Urinary Neurotransmitter Analysis

Utility in depression management

Given the pervasiveness of anxiety and depression in today’s society and the limitations of the current standard of care for many patients, there is a need for reliable neurobiological assessments that objectively identify underlying biochemical abnormalities as plausible therapeutic targets. Measurement of neurotransmitters is an excellent candidate to serve as an objective framework of diagnosis, prognosis, and response to treatment in psychiatry; neurotransmitters can be measured in various biological fluids including blood serum, platelets, cerebral spinal fluid (CSF), saliva, and urine. Currently biomarkers for depression and anxiety are not utilized in standard medical practice, and tools upon which to base treatment decisions are restricted to the evaluation of subjective clinical symptoms. Without information yielded from objective testing, selection of the best treatment for each person with a mood disorder remains challenging, and is often determined through a time-consuming process of trial and error. Urinary neurotransmitter testing is rapidly becoming the preferred bodily fluid for objective neurobiological assessment since a) urine is the primary route of neurotransmitter elimination, and b) it is a non-invasive and cost effective testing method.

Introduction

The nervous system is the central control mechanism for nearly every bodily process. Within the nervous system neurotransmitters serve as chemical messengers for trillions of connections between the brain and target tissues, and they are essential for maintaining body homeostasis (Marc 2011). In response to prolonged stress however, the biological system begins to lose its ability to maintain chemical homeostasis; as a result, disease processes may be initiated (Lurie 1991). Imbalances in neurotransmission, due to excessive or deficient levels of certain neurotransmitters, are associated with depression, anxiety, insomnia, behavioral disorders, memory disorders, and a spectrum of other neurological disorders (Marc 2011). Since neurotransmitters are thought to play an integral role in mediating these disease processes, the measurement of specific imbalances may be of use in guiding targeted interventions that are aimed at correcting the individual excess or deficiency in question.

The importance of effectively assessing and treating depression cannot be overstated. The World Health Organization’s Global Burden of Disease Study places unipolar affective disorder among the 10 leading medical causes of disability in the world, second only to ischaemic heart disease (Murray 1997).

The current standard of care for depression is to embark upon a course of treatment, most likely a pharmaceutical intervention, based on its efficacy in randomized clinical trials, its specificity or “match” for the patient’s symptomatology, or the patient’s previous response(s) to treatment (Fava 2005). The patient is then monitored for a good outcome, allowing for course correction if there is no improvement. Both steps fundamentally rely on clinical findings and subjective data. Clearly, objective data would further enhance the medical assessment, if available (Holsboer 2008).

Clinical Utility of Biomarkers

As in any other disease state, a primary goal in the research of mental health disorders is the identification of specific biomarkers that could enhance the ability to develop targeted patient treatments in order to enhance patient management and to improve treatment success (Holsboer 2008).

Biomarkers are commonplace in most branches of medicine. For example, if a patient has cancer, heart failure or even a bladder infection, a medical practitioner will likely intervene with the appropriate laboratory testing before committing to any course of treatment. Tumor markers are used in oncology (Srinivas 2001), troponin is a biomarker in cardiology (Sato 2012), C-reactive protein (CRP) and Rheumatoid Factor (RF) are biomarkers in rheumatology (Dasgupta 2012), and prostatespecific antigen (PSA) is used in the detection and management of prostate cancer (Chang 2012). In the context of these conditions, biomarkers are important for diagnosis, early detection, patient monitoring, prognosis, prediction of safety and appropriate dosing.

With so many biomarkers available for other medical conditions, why is there no formalized biomarker in psychiatry? In the past, biomarkers for brain health assessments have been viewed as irrelevant to symptomatology because measures had included peripheral biological fluids (blood, urine, and saliva) as opposed to central nervous system markers, such as cerebrospinal fluid and brain tissue, which are far too costly and invasive to use in clinical practice (Roy 1988).

A common misconception about peripheral biochemistry is that it cannot serve as a biological indicator of central nervous system (CNS) activity due to the presence of the blood-brain-barrier, which limits the transport of neurotransmitters from the peripheral nervous system (PNS) to the CNS and vice versa. The CNS and PNS must not be viewed as separate entities, however. In reality, central neurotransmitters are carried to the periphery via specific blood brain barrier (BBB) transporters followed by renal filtration with subsequent excretion of neurotransmitters in the urine (Lechin 2006). A study by Lechin demonstrated that specific CNS nuclei can manipulate peripheral neurochemistry, and peripheral neurochemistry can affect central pathways (e.g. vagal afferents from periphery to CNS) (2006). The CNS and PNS also communicate via direct neuronal projections (Moreira 2011). Finally, animal studies have suggested a relationship between urinary and CNS neurotransmitters (Lynn-Bullock 2004).

The kidneys also have neurotransmitter transport mechanisms. Circulating neurotransmitters are filtered from the blood by nephrons and subsequently excreted in the urine through glomerular filtration and by active transport via organic cation transporters (OCT’s) (Moleman 1992). Two mechanisms of neurotransmitter transport in the kidneys have been well established: (1) monoamine neurotransmitters are excreted by ultrafiltration from arterial blood into the glomeruli, secreted in the proximal tubules, subsequently distributed through the collecting duct to the urinary bladder and excreted in the urine (Graefe 1997); (2) in the luminal and basolateral membranes of the renal proximal tubules, OCT2 is responsible for the reabsorption and secretion of endogenous compounds, including monoamine neurotransmitters (Koepsell 1998).

According to Cook, certain criteria must be met for a biomarker to be considered for psychiatric management (2008). First, the biomarker must be timely, clinically useful, and cost-effective. Second, the technology needed to assess the biomarker must be well tolerated by the target patient population. Third, methods that can be easily integrated into the practitioner’s current practice patterns are more likely to be accepted than those that require a major change in the delivery of care. Urine testing of neurotransmitters satisfies all three of these criteria.

Value of Urinary Neurotransmitter Testing

Many people, patients and practitioners alike, are familiar with the role of serotonin in depression (Genung 2012), and most antidepressant medications target the extracellular availability of serotonin and/or norepinephrine (Rozas 2009). Practitioners are less likely to investigate or address other important neurotransmitters/ neuromodulators involved in brain processes such as GABA, glycine, asparagine, glutamate, taurine, dopamine, epinephrine, norepinephrine, cortisol, phenylalanine, tyrosine, melatonin and histamine (Duncan 2012, Fu 2012, Hovelso 2012, Tamatam, 2012) (See Figures 1,2, and 3). The considerable variability in neurotransmitters/ neuromodulators may provide a possible explanation for why so many people using anti-depressant medications or supplements sometimes find little-to-no relief from their treatment (Eby 2010, Fournier 2010, Rozas 2009); in fact, sixty percent of cases of clinical depression are considered to be treatment-resistant (Eby 2010). The assessment of urinary neurotransmitter testing offers the possibility of improving treatment outcomes in these cases by allowing correction of the underlying imbalance. In addition, pharmacological based antidepressant intervention can take weeks or months to tweak and perfect, if indeed this can be achieved at all (Hamer 2011, Oyebode 2012, Waterreus 2012), and the use of biomarkers to select the most appropriate intervention and monitor treatment response may possibly expedite this process. Indeed, a current trend in psychiatry includes the selection of anti-depressants that address several targets at once, due to the modest to negligible efficacy of highly selective drugs (Razas 2009); and this trend speaks to the difficulty of correctly selecting highly selective agents based on symptomatology alone.

Studies show depression to be a long term, relapsing condition associated with significant tendency towards chronicity. Three quarters of patients with depression experience more than one episode of depression and the risk of recurrence is higher if the first episode occurs at a younger age and if there is a family history of depression (Hollon 2006). The risk of recurrence increases with each new episode and as the number of depressive episodes increases, the influence of life stress on recurrence wanes (Kendler 2000). Given these findings, the need for effective treatment in the first episode of depression is obvious (Palazidou 2012).

Targeted testing can help to determine exactly which neurotransmitter levels are out of balance, and in turn, which therapies are best suited for an individualized treatment plan (Holsboer 2008). There exists a welldeveloped body of literature correlating urinary levels of various neurotransmitter metabolites to mood disorders, the focus of the following review being depression. With objective evidence of the specific neurotransmitter imbalances of a particular patient, a clinician is much better equipped to individualize a treatment plan targeting the imbalances unique to each presenting case.

Neurotransmitter Assessment in Depression

Thirty-six depressed patients seen at the National Institute of Mental Health were assessed for urinary and cerebrospinal fluid (CSF) measures of dopamine and dopamine metabolites. Episodes of suicidality among the patients were documented five years later. Baseline CSF and urinary assessment of dopamine (and metabolites) was then compared. The team showed urinary measures of dopamine and metabolites to be a much more powerful predictor of suicide attempt than CSF measures (Roy 1994).

Seventy-five female patients with purging bulimia nervosa (BN) and 30 healthy controls were compared for psychopathology (impulsivity, borderline personality traits, depressive symptoms and self-defeating personality traits) and neurobiological parameters reflecting hypothalamicpituitary- adrenal axis activity (morning serum cortisol before and after dexamethasone) and monoamine activity (24-hour urinary excretion of norepinephrine, serotonin, dopamine, and their main metabolites: 3-methoxy-4- hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid). BN patients had lower 24-hour excretion of serotonin and dopamine than controls, as well as lower ability to suppress cortisol (Val-Leal 2011).

Roy and colleagues examined subsets of unipolar depressed patients and compared these subjects to non-depressed controls. They found that depressed patients had high urinary norepinephrine and its metabolite normetanephrine, but lower urinary output of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) compared to controls. They concluded that high urinary output of norepinephrine and normetanephrine reflected abnormal sympathetic nervous system activity and suggested that urinary neurotransmitter testing may be helpful in determining subsets of depression (Roy 1986). Other studies also confirmed these findings, which reported elevations in urinary norepinephrine output in depressed and anxious individuals (Grossman 1999, Koslow 1983, Roy 1988). Otte and colleagues likewise demonstrated elevated urinary excretion of norepinephrine in depressed patients, yet found no difference in levels of urinary dopamine and metabolites relative to healthy controls (Otte 2005).

Hughes (2004) observed that higher levels of depressive symptoms, as assessed by the Beck Depression Inventory (BDI), were associated with increased norepinephrine and cortisol excretion in the urine. In this study, ninety one women aged 47-55 years were evaluated and 24-hour urine collections were assayed for epinephrine, norepinephrine and cortisol. Depressed women (n=17, BDI scores >/=10) exhibited a 25% higher rate of urinary norepinephrine excretion than women with BDI scores <10 (n=74), P=.007. Higher levels of state anxiety were also related to greater NE excretion; likewise, cortisol excretion was related to both depression and anxiety. Interestingly, depression and anxiety symptoms were unrelated to urinary epinephrine excretion.

Of tremendous significance to a discussion of the clinical utility of assessment of urinary neurotransmitter levels is the response of these levels to various types of treatment; do abnormal levels of urinary neurotransmitters correct themselves following various treatments for depression, natural or prescription? Do treatment responders differ in the impact of treatment to urinary levels of neurotransmitters from treatment non responders?

Nichkova et al (2012) evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. Results demonstrated significantly lower serotonin levels in depressed patients (87.53±4.89 μg/g Cr; n=60) than in non-depressed subjects (153.38±7.99 μg/g Cr). This study also demonstrated that urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by the dietary supplement 5-HTP and/or selective serotonin re-uptake inhibitor. Similarly, a double-blind, placebocontrolled, block-randomized, two-way crossover study designed to assess vascular safety administered 20 mg/d of the SSRI paroxetine. Urinary serotonin excretion increased significantly (89%) when compared to placebo 24-hours after oral administration (Kotzailias 2004).

Thirty-six un-medicated depressed patients were assigned to one of four groups; placebo, fluoxetine, duloxetine, or St John’s Wort. At baseline and following eight weeks of treatment, patients were assessed for urinary levels of melatonin. As anticipated, all three antidepressant treatments significantly increased urinary melatonin levels, while placebo had no effect (Carvalho 2009).

Thirty- five subjects aged 55-75 were assigned to receive tryptophan- fortified cereal (60mg per 30g cereal) and followed for a three- week period. Tryptophan increased sleep efficiency, actual sleep time, immobile time, and decreased total nocturnal activity, sleep fragmentation index, and sleep latency. Urinary 6-sulfatoxymelatonin (melatonin metabolite), and 5-hydroxyindoleacetic acid (serotonin metabolite) levels increased respectively. Anxiety and depression symptoms were also improved (Bravo 2012). A novel tart-cherry product has similarly been shown to reduce urinary levels of cortisol and increase urinary levels of 5-hydroxyindoleacetic acid, suggesting a role for the cherry in stress control/ mood regulation (Garrido 2012).

Of note is an investigation by Linnoila and colleagues (1984). After a series of studies evaluating urinary catecholamine levels in depressed patients, and their response to treatment, the team followed a small subset of depressed patients (21) regarding urinary serotonin and 5-hydroxyindoleacetic acid (5-HIAA). Three of the patients suffered from rapid cycling bipolar disorder (RCBD), a rare and difficult to treat variant of bipolar. The patients with RCBD demonstrated quite dramatically elevated levels of urinary serotonin and 5-HIAA, that were corrected upon administration of lithium. This small sample of patients suggests low levels of urinary serotonin correlate to depression, while elevated levels of urinary serotonin may correlate to mania and mood cycling.

Conclusions

Overall, urinary neurotransmitter analysis can be a useful tool in clinical assessment and treatment for depression as well as other mood based disorders. The urinalysis is cost-effective, timely, non-invasive, and can easily be incorporated into any clinical practice. The ability to identify abnormality across specific areas of the catecholamine pathway, the serotonin pathway, and the GABA pathway allows the integrated healthcare provider to tailor a treatment plan to the specific areas identified. There exists a formulary of 30-40 medicines most integrated healthcare providers call upon for management of depression and other affective disorders. Trials evaluating the impact of such medicines on urinary neurotransmitter assessment among depressed patients would be of tremendous value.

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Male Fertility

Role of oxidant stress and antioxidant therapy

Infertility is a significant problem affecting 15% of couples worldwide. Forty percent (40%) of infertility cases are attributable to male factors. Chronic and excessive levels of oxidative stress are increasingly recognized to contribute to chronic disease development, and a growing body of evidence points to a significant role of oxidative stress in the pathogenesis of infertility in men. This article outlines the pathogenic role of oxidative stress in male infertility and provides an overview of the evidence supporting the therapeutic use of selected dietary antioxidants (L-carnitine and L-acetylcarnitine, selenium, N-acetyl-cysteine, folate, zinc and vitamin C) in the integrative care of men with this condition.

Male Factor Infertility

Male factor infertility accounts for 40% of all cases of infertility (Akmal 2006, Tremellen 2008). The most common form of male infertility is oligoasthenoteratospermia (OAT) (Cavallini 2006, Ross 2010). There is no accepted medical treatment for OAT, while mechanical techniques such as IVF-ICSI (in vitro fertilization/intra-cytoplasmic sperm injection) are used to circumvent it (Cavallini 2006, Ross 2010, Safarinejad 2009). However, these procedures fail to address the potentially reversible, underlying causes of OAT (Ross 2010) and are not without signifi cant physical, emotional and fi nancial consequences for couples.

While thirty percent (30%) of OAT cases are diagnosed as idiopathic (Cavallini 2006, Ross 2010), several factors including subtle endocrine abnormalities, environmental toxins, chronic infl ammation or infection, obesity, sperm autoimmunity, genetic or constitutional factors, and excessive levels of oxidative stress induced by reactive oxygen species (ROS), have been implicated in the pathogenesis of OAT (Cavallini 2006, Comhaire 2000, Ross 2010, Safarinejad 2009). Many of the aforementioned factors can independently increase oxidative stress, while additional oxidation resulting from psychological stress, chronic diseases such as diabetes, smoking, alcohol, certain drugs, low fruit and vegetable intake may further contribute to OAT (Tremellen 2008). In fact, up to 30% to 80% of cases of male infertility have been linked to oxidative stress (Tremellen 2008 citing McLachlan 2001).

Oxidative Stress and Male Infertility

Seminal plasma contains the highest concentration of antioxidants of any human fl uid (Cavallini 2006). In fact, sperm and seminal plasma are natural repositories for enzymatic and non-enzymatic antioxidants, including superoxide dismutase, glutathione peroxidase, catalase, vitamins C and E, glutathione and carnitine (Ross 2010), which are capable of protecting sperm from ROS-induced damage (Cavallini 2006). The principal generators of ROS in seminal fl uid are sperm cytoplasm and leukocytes (Ross 2010).

Spermatozoa produce ROS as part of normal metabolic processes, and are particularly vulnerable to oxidative stress due to their high cell membrane polyunsaturated fatty acid content, limited capacity for DNA repair, and the removal of most of their antioxidant-containing cytoplasm during sperm maturation (Ebisch 2007, Menezo 2007).

Despite the normally high seminal antioxidant content, excessive generation and/or decreased scavenging of ROS may lead to accumulation of ROS with consequent oxidative damage to sperm DNA, cell membranes and proteins, leading to apoptosis (and thereby oligospermia), atypical morphology (teratospermia) and impaired motility (asthenospermia) (Ebisch 2007, Ross 2010). Oxidative cell membrane damage also impairs spermoocyte fusogenic capacity (Tremellen 2008). Oxidative DNA damage, including that induced through certain assisted reproductive technologies, can compromise the paternal genomic contribution to the embryo and could result in decreased pregnancy rates, increased miscarriage risk and unknown genetic consequences in offspring (Comhaire 2003, Menezo 2007, Tremellen 2008).

ANTIOXIDANT THERAPY IN INFERTILITY: SELECTED AGENTS

Carnitine

In a review of prospective clinical trials, Cavallini et al. (2006) found L-carnitine (LC) and L-acetyl-carnitine (LAC) to be signifi cantly more effective than placebo at improving fertility in men with idiopathic OAT. Zhou et al. (2007) systematically reviewed nine randomized controlled trials involving a total of 862 infertile men aged 18-65 years, the majority diagnosed with OAT, who had taken either LC, LAC, both carnitines together, or carnitines with other agents (NSAIDs, vitamin E or vitamin C) at dosages of 2-3 g daily in single or divided doses for 24 weeks to six months (Zhou 2007). Metaanalysis of seven eligible trials identifi ed a markedly signifi cant increase in pregnancy rates with carnitines, reporting 55 pregnancies in the treatment group and nine in the control group (odds ratio=4.10, 95% CI: (2.08, 8.08)), (p<0.0001). Compared to placebo or other agents, carnitines also signifi cantly increased total sperm motility (p=0.01) and forward motility (p=0.04) and decreased atypical morphology (p<0.00001), but did not signifi cantly infl uence sperm concentration.

LC is concentrated by active transport from the systemic circulation into the epididymal lumen (Ahmed 2011) where it accumulates as both LC and LAC (Zhou 2007). During sperm maturation in the epididymis, spermatozoa themselves accumulate LC (Ahmed 2011). LC acts as an antioxidant in seminal fluid, protecting sperm membranes and DNA from various mechanisms of oxidative stress (Zhou 2007). LC is essential to mitochondrial beta-oxidation of long chain free fatty acids (Zhou 2007, Moradi 2010), removes excess intracellular toxic acetyl-CoA (Zhou 2007) and is believed to serve as a post-ejaculatory energy source for sperm (Ahmed 2011).

Ahmed et al. (2011) found seminal LC levels to be significantly lower in infertile men than fertile men, while higher seminal carnitine levels have been positively associated with improved sperm count, motility and normal morphology. Comparing the effects on sperm parameters of LC (2 g/day) to those of the anti estrogen, clomiphene citrate (25 mg/ day), Moradi et al. (2010) found LC to be superior to clomiphene at increasing semen volume and equally effective at increasing sperm count and motility after three months of therapy; LC induced no significant effect on sperm morphology (Moradi 2010).

Selenium and N-acetyl-cysteine

Essential to normal spermatogenesis, sperm motility and function, selenium (Se) is considered to improve semen quality and male fertility through the action of glutathione peroxidases (GPXs), Secontaining antioxidant enzymes in seminal fluid that decrease propagation of ROS by reducing H2O2 and lipid peroxides to alcohols and water (Cavallini 2006, Mistry 2012, Safarinejad 2009). The intracellular antioxidant glutathione (GSH) is a cofactor for GPX and reacts directly with ROS and cytotoxic aldehydes to protect sperm from the effects of lipid peroxidation (Atig 2012). N-acetyl-cysteine (NAC) is a derivative of L-cysteine and a precursor to GSH that also possesses direct free radical scavenging activity (Safarinejad 2009). An open prospective study involving 27 infertile men showed that 600 mg NAC plus fatty acid supplementation (1 g docosahexaenoic acid, 0.25 g gamma-linolenic acid and 0.10 g arachidonic acid) daily significantly reduced seminal ROS and increased sperm count in oligospermic men (Comhaire 2000). A recent prospective controlled study involving 250 infertile men identified significant positive correlations between seminal selenium and selenoenzyme concentrations and sperm motility (Atig 2012).

Investigating the effects of Se, NAC, or the two antioxidants in combination, Safarinejad and Safarinejad (2009) conducted a doubleblind, placebo controlled, randomized trial in a cohort of 468 infertile men with idiopathic OAT. Participants received either 200 μg Se (n=116), 600 mg NAC (n=118), 200 μg Se plus 600 mg NAC (n=116), or placebo (n=118) daily for 26 weeks. None of the participants had Se deficiency or low blood plasma NAC at the outset of the study. Compared to placebo, all three treatment groups showed significant improvements from baseline in sperm concentration, mean total sperm count (30% increase), motility (19% increase), and normal morphology (26% increase). Se and/or NAC also produced significant decreases in serum LH and FSH and increases in serum testosterone and inhibin B, a marker of Sertoli cell function (Cavallini 2006). Seminal plasma Se and NAC were significantly and positively correlated to sperm count, concentration, motility and percent normal morphology, with the sum effects of seminal Se and NAC showing stronger correlations with increased sperm concentration (r=0.67, p=0.01), motility (r=0.64, p=0.01) and percent normal morphology (r=0.66, p=0.01). No adverse events were reported with Se or NAC intake (Safarinejad 2009).

Folate and Zinc

Zinc (Zn) is concentrated in the prostate gland, seminal plasma and spermatozoa (Atig 2012, Ebisch 2007). It is a cofactor for more than 80 metalloenzymes involved in DNA transcription, translation and repair, underscoring its importance in sperm cell development (Atig 2012, Colagar 2009, Ebisch 2007). Zn has important antioxidant and anti-apoptotic properties and supports the function of steroid hormone receptors (Atig 2012, Ebisch 2007). Depleted seminal plasma Zn levels have been correlated with idiopathic subfertility and lower sperm counts in several studies (Ebisch 2007). Significant and positive correlations have been reported between seminal Zn levels and sperm count (r=0.32, p<0.01) and normal morphology (r=0.42, p<0.001), with significantly higher seminal Zn levels noted among fertile men than infertile men (Colagar 2009). A recent prospective trial of 250 infertile men found seminal Zn concentrations to be significantly and positively correlated with sperm motility and concentration (Atig 2012). Based on National Health and Nutrition Examination Survey (NHANES, 1999- 2000) (U.S.) data, 79% of men are estimated to consume less than the recommended dietary allowance (RDA) of Zn in their diets (Young 2008).

Zn is also a cofactor for the folate-metabolizing enzymes dihydrofolate reductase and gamma-glutamyl hydrolase, as well as for methionine synthetase and betaine-homocysteine methyltransferase, which indirectly support the folate cycle through methionine metabolism (Ebisch 2007). Both folate and zinc possess anti-apoptotic properties, although excessively high zinc levels can induce apoptosis and necrosis (Ebisch 2007). Zinc deficiency decreases the intestinal absorption and metabolism of folate (Ebisch 2007, Forges 2007).

Folate is essential to healthy reproduction due to its role in nucleic acid synthesis and thereby the proliferation of rapidly dividing cells including sperm cell precursors (Ebisch 2007, Forges 2007). Folic acid, the synthetic form of folate, scavenges free radicals and prevents lipid peroxidation in sperm cell membranes and protects DNA from oxidative damage (Ebisch 2007). Various folates are concentrated in seminal plasma and the concentration of non-methyltetrahydrofolate has been significantly positively correlated with sperm count and concentration (Forges 2007). Folate may have a beneficial effect on spermatogenesis through improving cohesion of seminiferous epithelial cells and thereby preventing premature release of immature sperm into the tubules (Forges 2007). Folate deficiencyinduced hypomethylation of DNA and phospholipids may impair testicular exocrine and endocrine functions (Forges 2007). Folate deficiency also precipitates hyperhomocysteinemia, a pro-inflammatory state that is linked with poor sperm quality and male infertility (Forges 2007). Used in combination with other antioxidants, folic acid has improved sperm concentration and pregnancy rates in studies involving assisted contraception (Ross 2010 citing Tremellen 2007 and Wong 2002). NHANES data estimate that 64% of men consume less than the RDA of dietary folate (Young 2008).

Dividing 94 subfertile (sperm concentration 5 x 106/ ml to 20 x 106/ml) and 99 fertile men into four treatment groups, folic acid (5 mg/day) and zinc sulfate (66 mg/ day) together, folic acid or zinc plus a placebo, or two placebos, were administered to participants for six months (Forges 2007 citing Wong 2002). Folic acid and zinc together produced a substantial 74% increase in sperm concentration and count in subfertile men compared to fertile controls, although none of the men had zinc or folate deficiency prior to treatment (Forges 2007 and Comhaire 2000 citing Wong 2002). No significant changes were observed with separate administration of folate or zinc (Forges 2007 citing Wong 2002).

Vitamin C

Higher dietary and supplemental intake of vitamin C has been correlated with higher sperm count, concentration and progressive motility (Eskenazi 2005).

In a small open study involving 13 infertile men 25 to 35 years of age, 1000 mg vitamin C taken twice daily for two months produced significant increases in mean sperm count from 14.3 x 106 to 32.8 x 106 sperm/mL (p<0.001), normal morphology from 43% to 66.7% (p<0.001), and sperm motility from 31.2% to 60.1% (p<0.001) (Akmal 2006). A group of 36 infertile men with asthenoteratozoospermia and leukocytospermia showed significant improvements in progressive motility and morphology with decreased necrosis and leukocytosis after treatment with 60 mg vitamin C, 10 mg vitamin E, 100 mg fermented papaya, 194 mg lactoferrin and 40 mg beta-glucan daily for three months (Piomboni 2008). Ménézo (2007) administered a combination of 400 mg vitamin C with 400 mg vitamin E, 18 mg beta-carotene, 500 μmol Zn and 1 μmol Se daily for 90 days to 58 men with sperm DNA abnormalities and a history of at least two IVF or ICSI failures. Treatment significantly decreased sperm DNA fragmentation, but paradoxically increased sperm head decondensation, a phenomemon that when exceeding a critical threshold of 28% is associated with greater risk of chromosome condensation abnormalities and poorer prognosis for pregnancy with IVF or ICSI (Ménézo 2007).

Conclusion

The majority of literature reviewed consists of relatively small but positive studies showing benefits to sperm quality and in some cases pregnancy rates with treatment of infertile men with various single or combination antioxidants, with few adverse effects. Recognizing the role of oxidative stress in the pathogenesis of numerous serious chronic diseases, reducing the oxidative burden in affected men is likely to benefit sperm quality and fertility as well as their overall health in the longer term.

References

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. Akmal M, Qadri JQ, Al-Waili NS, Thangal S, Haq A, Saloom KY. Improvement in human semen quality after oral supplementation of vitamin C. J Med Food. 2006 Fall;9(3):440-2.

Atig F, Raffa M, Habib BA, Kerkeni A, Saad A, Ajina M. Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men. BMC Urol. 2012 Mar 19;12:6.

Cavallini G. Male idiopathic oligoasthenoteratozoospermia. Asian J Androl 2006; 8(2):143-157.

Colagar AH, Marzony ET, Chaichi MJ. Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men. Nutr Res 2009;29:82-88.

Comhaire FH, Christophe AB, Zalata AA, Dhooge WS, Mahmoud AM, Depuydt CE. The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men. Prostaglandins Leukot Essent Fatty Acids. 2000 Sep;63(3):159-65.

Comhaire FH, Mahmoud A. The role of food supplements in the treatment of the infertile man. RBM Online 2003;7(4):385-391.

Ebisch IM, Thomas CM, Peters WH, Braat DD, Steegers-Theunissen RP. The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility. Hum Reprod Update. 2007 Mar- Apr;13(2):163-74.

Eskenazi B, Kidd SA, Marks AR, Sloter E, Block G, Wyrobek AJ. Antioxidant intake is associated with semen quality in healthy men. Hum Reprod. 2005 Apr;20(4):1006-12.

Forges T, Monnier-Barbarino P, Alberto JM, Guéant-Rodriguez RM, Daval JL, Guéant JL. Impact of folate and homocysteine metabolism on human reproductive health. Hum Reprod Update. 2007 May-Jun;13(3):225-38.

Ménézo Y. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. RBM Online 2007:14(4):418-421.

Mistry HD, Broughton Pipkin F, Redman CW, Poston L. Selenium in reproductive health. Am J Obstet Gynecol. 2012 Jan;206(1):21-30.

Moradi M, Moradi A, Alemi M, Ahmadnia H, Abdi H, Ahmadi A, Bazargan-Hejazi S. Safety and efficacy of clomiphene citrate and L-carnitine in idiopathic male infertility: a comparative study. Urol J. 2010 Summer;7(3):188-93.

Piomboni P, Gambera L, Serafini F, Campanella G, Morgante G, De Leo V. Sperm quality improvement after natural anti-oxidant treatment of asthenoteratospermic men with leukocytospermia. Asian J Androl. 2008 Mar;10(2):201-6

Ross C, Morriss A, Khairy M, Khalaf Y, Braude P, Coomarasamy A, El-Toukhy T. A systematic review of the effect of oral antioxidants on male infertility. Reprod Biomed Online. 2010 Jun;20(6):711-23.

Safarinejad MR, Safarinejad S. Efficacy of selenium and/or N-acetylcysteine for improving semen parameters in infertile men: a doubleblind, placebo controlled, randomized study. J Urol 2009;181:741-751.

Tremellen K. Oxidative stress and male infertility – a clinical perspective. Hum Reprod Update 2008;14(3):243-258.

Tremellen K, Miari G, Froiland D, Thompson J. A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment. Aust N Z J Obstet Gynaecol. 2007 Jun;47(3):216-21. (Cited in Ross 2010).

Wong WY, Merkus HM, Thomas CM, Menkveld R, Zielhuis GA, Steegers-Theunissen RP. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril. 2002 Mar;77(3):491-8.

Young SS, Eskenazi B, Marchetti FM, Block G, Wyrobek AJ. The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men. Hum Reprod. 2008 May;23(5):1014-22.

Zhou X, Liu F, Zhai S. Effect of L-carnitine and/or L-acetylcarnitine in nutrition treatment for male infertility: a systematic review. Asia Pac J Clin Nutr 2007;16(Suppl 1):383-390.

Novel Cholesterol Subtypes

Markers of Cardiovascular Risk

Implementation of aggressive treatment targets for LDL-C has been credited with materially reducing risk of all cause death, sudden coronary death, stroke, as well as reduced risk of non fatal major coronary events, principally among populations of patients in settings of secondary coronary prevention. None-theless, questions remain, notably the phenomena whereby many individuals suffering cardiovascular events do so while having very low levels of circulating LDL-C, the apparent failure of statin therapy to correct coronary artery calcification, and the controversy surrounding the role of statin therapy in settings of primary coronary prevention. A selection of novel lipid markers may be able to equip practitioners to better predict a patients cardiovascular risk; these markers may correct erroneous risk predictions based on LDL among a subset of patients for whom assessment of LDL-C has proven to be a poor predictor of risk, they may identify individuals at risk sooner than LDL assessment alone would detect risk, and they may show reduced risk among a subset of individuals with marginally elevated LDL-C, preventing over treatment. The following review will examine evidence surrounding the role of Lp-PLA2, Apo B, LDL-P, Lp(a), and LDL/ HDL subfractions in predicting risk of cardiovascular events.

Introduction

Guidelines for cholesterol testing to examine cardiovascular (CV) risk have primarily relied on measurements of lowdensity lipoprotein cholesterol (LDL-C) and secondarily on non-high-density lipoprotein cholesterol (HDL-C) (NCEP 2002). Patients are stratified by CV risk and then LDL-C treatment goals are set based on their classification. This LDL-C strategy has been successful in reducing the incidence of CV morbidity and mortality. Further analyses of clinical trial data have supported the idea that non-HDL-C is a better treatment target than LDL-C (Robinson 2009). Non-HDL-C includes both LDL-C and VLDL-C and it is derived from calculating total cholesterol minus HDL-C. However, measurements and treatment to non-HDL-C goals have not been utilized, largely as a result of knowledge gaps on behalf of physicians (Virani 2011). Even though statins and LDL-C reduction reduce CV events, there remains a residual risk for events in both primary and secondary prevention populations. Primary prevention refers to avoiding the occurrence of disease. Secondary prevention refers to when disease is already present but before it causes significant morbidity. Residual risk is present in those who are on statin therapy and it is most evident in patients with metabolic syndrome and diabetes (Drexel 2010 & Rosenson 2010). As a result, the use of lipid biomarkers is a high-interest topic that has a large potential for clinical utility and to possibly improve patient outcomes. This is especially important as the availability of generic statins has decreased the cost of treatment and has improved the cost-effectiveness of using lipid markers (Davidson 2011). This article will review the recent assessment of an expert panel of lipid specialists in their analysis of the following lipid markers: lipoproteinassociated phospholipase A2 (Lp-PLA2), apolipoprotein (Apo) B, LDL particle concentration (LDL-P), lipoprotein(a) [Lp(a)], and LDL and HDL subfractions (Davidson 2011). The evaluation of novel markers can provide valuable insight into a patient’s CV risk, especially where there is suspicion that a patient may be at higher risk than suggested by LDL-C alone.

Current Guidelines

Many epidemiological studies have confirmed that the following risk factors account for the majority of coronary artery disease (CAD) cases: age, male gender, cigarette smoking, diabetes mellitus, cholesterol (as assessed by total cholesterol and LDL-C), HDL-C, elevated blood pressure, a family history of premature CAD before the age of 60, inflammatory biomarkers such as hs-CRP, and overweight or obesity (Smith 2006). Other variables that increase risk are poor nutrition, caloric excess, physical inactivity, and psychological stress. Current cholesterol treatment targets are obtained from the data of clinical trials. Most studies measure the serum or plasma of LDL-C. The Cholesterol Treatment Trialists meta-analysis of 14 statin trials showed a dose-dependent relative reduction in cardiovascular disease (CVD) with LDL-C lowering (Baigent 2005). The CTT Collaborators found that every 1.0 mmol/L reduction in LDL-C is associated with a corresponding 20% to 25% reduction in CVD mortality and nonfatal myocardial infarction. Secondary targets include a total cholesterol to HDL-C ratio of less than 4.0, a non-HDL-C level of less than 3.5 mmol/L, an Apo B/Apo AI ratio of less than 0.80, a triglyceride level of less than 1.7 mmol/L and an hs-CRP level of less than 2.0 mg/L (Genest 2009). The current guidelines advocate optimizing these secondary targets in high-risk patients only after achieving LDL-C targets.

Lp-PLA2

Lp-PLA2 circulates bound to LDL particles, HDL particles, Lp(a), and triglyceride-rich remnant lipoproteins (Anderson 2008). It is produced by numerous cell types, including mast cells, macrophages, and liver cells (Braun 2010). Lp- PLA2 activity is up-regulated in atherosclerotic lesions and in rupture-prone fibrous caps (Koenig 2006). Lp- PLA2 is an enzyme responsible for the hydrolysis of oxidized phospholipids in LDL particles within the arterial intima and produces two highly inflammatory mediators (Anderson 2008). These mediators result in a cascade of events linked to atherosclerotic plaque formation, including the expression of cytokines and the production of foam cells (Braun 2010). Foam cells aggregate to form a fatty streak covered by a fibrous cap, while cytokines and proteases destroy the collagen within the fibrous cap, making it prone to rupture (Davidson 2011).

Lp-PLA2 levels have been identified as a significant predictor of CV events and stroke (Braun 2010). In primary and secondary prevention trials, patients with Lp-PLA2 in the upper tertile or upper quartile had an approximately 2-fold increase in risk for CV events (Anderson 2008). In addition, unlike LDL-C, epidemiological studies show that an elevation in Lp-PLA2 confers a 2-fold increase in both first and recurrent strokes (Gorelick 2008). A meta-analysis of 80,000 patients showed that Lp-PLA2 elevations caused an 8% to 16% relative risk increases in the following: coronary heart disease (CHD), ischemic stroke, and vascular mortality (Lp-LPA2 Collaboration 2010). Interestingly, omega-3 fatty acids and weight loss have been shown to reduce Lp-LPA2 (Tzotzas 2008).

Apo B

All triglyceride-rich lipoprotein particles secreted by the intestine or the liver have one molecule of Apo B (Elovson 1988). The Apo B encircles the particle, provides external structural integrity, and stays with the lipoprotein particle for its lifetime. Thus, plasma Apo B concentration is a direct indication of the total number of circulating Apo B-containing lipoprotein particles. Atherosclerosis is initiated and advanced by the trapping of Apo B-containing lipoprotein particles within the subintimal space of the arterial wall (Davidson 2011). LDL Apo B particles have a greater importance in driving atherosclerosis because they are in greater concentration than VLDL Apo B particles and are smaller so they can enter the arterial wall more readily. The more Apo B particles enter the arterial wall, the greater the increase in the number trapped in the subendothelial space, and this leads to the development and progression of atherosclerosis (Smith 1982).

LDL-C is not the best indicator of the risk attributable to LDL because risk correlates more closely with the number of circulating atherogenic particles than with the quantity of cholesterol carried by those particles (Ingelsson 2007). The amount of cholesterol per LDL particle varies significantly. To better understand the problem this creates, consider a patient whose LDL particles contain less cholesterol than normal. This patient will have LDL-C concentrations that will underestimate the number of LDL particles. In such a patient, the Apo B concentration will more accurately reflect the number of LDL particles and the LDLrelated CV risk. Next, consider the reverse situation: a patient whose LDL particles contain more cholesterol than normal. In this patient, the LDL-C concentration will overestimate the number of LDL particles. In this case too, the Apo B concentration will provide a more accurate representation of LDL particles (Sniderman 2007). Cholesterol-poor LDL particles are the dominant form of LDL in a substantial portion of patients who are in all major clinical risk groups for vascular disease (Davidson 2011). In these groups, Apo B better reflects CV risk and this has been supported by a meta-analysis (Sniderman 2011). As a bonus, fasting is not required for Apo B measurement.

LDL-P

LDL particles can move into the arterial wall and the greater the circulating concentration of LDL particles, the greater the rate of passive diffusion into the arterial wall and the greater the vesicular ferrying through endothelial cells (Nielsen 1996). LDL particles bind to arterial wall proteoglycans, become oxidized, and are taken up by macrophages to form foam cells (Tabas 2007). When serum LDL-P is high, there are a greater amount of LDL particles in circulation and a greater amount of particles may enter the arterial wall. Conversely, when LDL-P is low, there are fewer LDL particles and a decrease in the initiation and promotion of atherosclerosis.

LDL-P represents the number of LDL particles and is therefore an alternative way to quantify LDL, as oppose to relying solely only on LDL-C. For many patients, their LDL-C and LDL-P are highly correlated. However, because of variability of the cholesterol content and size of LDL particles, they are sometimes unrelated (Otvos 2002). In the general population, approximately 50% of subjects have discordance between LDL-C and LDL-P (Otvos 2011). In those with elevated triglycerides or low HDL-C, the discordance rates are higher and the same is true of those with type 2 diabetes mellitus or metabolic syndrome (Cromwell 2007, Otvos 2011). CV risk is more strongly associated with LDL-P than with LDL-C when these two measures are discordant (Otvos 2011). LDL-P and Apo B are both measures of particle number. As such, the decision to use one or the other is determined by availability, cost, and physician preference.

Lp(a)

Lp(a) is a modified LDL molecule with the addition of a protein made in the liver, known as the lipoprotein antigen (Koschinsky 2004). The lipoprotein antigen is highly polymorphic in size, which causes highly variable molecular weights and variable plasma concentrations in the population (Kronenberg 1999). Lp(a) is taken up in the arterial wall by scavenger receptors on macrophages called beta-integrin Mac-1 (Sotiriou 2006). Interestingly, homocysteine increases the Mac-1 interaction with Lp(a) antigen by up to threefold. Lp(a) also binds to fibrin and may enhance the clotting triggered by endothelial damage or plaque rupture (Koschinsky 2004). The number of molecules of Lp(a) appear to be the strongest determinant of related CV risk (Davidson 2011).

When examining study data, Lp(a) has positive predictive power that is additive to other measures of lipoprotein risk factors (Davidson 2011). Lp(a) is specifically associated with increased risk for CHD in a continuous nonthreshold manner. The association between Lp(a) and CHD risk is independent of LDL-C, non-HDL-C, and the presence of other CV risk factors (Nordestgaard 2010). This makes Lp(a) a useful tool for assessing clinical risk, especially when there is a strong family history of vascular events, since elevated plasma concentrations are controlled by features of the Lp(a) gene (Kamstrup 2009).

LDL and HDL subfractions

Every lipoprotein particle in the LDL fraction is atherogenic, regardless of size. LDL particles become trapped in the arterial wall, cause foam cell formation, and cause the expansion of the inflammatory response (Ross 1999). HDL particles are involved in reverse cholesterol transport and also possess antiatherogenic properties, including antioxidant and anti-inflammatory properties (Rosenson 2011). Therefore, there is physiological rationale for the links between both LDL and HDL subfractions and adverse CV outcomes.

LDL particles vary in size, density, and cholesterol content. Small LDL particles are often present in patients with features of metabolic syndrome, including those with CHD, diabetes, low HDL and high triglycerides, and in those with insulin resistance (Sacks 2003). However, the statistical associations between small, dense LDL and CHD outcomes are diminished or disappear altogether when adjusted for LDL-P. Currently, there are no patient subgroups that have been identified in which LDL subfractionation has supporting evidence showing benefit (Sacks 2003). HDL particles are also variable in terms of size, charge, density, and cholesterol content. Many antiatherosclerotic functions of HDL are not fully understood (Reilly 2007). Population studies support the notion that HDL-C has protective effects for CV risk and HDL subfractions also correlate with this risk (Williams 2011). Similar to LDL subfractions, there have been no patient subgroups in which there is evidence supporting the routine use of HDL subfractionation.

Conclusion

Focusing treatment goals on LDL-C has been successful in reducing the incidence of CV morbidity and mortality. However, LDL-C does not adequately assess risk in all population subgroups due to the variability of multiple associated factors. As a result, the use of lipid biomarkers has large potential for clinical applications and could improve patient outcomes. This article reviewed the recent assessment of the expert panel of lipid specialists in their analysis of multiple lipid markers. Lp-PLA2 elevations were shown to cause 8% to 16% relative risk increases in CHD, ischemic stroke, and vascular mortality. Apo B was shown to better reflect CV risk in a substantial portion of patients, especially in those patients with other major clinical risks for vascular disease. LDL-P was shown to be more strongly associated with CV risk than LDL-C, especially in patients with elevated triglycerides or low HDL-C, in those with type 2 diabetes mellitus, and in those with metabolic syndrome. Apo B and LDL-P are both measures of particle number and the merits of choosing one over the other or using both are unclear. Lp(a) was shown to be specifically associated with increased risk for CHD in a continuous nonthreshold manner, independently of many other risk assessment parameters. Finally, LDL and HDL subfractions were shown to be weaker predictors of CV risk, despite physiologic rationale that appeared promising for both. Overall, many of these lipid markers appear to be useful in certain patient subgroups. However, some controversies exist on their value and it is difficult to recommend when they should be used, or for which patients they would be most beneficial. Beyond that, it may also be difficult to determine how these markers may impact specific treatment goals or specific treatment decisions.

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