Abstract
Background
Smell function has been initiated with theophylline treatment in 63% of patients with Type II congenital smell loss. Based upon a systematic evaluation of the protein components of nasal mucus we have demonstrated that interactions among four chemical moieties in nasal mucus may play significant roles in this initiation. Prior to treatment three of these moieties, cAMP, cGMP and sonic hedgehog (Shh), were significantly decreased in concentration whereas one of these moieties, TNFalpha, was increased in concentration. The mechanism(s) responsible for initiation of smell function in these patients, not immediately apparent, may depend upon understanding interactions among these moieties.
Methods
Measurements of cAMP, cGMP, Shh and TNFalpha in nasal mucus by specific spectrophotometric immunoassays before and after treatment with theophylline.
Results
Before theophylline treatment cAMP, cGMP and Shh in nasal mucus, which act as growth factors to support olfactory receptor function, were significantly decreased below normal levels whereas TNFalpha which acts as a “death factor” to inhibit olfactory receptor function was significantly increased above normal. After theophylline treatment cAMP, cGMP and Shh increased significantly whereas TNFalpha decreased significantly.
Conclusions
These results indicate that there are specific biochemical changes associated with smell loss in patients with Type II congenital smell loss and that correction of these biochemical changes are associated with initiation of smell function in these patients. Understanding these relationships play an important role in understanding receptor action in smell function.
1
Introduction
There are about 500,000 patients in the United States with congenital loss of smell (hyposmia) . There are two patient types – Type I patients comprise about 12% of the group . These patients usually have a familial history of smell loss, have multiple defined congenital defects of brain, have multiple brain abnormalities of the olfactory system, have multiple somatic abnormalities involving several organ systems and commonly exhibit hypogonadotropic hypogonadism . Some of these patients have what has been labeled Kallmann syndrome . All patients have been unable to recognize any external odor since birth.
Type II patients comprise 88% of this group . They usually do not have a familial history of smell loss but smell loss occurs spontaneously. They exhibit no known genetic abnormalities but we have recently presented data which suggests that they exhibit such an abnormality on chromosome 1 . While anatomy of the olfactory system in their brain exhibits some changes from normal subjects all patients have olfactory bulbs and olfactory anatomy consistent with a functioning olfactory system . They do not exhibit any somatic abnormalities. These patients are as unable to recognize any external odor as are Type I patients.
No effective treatment has been determined for Type I patients due primarily to the multiple anatomical abnormalities present in their olfactory system. However, treatment of Type II patients has been effective in initiating smell function in 63% of these patients after use of the phosphodiesterase (PDE) inhibitor theophylline .
We have previously reported studies in which the major biochemical moieties present in nasal mucus which influence olfactory function were elucidated . We have also reported preliminary studies about biochemical abnormalities present in Type II patients related to their hyposmia . We now wish to explore these biochemical abnormalities in more detail. We also wish to present changes in these biochemical abnormalities associated with improvement in their smell function for the first time. These changes, associated with initiation of their smell function, relate to their treatment with the PDE inhibitor theophylline.
2
Methods
2.1
Patients
Nineteen patients aged 6–57 y (25 ± 5 y, Mean ± SEM) with Type II congenital hyposmia were studied at The Taste and Smell Clinic in Washington, DC. Each patient presented to the clinic in an effort to initiate their smell function. Results reflect data collected in all patients studied. Patients were five males aged 6–29 y (19 ± 5 y) and 14 females aged 7–57 y (27 ± 4 y). Studies were approved by the Institutional Review Board of the Chesapeake IRB of Columbia, MD.
Each patient stated they were never able to recognize any odor. Each had been evaluated prior to their visit to the clinic by otolaryngologists and/or neurologists; each was found to have no otolaryngological or neurological abnormalities associated with their smell loss although some patients had smaller than normal olfactory bulbs and olfactory clefts unilaterally . Computed tomography (CT) and/or magnetic resonance imaging (MRI) studies of brain exhibited some changes in olfactory cleft or bulb size, usually of a unilateral nature, but otherwise the olfactory system was anatomically intact . One patient had duplicated olfactory bulbs that were of smaller than normal size . All adult patients exhibited normal gonadal function. All prepubertal patients exhibited normal growth and development. No patient exhibited any somatic abnormality of any organ system.
All studies were performed in a single center, The Taste and Smell Clinic in Washington, DC, in an open label clinical trial. All patients and investigators were unmasked. During all assessments staff were masked to the study group at all patient visits.
All patients received oral theophylline at doses of 200–800 mg in two divided doses for periods of 2–36 months. Patients were initially evaluated on 200 mg after two months of treatment and evaluated at six month intervals thereafter based upon initiation or lack of initiation of smell function. If patients initiated smell function at 200 mg they were maintained on this dose and reevaluated at six month intervals. If they did not respond with smell initiation at 200 mg, their dose was increased to 400 mg. They were evaluated after six months of treatment. If they initiated smell function at 400 mg they were maintained at this dose and reevaluated at six month intervals. If they did not initiate smell function at 400 mg their dose was increased to 600 mg with the same sequence used at 600 mg and 800 mg.
Patients were reevaluated at the clinic at periods of 2–6 months at which time blood for serum theophylline , nasal mucus and olfactometry were obtained. Before treatment each patient was demonstrated by olfactometry to exhibit either anosmia, Type I hyposmia or Type II hyposmia .
Twenty-five normal volunteers aged 25–82 y (66 ± 4 y), 10 men aged 25–82 y (70 ± 6 y) and 15 women aged 25–77 y (63 ± 5 y) were also studied. Subjects were patients who presented to the clinic for clinical problems unrelated to smell dysfunction (e.g., tinnitus, oral pyrosis). Each patient stated that they had normal smell function. Smell function studies similar to those performed in the patients were performed in these subjects and were in the normal range. Nasal mucus was collected from each volunteer in the same manner as used for the patients and processed in the same manner.
2.2
Techniques
Nasal mucus was collected at the initial presentation to the clinic. Nasal mucus was collected in a 50 ml plastic tube over a period of 1–3 days and kept refrigerated overnight if collection extended longer than 24 h. Nasal mucus was transferred to 12 ml centrifuge tubes, centrifuged for 40–50 min at 18,000 rpm in a Sorvall RC-5C Plus centrifuge. Supernatant was transferred to a pcr tube and frozen at − 20 °C until processing.
Nasal mucus samples were evaluated by immunoassay by kits prepared by R&D Systems (Minneapolis, MN) for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and by Abcam (Cambridge, MA) for TNFalpha. All studies were performed independent of any clinical knowledge of the source of the sample. Results were not collated until all studies were completed.
2
Methods
2.1
Patients
Nineteen patients aged 6–57 y (25 ± 5 y, Mean ± SEM) with Type II congenital hyposmia were studied at The Taste and Smell Clinic in Washington, DC. Each patient presented to the clinic in an effort to initiate their smell function. Results reflect data collected in all patients studied. Patients were five males aged 6–29 y (19 ± 5 y) and 14 females aged 7–57 y (27 ± 4 y). Studies were approved by the Institutional Review Board of the Chesapeake IRB of Columbia, MD.
Each patient stated they were never able to recognize any odor. Each had been evaluated prior to their visit to the clinic by otolaryngologists and/or neurologists; each was found to have no otolaryngological or neurological abnormalities associated with their smell loss although some patients had smaller than normal olfactory bulbs and olfactory clefts unilaterally . Computed tomography (CT) and/or magnetic resonance imaging (MRI) studies of brain exhibited some changes in olfactory cleft or bulb size, usually of a unilateral nature, but otherwise the olfactory system was anatomically intact . One patient had duplicated olfactory bulbs that were of smaller than normal size . All adult patients exhibited normal gonadal function. All prepubertal patients exhibited normal growth and development. No patient exhibited any somatic abnormality of any organ system.
All studies were performed in a single center, The Taste and Smell Clinic in Washington, DC, in an open label clinical trial. All patients and investigators were unmasked. During all assessments staff were masked to the study group at all patient visits.
All patients received oral theophylline at doses of 200–800 mg in two divided doses for periods of 2–36 months. Patients were initially evaluated on 200 mg after two months of treatment and evaluated at six month intervals thereafter based upon initiation or lack of initiation of smell function. If patients initiated smell function at 200 mg they were maintained on this dose and reevaluated at six month intervals. If they did not respond with smell initiation at 200 mg, their dose was increased to 400 mg. They were evaluated after six months of treatment. If they initiated smell function at 400 mg they were maintained at this dose and reevaluated at six month intervals. If they did not initiate smell function at 400 mg their dose was increased to 600 mg with the same sequence used at 600 mg and 800 mg.
Patients were reevaluated at the clinic at periods of 2–6 months at which time blood for serum theophylline , nasal mucus and olfactometry were obtained. Before treatment each patient was demonstrated by olfactometry to exhibit either anosmia, Type I hyposmia or Type II hyposmia .
Twenty-five normal volunteers aged 25–82 y (66 ± 4 y), 10 men aged 25–82 y (70 ± 6 y) and 15 women aged 25–77 y (63 ± 5 y) were also studied. Subjects were patients who presented to the clinic for clinical problems unrelated to smell dysfunction (e.g., tinnitus, oral pyrosis). Each patient stated that they had normal smell function. Smell function studies similar to those performed in the patients were performed in these subjects and were in the normal range. Nasal mucus was collected from each volunteer in the same manner as used for the patients and processed in the same manner.
2.2
Techniques
Nasal mucus was collected at the initial presentation to the clinic. Nasal mucus was collected in a 50 ml plastic tube over a period of 1–3 days and kept refrigerated overnight if collection extended longer than 24 h. Nasal mucus was transferred to 12 ml centrifuge tubes, centrifuged for 40–50 min at 18,000 rpm in a Sorvall RC-5C Plus centrifuge. Supernatant was transferred to a pcr tube and frozen at − 20 °C until processing.
Nasal mucus samples were evaluated by immunoassay by kits prepared by R&D Systems (Minneapolis, MN) for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and by Abcam (Cambridge, MA) for TNFalpha. All studies were performed independent of any clinical knowledge of the source of the sample. Results were not collated until all studies were completed.
3
Results
TNFalpha levels in nasal mucus in untreated patients were over five times higher than those in normal subjects ( Table 1 ). After treatment with oral theophylline TNFalpha decreased to levels less than 19% in the untreated state ( Table 2 ) and to levels less than those measured in normal subjects.
| Subjects | Nasal Mucus (pg/ml) |
|---|---|
| Patients (19) | 19.4 ± 10.3 ⁎ |
| Normals (25) | 3.6 ± 0.3 |
| Patients | Nasal mucus (pg/ml) |
|---|---|
| Untreated | 19.4 ± 10.3 ⁎ |
| Treated | 1.0 ± 0.1 |
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