Nitric oxide branch of arginine metabolism in depression: effect of venlafaxine.
Abstract: Objectives

Major Depressive Disorder is an established independent risk factor for cardiovascular disease, but the precise pathophysiological mechanism remains obscure. The nitric oxide branch of arginine metabolism has been linked to vascular homeostasis.

Methods

Five plasma biomarkers of the nitric oxide branch of arginine metabolism were quantified in depressed patients (n = 22) and healthy controls (n = 17): total nitrite, nitrotyrosine, asymmetric dimethylarginine, agmatine, and myeloperoxidase. Thirteen of the depressed patients were restudied after 4-8 weeks of mood normalization with venlafaxine, a mixed serotonin/norepinephrine reuptake blocker.

Results

None of the biomarkers were altered in depressed patients compared to controls. However, treatment reduced agmatine and myeloperoxidase levels (p=0.02 each). A clear but non-significant rise in total nitrite and nitrotyrosine was also observed at week-4.

Conclusions

Despite no changes at pre-treatment, the reductions in agmatine and myeloperoxidase may result from serotonin and/or noradrenaline changes occurring with venlafaxine antidepressant therapy.

Keywords: Nitric oxide, Arginine, Nitrotyrosine, Agmatine, Depression, Antidepressants, Venlafaxine, Cardiovascular Disease
Article Type: Report
Subject: Cardiovascular diseases (Risk factors)
Cardiovascular diseases (Physiological aspects)
Endothelin (Physiological aspects)
Venlafaxine (Physiological aspects)
Nitric oxide (Physiological aspects)
Arginine (Physiological aspects)
Authors: Piletz, John E.
Halaris, Angelos
Iqbal, Omer
Hoppensteadt, Debra
Fareed, Jawed
Zhu, He
Sinacore, James
DeVane, C. Lindsay
Pub Date: 10/01/2009
Publication: Name: International Journal of Health Science Publisher: Renaissance Medical Publishing Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Renaissance Medical Publishing ISSN: 1791-4299
Issue: Date: Oct-Dec, 2009 Source Volume: 2 Source Issue: 4
Product: Product Code: 2813772 Nitric Oxide NAICS Code: 32512 Industrial Gas Manufacturing SIC Code: 2813 Industrial gases
Accession Number: 221654550
Full Text: INTRODUCTION

Major Depressive Disorder (MDD) is recognized as an independent risk factor for cardiovascular disease (CVD). (1) The cardiovascular burden that depression imposes towards CVD is clear, documented by every long-term prospective study we could find (now numbering more than 25 studies) where psychological predictors have been investigated (2-6), as well as by most retrospective studies (7) (for complete review see Lett et al, 2004 (1)). A relative risk of approximately 2.0 has been established for developing cardiac disease in those patients carrying a diagnosis of major depression. (8) Patients who experience clinical depression following myocardial infarction have an even higher relative risk for subsequent myocardial infarction and cardiac mortality within 6-12 months (RR = 1.7 - 3.5+). (9, 10) No unifying biological mechanism has yet been able to fully explain the link of MDD leading to CVD. Platelet hyperactivity, endothelial damage, and hypothalamic-pituitary-adrenal axis dysregulation have all been suggested as contributing factors. (11, 12)

Arginine (arg) is a nutritionally essential amino acid of critical importance to vascular tone and hemodynamics. (13) Arg metabolism is highly regulated and follows co-expressed competitive metabolic branches in humans. (13) The bulk of arg is normally metabolized by arginases, leading to ornithine and its downstream products (principally the polyamines). (14) Additional branches of arg metabolism are mediated by: (a) nitric oxide synthases (NOSs) leading to production of nitric oxide (NO) and downstream nitrogenous compounds, (b) arg decarboxylase leading to agmatine and its related byproducts, and (c) arg:glycine amidinotransferase leading to guanidinoacetic acid, the immediate precursor of creatine. Each enzyme is highly regulated under physiological or pathological conditions. (13) The production of NO is often placed "center stage" due its key role in vascular homeostasis (15) (Fig. 1). Decreased metabolism of arg to form NO (plus forming citrulline as byproduct) has also been associated with cardiovascular risk. (16) Rising NO and nitrate levels are the main actions of the therapeutic agent, nitroglycerine. (17) Two stable NO metabolites, nitrite and nitrate, collectively designated NOx, also have healthy vascular properties. However, a further downstream product, peroxynitrite, is a reactive metabolite that is cytotoxic. (18) Myeloperoxidase is the main plasma enzyme that oxidizes nitrites and nitrates to peroxynitrite. (19) Peroxynitrite reacts avidly with free tyrosine and/or proteinacious tyrosine, to produce nitrotyrosine (NO(2)Tyr). High NO(2)Tyr is considered an index of oxidative stress if found in conjunction with elevated myeloperoxidase activity. (18,19) Completing the circuit, NO production is also partly regulated by proteinacious tyrosine. Asymmetric dimethylarginine (ADMA) derived from protein's breakdown during tissue damage is a negative modulator of NOS. (16) In this report, we focused on five key biomarkers of arg metabolism that collectively reflect NO and its down stream products in the endothelium, as shown in Fig 1.

[FIGURE 1 OMITTED]

A handful of previous reports (20-23) have provided some evidence in depression for lowered NO production, with relatively more arg going to non-NO/citrulline products in the blood of depressed patients. There is, however, one report (24) showing no change in plasma NO in depressed patients, plus two reports (25, 26) showing increased NOx in arguably the most severe form of depression: suicide attempters. A genetic study has provided additional tentative evidence that gene variants of neuronal NOS (nNOS) are associated with suicide attempters while other gene variants of endothelial NOS (eNOS) may be "protective" from suicidal behavior. (27) To further study NO in depression, we designed and implemented the present study. We chose five biomarkers of the NO branch of arg metabolism, whereas previous studies generally measure only one or two of the NO biomarkers in each study. Secondly, we chose a group of depressed patients with low suicidal risk and low (or nil) signs of CVD in an attempt to rule out potential confounds. These patients were part of our earlier study showing elevated pro-inflammatory cytokines. (28)

The biomarkers chosen for the present study were NOx, NO2(Tyr), ADMA, agmatine, and myeloperoxidase. Subjects were selected based on being free from concurrent medical problems--with special attention given to having few if any traditional risk factors for CVD. Following baseline measurements, the effects of 4 and 8 weeks of treatment with venlafaxine were studied in 13 of the patients. Venlafaxine is a serotonin and norepinephrine reuptake inhibitor (SNRI) at therapeutic doses (lowest doses are predominantly serotonin blocking). (29) Our study may fall in line with that of Ikenouchi-Sugita and colleagues (23) who demonstrated treatment effects with another SNRI (milnacipran), which led to significant elevations in total blood nitrite levels.

MATERIALS AND METHODS

This study was approved by the appropriate Institutional Review Board. Subjects were identified through advertisements and/or hospital referrals. Written informed consents were obtained prior to enrollments and anonymity was preserved. Enrollment period was the same for patients and controls in order to minimize ordering effects or possible seasonality of the biomarkers. All subjects were seen as out-patients. A modest compensation plan was used to reimburse travel expenses. Patients opting to stay in the study beyond baseline measurements, received venlafaxine free-of-charge.

The Structured Clinical Interview was administered at baseline to arrive at a diagnosis of MDD for each patient. (30) After MDD was diagnosed, the 21-item Hamilton Depression Scale (HAM-D (31)) was administered. Inclusion criteria for patients required a diagnosis of primary MDD, unipolar type, recurrent or first-episode lasting 2-6 months, and a minimum score of 19 on the HAM-D. None of the patients had active suicidal ideations at any point in the study. Other rating scales were also administered to gain further insight about the depressions of these patients: the Beck Depression Inventory, Profile of Mood States, Life Events Questionnaire, and Covi Anxiety Inventory. Scores on these additional rating scales were fully compatible with a non-suicidal MDD diagnosis. Healthy control subjects were ascertained psychologically by the Minnesota Multiphasic Personality Inventory. (32) Prospective healthy subjects scoring two or more standard deviations from the mean of any item on the MMPI were excluded. Healthy controls were also required to have no personal or family history of psychiatric illnesses amongst first-degree relatives.

In many regards, the patients and controls were treated alike: (a) a physical examination with measures of blood pressure, heart rate and electrocardiogram, and (b) laboratory tests including complete blood count, chemistry panel, lipid profile, thyroid function, urinalysis, and a pregnancy test if suspect. A first category of exclusion criteria was any abnormalities revealed by the clinical assessments (including high cholesterol). Other exclusion criteria were histories of metabolic syndrome, allergies to antidepressant medications (if an MDD patient), heart disease, vascular disease, diabetes, arthritis, hypertension, or bleeding diathesis of any etiology. Most psychiatric and/or neurological conditions were also exclusion criteria (including histories of substance abuse/dependence, post-traumatic stress disorder, psychosis of any etiology, and seizures). Nine of the MDD patients who enrolled in the study had had prior antidepressant treatment(s) for recurrent depressive episodes, but were medication free for at least three weeks prior to beginning the study. Five of the recurrent depressives went on to be included in the venlafaxine treatment group, which totaled 14.

Several baseline parameters related to CVD were assessed in the subjects. Already mentioned is the fasting lipid profile in normal range. None of the subjects exhibited supine heart rates above 95 beats per minute (bpm) or supine blood pressures above 145 mm Hg (systolic) or 90 mm Hg (diastolic). None of the subjects had family histories of early-onset heart or vascular disease. Regarding body mass indices (BMIs) at baseline, 7 of the 22 depressed patients, compared to 4 of the 17 healthy control subjects, were categorized as obese with BMIs [greater than or equal to] 30. Smoking habits were recorded using a questionnaire. None of the subjects were heavy smokers, but a few smoked occasionally (< 1 pack cigarettes per day). In no case was smoking allowed within 1.5 h of the morning blood drawing. Based on the questionnaires, 4 of 22 depressed patients at baseline were this type of smoker, compared to none of 17 healthy controls at baseline. One of the smokers went on to enter and complete venlafaxine treatment (the other 3 opted for other treatments).

Participants were reminded throughout the study to report even minor medical developments. In this way, none presented with signs or symptoms of inflammation at the time of blood drawing. Blood drawings were rescheduled if/when physical symptoms were reported. Subjects were expected to refrain from alcohol consumption for 24 hours prior to each blood drawing, and to refrain from vitamins C or E, fish oils, sleeping pills, anti-inflammatory agents, or antihistamines for 3 days prior to each blood drawing. Females were not scheduled for blood drawings immediately before or during their menses. However, menstrual stages were not recorded.

Subjects

At baseline, there were 22 MDD patients and 17 healthy controls of similar age and sex distribution. Of the 22 MDD patients enrolled at baseline, 14 went on venlafaxine treatment (8 opted to receive other treatment modalities). Venlafaxine was dispensed in open-label fashion and its dose was adjusted over the first four weeks between 150-225 mg/day according to patient tolerance and response. Antidepressant blood levels were also measured. The night before the week-4 and week-8 blood assessments, patients were expected to take venlafaxine at 23:00 hours. Subjects arrived the next morning in fasting state for blood drawings. Thirteen patients completed the 8 weeks of venlafaxine treatment. The drop-out was due to blood pressure rise. By power analysis, it was calculated from comparable studies by Leo et al (33) and Ikenouchi-Sugita et al (23) that n=13 subjects/group should be sufficient for detecting antidepressant effects with 80% power at the 5% level of confidence.

Blood Processing

Blood samples were obtained between 9:00-11:00 hours. This occurred at baseline (week 0) and after 4 and 8 weeks of treatment. They were not allowed to engage in strenuous physical activity or consume caffeine products since 23:00 hours the evening before. With the hope of further minimizing artifact, an 18-gague angiocatheter-needle was inserted into the antecubital vein followed by the subjects resting in supine position, with the line patent, for at least 20 min before each blood collection. The first 3 ml of blood was discarded to avoid tissue thrombotic effects. Experimental blood samples (20 ml) were then drawn slowly with a syringe followed by dispensing into sterile tubes.

Within 15 min of collection, the blood was centrifuged (2,000 x g, 4[degrees]C, 15 min). The fraction used for agmatine analysis (10 ml) was immediately mixed with ice-cold trifluoroacetic acid (0.5 ml/10 ml plasma), vortexed, and the mixture allowed to sit for 1 hr on ice before another centrifugation (10,000 x g, 4[degrees]C, 15 min). (34) As soon as each sample was prepared, it was snap-frozen in dry-ice/ethanol slurry. Agmatine assays were performed on thawed samples within 2 months. The other biomarker assays were performed after samples had been stored at -80[degrees]C for 3-5 years.

Biomarker Assays

Agmatine was assayed by isocratic high performance liquid chromatography (HPLC) as described by Feng et al (21, 34) with one improvement: 1-(3-aminopropyl)2-pipecodine (0.8 ng per injection) was used as internal standard. 1-(3-aminopropyl)2-pipecodine is cleanly resolved from agmatine (and other HPLC peaks) and is therefore better-suited than the internal standard used in our original paper. (34) The lower limit of detection of agmatine was found to be 0.5 ng/ml. Whenever agmatine was found near this level, we repeated the HPLC chromatography (up to 7 repeats) to obtain a reliable average value. The reliability of the assay was determined with spiked plasma samples from the local blood bank (spiked with 25 ng/ml of agmatine). The assay yielded intra-assay and inter-assay CVs of less than 10%.

Plasma total nitrite was assayed subsequent to conversion of nitrate to nitrite by the enzyme nitrate reductase. Detection of this nitrite level (called NOx in this context) was determined as the colored azo-dye product of Greiss Reaction that absorbs light at 540-570 nm (kit from R&D Systems Inc., Minneapolis, MN). Duplicate values were averaged. The lowest detection limit of the NOx assay was 1.35 [micro]mol/L. The intra-assay and inter-assay CVs for NOx were 5.3% and 7.0%, respectively.

Plasma NO2(Tyr) levels were measured using the BIOXYTECH[R] Nitrotyrosine EIA-assay kit (OXIS International Inc., Portland, OR). Duplicate values were averaged. The lowest detection limit of the assay was 2 nM. The intra-assay and inter-assay CVs were 2.32% and 11.17%, respectively.

ADMA levels were determined by sandwich ELISA procedure using an ADMA ELISA assay kit (Cardio-Vasics, Palo Alto, CA). Duplicate values were averaged. The lowest detection limit of ADMA by this method was 0.2 [micro]M. Intra-assay and inter-assay CVs were not more than 5%.

Myeloperoxidase was assayed by an enzyme immunometric kit (Assay Designs Inc., Ann Arbor, MI). Duplicate values were averaged. The lowest detection limit of the assay was 0.13 ng/ml. The intra-assay and inter-assay CVs were 2.8% and 5.9%, respectively.

Plasma antidepressant levels were determined after shipment on dry ice to the Medical University of South Carolina (C. Lindsey DeVane). HPLC was used to measure venlafaxine and its metabolite o-desmethylvenlafaxine as previously described. (28)

Statistical Analysis

Initial descriptive analyses revealed non-normal distributions for most of the blood parameters. To approximate normal distributions, the values were logarithmically transformed. Aside from this transformation during the statistical analysis, the means and standard errors of each variable are expressed unchanged (non-logarithmic) throughout the manuscript. Two-group comparisons were performed using Student's paired and unpaired t-test from related and unrelated groups, respectively. In those analyses where BMI was deemed necessary to be controlled, an analysis of covariance was conducted with BMI as covariate. Change scores were calculated by subtracting the pre-treatment values from the post-treatment values. Changes in the biomarkers over the course of treatment were tested with repeated measures analysis of variance (ANOVA). Correlations were assessed with the Pearson Correlation Coefficient.

RESULTS

The baseline CVD risk factors (age, sex, smoking status, BMI, and blood pressure) were roughly equivalent in the two groups under study: depressed patients compared to healthy control subjects (Table 1). Thus, only the presence of depression (i.e., high HAM-D scores) stood out as a distinguishing factor between patients and controls (Table 1). For simplicity sake, Table 1 has also been limited to just those depressed patients that actually completed pre- and post-treatment. The values were super-imposable for the larger group that included depressives who opted-out before and the one who dropped-out during treatment.

Table 2 displays biomarker values from the 13 MDD patients who completed treatment with venlafaxine. The baseline biomarker data on the larger group is shown in Fig. 2 for comparison. No significant differences were observed at baseline between untreated MDD patients versus healthy controls even though levels of NOx, NO2(Tyr), and agmatine tended higher in MDD (Table 2, Fig. 2). Correcting for smoking incidence did not result in significant skewing of the data according to subsequent statistical testing. But, a weak association between BMI and NO2(Tyr) levels was of concern (ANOVA revealed p = 0.10 covariant influence of BMI on NO2(Tyr) levels). When BMI was included in the covariate analysis, there was still no significant difference between MDD patients and healthy subjects at baseline.

Treatment with venlafaxine produced two biochemical findings. Firstly, agmatine was significantly reduced after venlafaxine treatment (Fig. 3c). From a level of 31.9 [+ or -] 6.3 ng/ml at baseline in untreated MDD patients (n = 13), the concentration of agmatine lowered to 16.0 [+ or -] 3.5 ng/ml after 4 weeks and stayed low at 17.2 [+ or -] 4.8 ng/ml after 8 weeks on venlafaxine (F = 6.69, p = 0.02: repeated measures ANOVA). Secondly, myeloperoxidase was significantly reduced after venlafaxine treatment. In untreated MDD patients (n=13) the concentration of myeloperoxidase was 22.9 [+ or -] 4.1 ng/ml at baseline. This lowered to 18.3 [+ or -] 2.0 ng/ml after 4 weeks and 16.4 [+ or -] 1.7 ng/ml after 8 weeks of treatment (F = 4.01, p = 0.02: repeated measures ANOVA). There was a trend for plasma NOx and NO2(Tyr) to be elevated at week-4 during venlafaxine treatment (Fig. 3a,b), but this failed to reach statistical significance.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Correlation Analyses

None of the biomarkers, whether expressed as absolute levels or as change scores with treatment, were correlated with HAM-D. On the other hand, some positive correlations were observed between agmatine and diastolic blood pressures at each time point of the study (r= 0.55, p = 0.04; r = 0.41, p = 0.14; r = 0.57, p = 0.03, respectively) for patients at weeks 0, 4 and 8 of treatment, but not with systolic blood pressures at baseline (r = 0.14, p = 0.62) or other time points. A positive correlation was likewise obtained between agmatine and heart rates (bpm) at baseline (r = 0.57, p = 0.03), but not at other times of treatment. None of the other biomarkers were found to correlate with cardiovascular measures.

DISCUSSION

Depression is considered an independent risk factor for developing CVD, but no precise pathophysiological mechanism can be pointed to. A major review article by Pinto et al. (35) focused on the possible role of NO in this pathophysiology. One of the first studies was by Selley (20), showing low NOx in the plasma of patients with MDD compared to controls. They also found plasma ADMA to be higher in MDD compared to controls (20). In accord with this, Chrapko et al. (36) found low plasma NOx and endothelial nitric oxide synthase (eNOS) activity in MDD patients compared with healthy controls. They later reported (37) that the SSRI antidepressant, paroxetine, restored the levels of plasma NOx. More recently, Ikenouchi-Sugita and colleagues (23) found lower plasma NOx in MDD patients compared with healthy controls. A negative correlation between baseline HAM-D and baseline plasma NOx was also noted in the depressives. Our group has instead studied agmatine, which is a negative modulator of NOS (Fig. 1). We've previously found agmatine high in the plasma of untreated MDD patients and lowered after 8 weeks of antidepressant, bupropion, treatment (21). Another study reported (22) high serum arginase in MDD patients compared with healthy controls. These observations have collectively suggested that the non-NO branches of arg metabolism may predominate in depression, perhaps indicative of decreased substrate availability for NO formation. But, Herken and colleagues reported (24) no difference in NOx in depression, and there are two reports (25,26) of increased NOx levels in arguably the most severe form of depression: suicide attempters. Thus, the literature has not reached consensus.

We could find no difference in levels of NOx, NO2(Tyr), ADMA, agmatine, and/or myeloperoxidase between untreated depressed patients and healthy controls. Thus, we did not replicate the aforementioned studies. However, it should be emphasized that we corrected for BMI. Obesity is an accepted risk factor for endothelial dysfunction and the metabolic syndrome. Not mentioned under the Methods section was a statistical exploratory analysis we did. In this exploratory analysis, our entire 39-subject pre-treatment group of subjects was solely divided on the presence or absence of obesity (BMI >30). When re-analyzed in this BMI-centric manner, the NOx of obese subjects (independent of diagnosis) was statistically higher: 23.5 [+ or -] 5.3 (n = 15) versus 15.3 [+ or -] 0.9 in non-obese subjects (n = 31; significant at p = 0.05). Likewise, the concentration of NO2(Tyr) in the obese subjects tended to be higher independent of diagnosis: 64.4 [+ or -] 26.0 (n = 15) compared to 16.8 [+ or -] 10.0 in non-obese subjects (n = 31; NS, p = 0.16). Thus, BMI seems to be a covariate that cannot be ignored in measuring plasma NOx-related compounds, and correcting for BMI is one of the main ways our study differs from previous studies of depression.

Despite finding no change at baseline in MDD patients, treatment with venlafaxine produced a couple of interesting effects on two biomarkers. Firstly, venlafaxine led to a decrease in plasma agmatine (Fig. 3). This confirms our earlier report with the antidepressant bupropion. (21) Secondly, venlafaxine led to a down-regulation of the key enzyme, myeloperoxidase. Reductions in agmatine and myeloperoxidase were seen by week-4 of treatment and remained consistently low at week-8 (Fig. 3). Change scores for these biomarkers were uncorrelated with each other. Due to a lack of correlation between the change scores for agmatine and myeloperoxidase, it seems unlikely the substances are closely linked. Interestingly, at week-4, there was also a trend for total nitrate/nitrite and NO2(Tyr) to be up-regulated by venlafaxine (Fig. 3), possibly reflecting diversion of arg metabolism away from agmatine and towards NO formation. Although this trend did not reach statistical significance, it would also point in the direction of a protective effect of venlafaxine. This has been previously reported (38) for venlafaxine and other antidepressant treatments in an animal model of chronic stress.

CONCLUSIONS

These studies were performed because it seemed likely that some alterations in plasma NO might exist even in physically healthy depressed patients indicative of a compromised endothelium and early-stage CVD, with the added question of whether venlafaxine could modify this compromised state. We found no evidence of altered NO in the depressed patients, but some changes were observed in response to venlafaxine that might actually improve cardiovascular status.

Acknowledgements

The authors wish to thank Drs. Jeffery Ali for clinical assistance. We also thank Wyeth Pharmaceuticals Inc. for providing venlafaxine. The study was supported by grant MH57601 from the National Institute of Mental Health, and an intramural grant from the Stritch School of Medicine, Loyola Medical Center of Chicago.

Statement of Interest

No conflicts of interest.

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John E. Piletz [1], Angelos Halaris [1], Omer Iqbal [2], Debra Hoppensteadt [2], Jawed Fareed [2], He Zhu [2], James Sinacore [3], C. Lindsay DeVane [4]

Departments of Psychiatry [1], Pathology [2] and Epidemiology [3], Loyola University Stritch School of Medicine, Maywood, Illinois; and Department of Psychiatry, Medical University of South Carolina [4], Charleston, South Carolina

Corresponding author: Dr. John Piletz Department of Psychiatry Stritch School of Medicine, Loyola University Chicago 2160 South First Ave. Building 105, Room 1940 Loyola University Medical Center Maywood, IL 60153 USA 708-216-3276 e-mail: jpiletz@lumc.edu
Table 1--Baseline Characteristics of Those Patients Completing
Venlafaxine Treatment in Comparison to Healthy Controls

Subjects                  Age (yr)           Sex       Smoking *

MDD pre-Venlafaxine   39.5 [+ or -] 2.1    2 males,     1 of 13
(n = 13)                                  11 females

Healthy Controls      39.7 [+ or -] 2.1    3 males,     0 of 17
(n = 17)                                  14 females

                                             Systolic BP
Subjects                     BMI                (mm)

MDD pre-Venlafaxine   28.9 [+ or -] 2.0   118 [+ or -] 3.8
(n = 13)

Healthy Controls      26.2 [+ or -] 1.5   116 [+ or -] 4.2
(n = 17)

                        Diastolic BP            HAM-D
Subjects                    (mm)              scores **

MDD pre-Venlafaxine   70.1 [+ or -] 2.1   26.1 [+ or -] 1.0
(n = 13)

Healthy Controls      67.5 [+ or -] 2.8    1.0 [+ or -] 0.4
(n = 17)

* There was one occasional smoker (<1 pack/day) in the pre/post
treatment comparison group, who went through a routine of one
cigarette at 1.5 hour prior to each morning visit to the clinic.

** Patient's HAM-D scores were highly significant compared to
healthy controls (p < 0.0001). None of the other comparisons
between groups reached statistical significance.

Table 2--Baseline Plasma Biomarkers of Those Patients Completing
Venlafaxine Treatment in Comparison to Healthy Controls

                               NOx             Nitro-Tyrosine
Subjects                ([micro]mol/L) *           (ng/ml)

MDD pre-Venlafaxnie     16.4 [+ or -] 1.8      6.8 [+ or -] 1.9
(n = 13)

Healt hy Subjects       14.2 [+ or -] 1.3     10.2 [+ or -] 4.5
(n = 17)

                           Asymmetric
                            Dimethyl              Agmatine
Subjects                Arginine (ng/ml)           (ng/ml)

MDD pre-Venlafaxnie     2.1 [+ or -] 0.3      31.9 [+ or -] 6.3
(n = 13)

Healt hy Subjects       2.1 [+ or -] 0.3      25.8 [+ or -] 3.5
(n = 17)

                         Myeloperoxidase
Subjects                     (ng/ml)

MDD pre-Venlafaxnie     18.3 [+ or -] 2.0
(n = 13)

Healt hy Subjects       19.8 [+ or -] 2.8
(n = 17)

* NOx was calculated from the summation of nitrate and nitrite
levels which individually were not significantly different
between patients and controls.

None of the comparisons between groups reached statistical
significance.
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