Plasma homocysteine in obstructive sleep apnoea

Anna Svatikovaa, Robert Wolka, Mark J Mageraa, Abu S Shamsuzzamana, Bradley G Phillipsb and Virend K Somersa,*

a Division of Cardiovascular Disease, Mayo Clinic and Mayo Foundation, 200 1st St. SW, Rochester, MN 55901, USA
b University of Iowa, Iowa City, IA, USA

* Corresponding author. Tel.: +1-507-255-1144; fax: +1-507-255-7070
E-mail address: somers.virend{at}mayo.edu

Received 1 March 2004; revised 28 April 2004; accepted 18 May 2004 See page 1281 for the editorial comment on this article1

Abstract

Aims Whether increased homocysteine is one mechanism linking obstructive sleep apnoea (OSA) to cardiovascular abnormalities is unclear. We hypothesised that plasma homocysteine would be higher in OSA patients than in control subjects, would increase further during sleep, and decrease after treatment with continuous positive airway pressure (CPAP).

Methods and Results For study A, homocysteine was measured in 22 OSA patients and 20 controls first before sleep, then after 5 h of untreated OSA, and then in the morning after CPAP treatment. Homocysteine was similar in the OSA and control subjects at all three time points, and declined overnight in both groups (, , respectively). To further assess this diurnal variation, we studied plasma homocysteine under a full-night protocol in 10 OSA patients and 12 controls (study B). Homocysteine was measured before sleep, in the morning after sleep, and at noon. Results in both OSA and control groups showed an overnight decline in homocysteine which was reversed by noon (repeated measures ANOVA: OSA, ; controls, ). Study C showed that disturbed sleep did not affect homocysteine levels in normal subjects.

Conclusion There is a significant diurnal variation in plasma homocysteine, so that homocysteine is lower in the morning after waking. Neither OSA nor disturbed sleep elicit acute or chronic changes in homocysteine.

Key Words: Homocysteine • Sleep apnoea • CPAP

Introduction

Patients with severe obstructive sleep apnoea (OSA) are subject to repetitive episodes of profound nocturnal hypoxaemia and have decreased nitric oxide (NO) production,1,2 endothelial dysfunction,3 insulin resistance,4 and an increased risk of cardiac and vascular damage,5 stroke,6 and dementia.7 The mechanisms linking OSA to these endothelial, metabolic, cardiovascular, and neural abnormalities are not known.

Plasma homocysteine has recently emerged as an independent vascular risk factor.8 Elevated homocysteine is associated with increased risk of coronary heart disease, hypertension, atherosclerosis, stroke, and dementia.9–11 High plasma homocysteine is connected with endothelial dysfunction, arterial intimal-medial wall thickening, and a pro-thrombotic state.9,12–14 The cardiovascular risk associated with OSA may conceivably be related in part to increased plasma homocysteine levels. Homocysteine may increase the magnitude of oxidative stress induced by the episodes of hypoxia in OSA.15,16

Only one prior study has directly addressed the question of homocysteine levels in OSA patients17 and noted higher levels of homocysteine in OSA patients with cardiovascular diseases but no differences between OSA patients and healthy controls. Limitations of the previous study include, first, that homocysteine was only measured in the morning hours, whereas there is a marked diurnal variation in homocysteine levels, with the peak occurring late in the evening.18 Second, over 30% of the control and OSA patients in that study were smokers. Third, the control subjects tended to be less obese than OSA patients. Both smoking and obesity may affect homocysteine levels.19–23 Last, many subjects in the prior study were on some kind of medication. In the present study, we compared homocysteine levels in OSA patients to normal control subjects with similar characteristics. All patients and subjects were non-smokers and on no medications. OSA patients were previously undiagnosed and otherwise healthy. Normal subjects underwent complete overnight polysomnography to exclude occult OSA. We investigated the overnight and late morning changes in homocysteine levels in control and OSA patients to test whether homocysteine levels might be different in the two groups in the evening (the time when homocysteine levels are known to peak), and whether homocysteine may be affected acutely by hypoxia, oxidative or other stresses of sleep apnoea.

Methods

We compared 32 male subjects with newly diagnosed moderate to severe OSA (Apnea-Hypopnoea Index (AHI)>=20) to 32 healthy men with similar age and body mass index (BMI), in whom occult OSA was excluded (AHI<= 5). All patients and healthy controls were free from other diseases and were taking no medications. OSA patients had not been previously treated for OSA. All participants were non-smokers and were not taking any supplemental vitamins. The study was approved by the Human Subjects Review Committee. Informed written consent was obtained from each subject.

The presence and severity of sleep apnoea were determined by standard overnight polysomnography. Apnoea was defined as complete cessation of airflow for at least 10 s. Hypopnoea was defined as a reduction of respiratory signals for at least 10 s associated with oxygen desaturation of >=4%. AHI was calculated as the total number of respiratory events per hour of sleep.

The sleep studies followed either a split-night (study A) or a full-night (study B) protocol. Subjects undergoing the split-night protocol, as dictated by our institutional clinical guidelines, were recruited from the Mayo Clinic Sleep Disorders Center. The night before polysomnography was performed, study personnel identified subjects who did not have any co-morbidities, who were taking no medications, and were non-smokers. The following morning, after completing overnight polysomnography and obtaining the subject's complete sleep report, we identified the subjects either as healthy controls (AHI<=5 events/h) or OSA (AHI>=20 events/h). We excluded subjects with mild OSA (AHI between 5 and 20 events/h).

Study A, the split-night protocol, was designed to compare the acute effects of untreated OSA and subsequent CPAP therapy, on homocysteine levels. The first half of the study was for the diagnosis of OSA severity. Therapy with CPAP was administered in the second half of the night. Plasma homocysteine levels in 22 OSA patients were measured at 9:00 p.m. (before sleep), at 2:00 a.m. (after 5 h of untreated OSA and before CPAP therapy started), and at 6:00 a.m. (after waking in the morning, after 4 h of CPAP treatment). Measurements were obtained at similar times in 20 control subjects. The results of the split-night protocol led us to further investigate plasma homocysteine levels during uninterrupted sleep (study B – the full-night protocol) and during wakefulness. Therefore, for the full-night part of the study, we recruited additional subjects from the local Rochester community. They underwent overnight polysomnography in the General Clinical Research Center, Saint Mary's Hospital, Mayo Clinic.

The full-night protocol, study B, was designed to evaluate the presence and magnitude of any diurnal variation in homocysteine and any influence of OSA on this variation. Ten untreated OSA patients (without CPAP) and 12 controls slept continuously through the night and homocysteine levels were measured in the evening (9:00 p.m.), morning (6:00 a.m.), and at noon (12:00 p.m.). Finally, we studied the effect of disturbed sleep on homocysteine levels in healthy control subjects without OSA (study C). The severity of sleep disturbance was indicated by the arousal index (number of arousals per hour of sleep). We compared homocysteine levels before and after sleep in healthy subjects with a high arousal index (increased sleep disturbance) to a group with a low arousal index.

Plasma homocysteine concentrations were measured by a highly sensitive and specific liquid chromatography electrospray tandem mass spectrometry method (LC–MS/MS).24 Because of consistent data showing increased homocysteine in obese individuals,20–23 we also calculated a homocysteine/BMI ratio in all subjects.

Results are reported as means±SEM. All statistical tests were two-sided. Since we used three different study protocols (A, B, and C), statistical analysis was performed separately for each study protocol. In each protocol, we had two groups; we therefore used an unpaired two-sided Student's t-test to analyse any differences in demographic and haemodynamic characteristics between these two groups at a specific time point. In order to analyse changes within each group over time (three time points), we used analysis of variance (ANOVA) for repeated measures, followed by Newman–Keuls post hoc tests. Time points were included as a class variable, and homocysteine as a continuous variable. Statistical significance was defined as .

Results

Baseline (evening) levels of plasma homocysteine were similar in 32 severe OSA patients and 32 healthy control subjects () (Fig. 1). Homocysteine/BMI ratio was virtually the same in OSA patients and in healthy controls (Table 1).



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Fig. 1 Baseline levels of plasma homocysteine in OSA patients and healthy controls. Plasma levels of homocysteine measured at baseline (9:00 p.m.) in 32 OSA patients and 32 control subjects. . Data represent means±SEM.

 

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Table 1 Baseline characteristics of OSA patients and controlsa

 
Study A – split night protocol
The OSA and control groups were very similar with regard to demographics and haemodynamics (Table 1). In OSA patients, AHI was significantly higher compared to controls (48±3 vs. 3±3 events/h, ). With CPAP treatment, AHI decreased from 48±3 to 6±3 events/h ().

Plasma homocysteine was similar in OSA and control subjects at all three time points, (evening: 8.8±0.5 vs. 8.2±0.5 µmol/L, ; middle of the night 8.5±0.5 vs. 7.8±0.5 µmol/L, ; morning 7.8±0.3 vs. 7.6±0.3 µmol/L, ). However, in both the OSA and the control groups, there was a gradual overnight decrease in homocysteine levels (; , respectively) (Fig. 2), without any significant difference between the two groups.



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Fig. 2 Plasma homocysteine levels in OSA patients and healthy controls during split-night protocol. Plasma levels of homocysteine measured in 22 OSA patients and 20 healthy controls before sleep (9:00 p.m.), after 5 h of untreated OSA (2:00 a.m.), and after 4 h of CPAP treatment (6:00 a.m.). Data represent means±SEM.

 
Study B – full night protocol
This diurnal variation was further studied under the full-night protocol. We measured plasma homocysteine in another 10 OSA patients and 12 healthy controls with similar characteristics. Homocysteine was measured first before sleep, then in the morning after awakening, and a third measurement at noon. In the OSA patients, AHI was significantly higher compared to controls (55±7 vs. 3±6 events/h, ; Table 1), and OSA patients experienced significant nocturnal hypoxaemic desaturation (mean desaturation nadir of 73±4%). Plasma homocysteine was again similar in the OSA and control subjects at all three time points (evening: 9.2±0.7 vs. 8.2±0.6 µmol/L, ; morning: 7.9±0.5 vs. 7.4±0.4 µmol/L, ; and noon: 8.7±0.6 vs. 7.6±0.6 µmol/L, , respectively). Consistent with the results from the split-night study, homocysteine levels decreased overnight. This overnight decrease was reversed during the day, when homocysteine levels approached baseline evening values (ANOVA for repeated measures: OSA, ; controls, ) (Fig. 3). The magnitude of this early morning reduction in homocysteine was similar both in the normal subjects and the OSA patients.



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Fig. 3 Plasma homocysteine levels in OSA patients and healthy control subjects during full-night protocol. Plasma levels of homocysteine in 10 OSA patients and 12 healthy control subjects measured before sleep (9:00 p.m.), in the morning (6:00 a.m.), and at noon (12:00 p.m.). Data represent means±SEM.

 
Study C – effect of sleep disturbance
We compared plasma homocysteine levels in six healthy subjects with high sleep disturbance (arousal index: 32±1 events per h) to six healthy subjects with low sleep disturbance (arousal index: 14±1 events/h). These two groups of subjects were very similar with regard to demographics and haemodynamics (Table 1). Plasma homocysteine levels were similar between these two groups both at baseline, before sleep, and in the morning, after awakening (controls with high arousal index vs. controls with low arousal index; baseline: 8.1±0.7 vs 8.3±0.7 µmol/L, and after awakening: 7.1±0.5 vs. 7.1±0.5 µmol/L, ; between group comparison ). Therefore, increased sleep disturbance did not affect either baseline homocysteine levels or the morning decline in homocysteine (Fig. 4).



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Fig. 4 Effect of sleep disturbance on plasma homocysteine levels. Plasma levels of homocysteine in six healthy control subjects with high arousal index (high sleep disturbance) measured before sleep (9:00 p.m.) and in the morning (6:00 a.m.) compared to six healthy control subjects with low arousal index (low sleep disturbance). The differences between the two groups were not significant (). Data represent means±SEM.

 
Discussion

Our studies of plasma homocysteine levels during normal and apnoeic sleep indicate the following: first, homocysteine levels are not chronically elevated at any time point studied in otherwise healthy patients with OSA, compared to control subjects with similar characteristics. This suggests that alterations in homocysteine are unlikely to play a key role in the cardiovascular pathophysiology of OSA. Second, both in the control and OSA groups, there was a diurnal variation in plasma homocysteine levels, with an overnight decrease followed by a subsequent increase during the day. Third, neither acute untreated OSA nor CPAP treatment obscure the diurnal changes in plasma homocysteine levels. Fourth, disturbed sleep also does not significantly affect homocysteine levels.

Our study provides some novel and additional information regarding plasma homocysteine levels in OSA patients without other diseases. Homocysteine is an amino acid released as the body digests proteins. Total homocysteine levels in adults follow a circadian variation.18 There is no report on circadian or diurnal variation in the setting of repetitively disturbed apnoeic sleep. Lavie et al.17 report no differences in morning homocysteine levels between OSA patients and controls. We built further upon this important observation and investigated the presence of a diurnal variation in OSA patients and examined whether there were any chronic differences in homocysteine between OSA and control subjects in the late evening, when homocysteine reaches its highest levels.

Furthermore, our results may have an important implication for understanding the relationship between obesity and homocysteine levels. Namely, it has been suggested that obesity may be associated with increased plasma homocysteine levels.20–23 Obesity is frequently accompanied by OSA and the presence of OSA can explain several abnormalities traditionally ascribed to obesity.25 In this present study we, however, have shown that occult OSA is unlikely to explain any association between obesity and hyperhomocysteinaemia.

Important strengths of our split and full-night studies are, first, that we included only normotensive OSA patients who had no co-existing disease conditions apart from OSA. Second, patients and controls were not taking any medications, and had not been previously diagnosed or treated for sleep apnoea. Third, the control subjects had similar age and BMI, thus ruling out any potential confounding influence of age or obesity on our data. Fourth, the overnight polysomnographic recordings excluded any effects of occult sleep apnoea in our obese control subjects. Last, we used a highly specific methodology utilising LC–MS/MS for measurements of plasma homocysteine.24

In conclusion, several cardiovascular and neuronal diseases have been associated with increased plasma homocysteine levels.8–11 We show that plasma levels of homocysteine are not chronically elevated in patients with OSA in the absence of any of these conditions, and that neither acute untreated OSA, nor treatment with CPAP, nor disturbed sleep affect plasma homocysteine levels or obscure its diurnal variation. Therefore, homocysteine per se is unlikely to contribute to cardiac and vascular morbidity in otherwise healthy OSA patients.

Acknowledgments

This work was supported by the NIH Grants: NIH, HL-65176, HL-61560, HL-70602, and M01-RR00585.

Footnotes

1 doi:10.1016/j.ehj.2004.06.012. Back

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