. Over the range of protein
concentrations used in these studies, optical density was proportional
to the amount of radioactivity associated with each antigen band. In
all cases presented, there was a linear relationship between the amount
of membrane protein loaded onto the gel and the amount of
immunoreactive signal detected.
Olfactory test procedures
The patients and matched controls were administered standardized tests of odor identification, detection, and odor memory. The order of presentation of the tests was randomized across subjects. The test session lasted approximately an hour and a half, and the subjects were allowed to take rest breaks between the administration of the tests.
The first test that we administered was the University of Pennsylvania Smell Identification Test (UPSIT; commercially available as the Smell Identification Test, Sensonics, Inc., Haddon Hts., NJ). This 40-item microencapsulated test is the most widely-administered test of olfactory function in North America (having been administered to an estimated 30,000 patients) and is described in detail elsewhere (22, 23). Briefly, a subject is required to identify, in a 4-alternative multiple choice format, each of 40 odorants presented on microencapsulated "scratch and sniff" labels. For example, one of the test items reads, "This odor smells most like: (a) chocolate; (b) banana; (c) onion; or (d) fruit punch." The subject must provide a response even if no odor is perceived (i.e. the test is forced-choice). The number of items out of 40 that were answered correctly served as the dependent measure.
The second test we administered was the phenyl ethyl alcohol (PEA) odor detection threshold test. In this test, detection threshold values for PEA, a rose-like smelling odorant with minimal intranasal trigeminal nerve activity, were determined using a modified single staircase procedure described in detail elsewhere (22). In the present study, the staircase was begun at the -6.50 log concentration step of a half-log step (vol/vol) dilution series extending from -10.00 log concentration to -2.00 log concentration and was moved upward in full log steps until correct detection occurred on five sets of consecutive trials at a given concentration. If an incorrect response was given on any trial, the staircase was moved upward a full log step. When a correct response was made on all five trials, the staircase was reversed and subsequently moved up or down in 0.50 log increments or decrements, depending upon the subject’s performance on two pairs of trials (each pair consisting of a choice between a blank and an odorant) at each concentration step. The geometric mean of the four staircase reversal points following the third staircase reversal was used as the threshold measure.
The third test of the test battery was one of odor recognition memory. On a given trial of this 12-trial test (see ref. 24 for details), a microencapsulated "target odorant" is presented to a subject, followed by four odorants from which the subject is instructed to select the one identical to the target stimulus. On four trials, a 10-second interval is interspersed between the sampling of the target stimulus and the presentation of the first of the four alternatives. On four others, a 30-second interval is enforced, whereas on the other four trials a 60-second period intervenes. In the present study the number of trials in which the target odor was correctly identified, irrespective of delay interval, served as the dependent measure.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Matched-pairs
t-tests revealed that the Type Ia PHP subjects performed
significantly more poorly than their matched controls on all three
tests [t-tests with Bonferroni-corrected levels: UPSIT
t (12) = 11.08, P < 0.0001; PEA
t (11) = 3.46, P < 0.05; memory
t (11) = 6.20, P < 0.0001]. The Type Ib
PHP patients were also less sensitive than the controls on the PEA odor
detection threshold test [PEA t (7) = 6.64,
P = 0.005] and on the odor memory test [t
(7) = 3.48, P < 0.05], but not on the UPSIT
[t (7) = 1.49, P > 0.20]. None of the
scores of the PPHP patients differed significantly from those of their
matched controls (all Ps > 0.50).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein deficient PPHP patients have relatively
normal olfactory function. Taken together, these observations throw
into question the hypothesis that the olfactory dysfunction associated
with PHP Type Ia is caused by generalized Gs protein
deficiency. Furthermore, these data clearly demonstrate that the loss
is not the result of AHO, per se. Although the basis for the olfactory deficits in Type Ia and Type Ib PHP patients is unknown, several possibilities are worthy of consideration. First, it is conceivable that these types of PHP patients have, perhaps to differing degrees, decreased function in the primary olfactory G protein, Golf, or in the type III olfactory adenylyl cyclase that is coupled by Golf to odorant receptors (5, 6). Second, in as much as the olfactory adenylyl cyclase behaves as a coincidence detector that can be stimulated by both Golf and Ca2+ (5), it is possible that mild hypocalcemia, which is common in patients with Type Ia and Type Ib PHP, may reduce generation of cAMP in olfactory neuroepithelium and thereby impair olfaction. Third, given that PHP Type Ia patients (i) seem to have greater odor identification dysfunction than PHP Type Ib patients and (ii) exhibit a wider range of hormone resistance than do PHP Type Ib patients, the difference in odor identification ability may be related to the degree of target tissue resistance to hormones that act via stimulation of the cAMP second messenger system. The failure to find this degree of difference on olfactory tests other than odor identification may reflect their relatively lower reliability and sensitivity, rather than the sampling of different neural substrates (25). Fourth, given the normal olfactory function of the PPHP patients, who do not exhibit PTH resistance, the decreased olfactory function in the Type Ia and Ib PHP patients could reflect PTH resistance, per se. However, since no olfactory disturbance has been reported in other patients with PTH deficiency, such as patients with hypoparathyroidism, it is possible that PTRrP resistance, rather than PTH resistance, is the mechanism responsible for the decreased olfactory function.
Whatever the basis for the olfactory alterations observed in Type I PHP, the present study clearly demonstrates that such losses are observed on several types of olfactory tests. While the influence of Type I PHP on nominally disparate olfactory tests could reflect the involvement of cAMP-related biochemical changes at multiple levels within the nervous system, alterations within limited sectors of the olfactory pathway could also explain this result. For example, if detection ability is altered as a result of biochemical changes within the olfactory epithelium, poor performance would also occur on tests of odor memory and identification, because no odor sensation would be available for encoding the percept needed for such tasks (26). Thus, in such cases, underlying neural circuits needed for odor memory or identification could still be intact. Clearly, research is needed to determine if the PHP-related changes in olfactory function reflect, in fact, alterations in the olfactory receptor cells themselves, or if alterations in other elements of the olfactory pathway are also present.
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Acknowledgments |
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Footnotes |
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Received August 1, 1995.
Revised July 9, 1996.
Accepted August 27, 1996.
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References |
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