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List of abbreviation:
DTNB- 5,5- dithiobis,2-nitrobenzoic acid
t-BHP- Tertiary butyl hydroperoxide
GSH-Reduced glutathione
GSSG- Oxidised glutathione
Objective: The study was taken up to see the effect of melatonin on the alteration of reduced glutathione level in red blood cells.
Pineal melatonin is involved in many physiological functions, the most important among them being sleep promotion and circadian regulation. This pineal product exhibits characteristic diurnal rhythm of synthesis and secretion, which attains its peak at night followed by a gradual decrease during the daytime. Melatonin detoxifies highly toxic hydroxyl and peroxyl radicals in vitro, scavenges hydrochlorous acid, as well as peroxynitrite. It has also been reported to increase the synthesis of glutathione and of several antioxidant enzymes [1].
Method: The present study was undertaken to understand the modulation of intracellular reduced glutathione (GSH) by melatonin in human red blood cells according to the oscillatory circadian changes in levels of this hormone.We have also studied the dose-dependent effect of melatonin on GSH in erythrocytes obtained from blood at two different times, subjected to oxidative stress by incubating with tert-butyl hydroperoxide (t-BHP) [2]. We used t-BHP as pro-oxidant [3]. Erythrocyte GSH was measured following the method of Beutler [4]. The method was based on the ability of the –SH group to reduce 5,5- dithiobis,2-nitrobenzoic acid (DTNB) and form a yellow coloured anionic product whose OD is measured at 412 nm.
A suspension of packed red blood cells in phosphate-buffered saline (PBS) containing glucose was treated with melatonin taken at different concentrations. A stock solution (10mM) of melatonin was prepared in absolute ethanol; further dilutions (100 uM–10 nM) were done with PBS. The concentration of ethanol was alwaysThe in vitro effect of melatonin was evaluated by incubating erythrocytes with melatonin at different doses (10 uM –1 nM final concentration) of melatonin for 30 minutes at 37°C. After washing the erythrocytes with the buffer, to remove any amount of the compound, and finally, packed erythrocytes were used for the assay of GSH. In parallel control experiments, blood was incubated with ethanol (final concentration not more than 0.01% (v/v)) but without melatonin.Oxidative stress was induced in vitro by using tert-butyl hydroperoxide both in presence and absence of melatonin. Use of TBHP is in accordance with the published reports [5].
Results and Discussion: The experiment demonstrated that erythrocyte GSH level increased in nocturnal samples which highlights the role of endogenous melatonin in the circadian changes in cellular glutathione level. Exogenous melatonin demonstrated a protective effect against t-BHP-induced peroxidative damage in both diurnal and nocturnal samples, the effect being more pronounced in aliquots containing very low concentration of melatonin (10 nM – 1 nM) [6]. Melatonin was found to inhibit GSH oxidation in a dose-dependent manner.
Melatonin has been found to upregulate cellular glutathione level to check lipid peroxidation in brain cells [7]. We may say that the incubation of the red cells with melatonin for an extended period (more than 30 minutes) may not have the same effects on the level of glutathione in these cells [12]. Melatonin may act as pro-oxidant in the cells exposed to the indoleamine for longer time. Also, the half-life period of pineal melatonin is for 30 to 60 minutes, as reviewed by Karasek and Winczyk [11].The recycling of glutathione in the cells depends on an NADPH-dependent glutathione enzyme system which includes glutathione peroxidise, glutathione reductase, and γ-glutamyl-cysteine synthase forming a meshwork of an antioxidative system. The stimulatory effect of melatonin on the regulation of the antioxidant enzymes has been reported [8].Since melatonin has an amphiphilic nature, its antioxidative efficiency crosses the cellular membrane barriers in a non-receptor-mediated mechanism. Another explanation of melatonin’s antioxidative activity may be based on its role in the upregulation of some antioxidant enzymes directly. Blanco et al had reported that glutathione reductase and glutathione peroxidase, the major constituents of the glutathione-redox system being stimulated by melatonin [9]. The plasma GSH/GSSG redox state is controlled by multiple processes, which includes synthesis of GSH from its constitutive amino acids, cyclic oxidation and reduction involving GSH peroxidase and GSSG reductase, transport of GSH into the plasma, and the degradation of GSH and GSSG by γ-glutamyltranspeptidase. The increase in erythrocyte GSH concentration after melatonin administration can be related Blanco et al’s report on the known stimulation of γ-glutamylcysteine synthase,a rate-limiting enzyme in reduced glutathione synthesis, by melatonin [10].
Conclusion: On the basis of our study, we may conclude that melatonin affects the glutathione level in red blood cells in a circadian manner. The rhythmic pattern of glutathione level confirms the relationship between physiological melatonin and erythrocyte GSH level and pharmacological dosage of the drug. The role of melatonin as an antioxidant and its activity in relation to these biomarkers has been studied in the above experiments.
Introduction: The following document is a seminal article concerning the relationship between hyoerhomocysteinemia and cardiovascular and other diseases. It provides a new insight based on the metabolism of S8 and geographic factors affecting the distribution, the differences of plant and animal sources of dietary intake,
and the great impact on methylation reactions. The result is the finding that hyperhomocysteine is a “signal”, just as CRP is a measure of IL-6, IL-1, TNFa -mediated inflammatory response. A deficiency of S8 due to the unavailability of S8, leads to CVD, and is seen in sulfur deficient regions with inadequate soil content and with veganism. Hyperhomocysteinemia is also an indicator of CVD risk in the well fed populations, and that gives us a good reason to ASK WHY?
I have trimmed the content to make the necessary points that would be sufficient for this content. The article can be viewed at OCCJ online.
The Oxidative Stress of Hyperhomocysteinemia Results from Reduced Bioavailability of Sulfur-Containing Reductants
Yves Ingenbleek*
Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur Strasbourg, France
Abstract
A combination of subclinical malnutrition and S8-deficiency
maximizes the defective production of Cys, GSH and H2S reductants,
explaining persistence of unabated oxidative burden.
The clinical entity
increases the risk of developing cardiovascular diseases (CVD) and stroke
Impairment of CbS activity in protein malnutrition, entails
supranormal accumulation of Hcy in body fluids,
stimulation of (5) activity and maintenance of Met homeostasis.
This last beneficial effect is counteracted by
decreased concentration of most components generated downstream to CbS,
explaining the depressed CbS- and CbL-mediated enzymatic production of *H2S along the TS cascade.
The restricted dietary intake of elemental S is a limiting factor for
its non-enzymatic reduction to **H2S which contributes to
downsizing a common body pool (dotted circle).(Fig 1)
Combined protein- and S-deficiencies work in concert
to deplete Cys, GSH and H2S from their body reserves,
impeding these reducing molecules from countering
the oxidative stress imposed by hyperhomocysteinemia.
Hyperhomocysteinemia
Hyperhomocysteinemia (HHcy) is an acquired metabolic anomaly first identified by McCully [1]
The current consensus is that dietary deficiency in any of three water soluble vitamins may operate as causal factor of HHcy.
PLP–deficiency may trigger the upstream accumulation of Hcy in biological fluids [2] whereas
the shortage of vitamins B9 or B12 is held responsible for its downstream sequestration [3,4].
HHcy is regarded as a major causal determinant of CVD
working as an independent and graded risk factor
unrelated to the classical Framingham criteria such as
hypercholesterolemia,
dyslipidemia,
sedentary lifestyle,
diabetes and
smoking.
Hcy may invade the intracellular space of many tissues and locally generate [5]
endothelial dysfunction working as early harbinger of blood vessel injuries and atherosclerosis.
Most investigators contend
that production of harmful reactive oxygen and nitrogen species (ROS, NOS), notably
hydrogen peroxide (H2O2), superoxide anion (O2 .-) and peroxinitrite (ONOO.-),
constitutes a major culprit in the development of HHcy-induced vascular damages [7-10].
Accumulation of ROS associated with increased risk for
cardiovascular diseases [11]
stroke [12],
arterial hypertension [6],
kidney dysfunction [13],
Alzheimer’s disease [14],
cognitive deterioration [15],
inflammatory bowel disease [16] and
bone remodeling [17].
These effects overlook the protective roles played by
extra- and intracellular reductants such as cysteine (Cys) and glutathione (GSH)
in the sequence of events leading from HHcy to tissue damage.
Hydrogen Sulfide (H2S)
After the discovery of nitric oxide (NO) and carbon oxide (CO), hydrogen sulfide (H2S) is the
third gaseous signaling messenger found in mammalian tissues [18].
H2S is a reducing molecule displaying strong scavenging properties
as the gasotransmitter significantly attenuates [19, 20] or
even abolishes [21,22] the oxidative injury imposed by HHcy burden.
The endogenous production of the naturally occurring H2S reductant depends on
Cys bioavailability through
the mediation of TS enzymes [23,24].
H2S may also be produced in human tissues starting from elemental sulfur,
by a non-enzymatic reaction requiring the presence of Cys, GSH, and glucose [25,26].
It would be worth disentangling the respective roles played by
Cys,
GSH
H2S
for the prevention and restoration of HHcy-induced oxidative lesions.
but the plasma concentration of Cys and GSH is severely depressed in
subclinically malnourished HHcy patients [27],
impeding appropriate biogenesis of H2S molecules.
The present paper reviews the biological consequences
resulting from the complex interplay existing between the 3 reducing molecules,
to gain insight into the pathophysiologic mechanisms associated with HHcy states.
CLINICAL BACKGROUND
Numerous surveys have conclusively shown that the water soluble vitamin deficiency concept,
provides only partial causal account of the HHcy metabolic anomaly.
The components of body composition, mainly
the size of lean body mass (LBM),
constitutes a critical determinant of HHcy status [28,29].
Because nitrogen (N) and sulfur (S) concentrations
maintain tightly correlated ratios in tissues, we hypothesize
defective N intake and accretion rate would cause concomitant and
proportionate depletion of total body N (TBN) and total body S (TBS) stores [30].
Our clinical investigation undertaken in Central Africa in apparently healthy but
nevertheless subclinically malnourished vegetarian subjects has
documented that reduced size of LBM could lead to HHcy states [27].
The field study conducted in the Republic of Chad, populated by the Sara ethnic group [27], is a semi-arid region and
the staple food consists mainly of cassava, sweet potatoes, beans, millets and groundnuts.
Participants were invited to fill in a detailed dietary questionnaire whose results were compared with values reported in food composition tables [32-34] [27].
The dietary inquiry indicates that participants
consumed a significantly lower mean SAA intake (10.4 mg.kg-1.d-1)[27]
than the Recommended Dietary Allowances (RDAs) (13 mg.kg-1.d-1)[33,34].
Blood Analytes
The blood lipid profiles of rural subjects were confined within normal ranges
ruling out this class of parameters as causal risk factors for CVD disorders.
The normal levels measured for pyridoxine, folates, and cobalamins
precluded these vitamins from playing any significant role in the rise of Hcy
plasma concentrations [27]. Analysis of plasma SAAs revealed
unmodified methioninemia, significantly
elevated Hcy values (18.6 umol/L)
contrasting with significantly decreased plasma Cys and GSH values [27].
The significant lowering of classical
anthropometric parameters
(body weight, BW;
body mass index, BMI)
together with that of the main plasma and urinary biomarkers of
metabolic (visceral) and
structural (muscular) compartments point to
an estimated 10 % shrinking of LBM [27].
Transthyretin (TTR) and Lean Body Mass (LBM)
We have attached peculiar importance to the measurement of plasma transthyretin (TTR)
this indicator integrates the evolutionary trends outlined by body protein reserves [35],
providing from birth until death an overall and balanced estimate of LBM fluctuations [29].
In the absence of any superimposed inflammatory condition,
LBM and TTR profiles indeed reveal striking similarities [29].
Scientists belonging to the Foundation for Blood Research (Scarborough, Maine, 04074, USA) have recently published a large number of TTR results recorded
in 68,720 healthy US citizens aged 0-100 yr which constitute a comprehensive reference material to follow the shape of LBM fluctuations in relation with sex and age [29].
TTR concentrations plotted against Hcy values reveal a strongly negative correlation (r = –0.71) [29,30], confirming that
the depletion of TBN and TBS stores plays a predominant role in the development of HHcy states.
The body of a reference man weighing 70 kg contains 64 M of N (1,800 g) and 4,400 mM of S (140 g) [36]. Our vegetarian subjects consume diets providing
low fat and high fiber content conferring a large spectrum of well described health benefits notably for the prevention of several chronic disorders such as
cancer and diabetes, together with an effective protection against the risk of hypercholesterolemia-induced CVD [37,38].
Plant-based regimens, however, do not supply appropriate amounts of
nitrogenous substrates of good biological value which are required to adequately fulfill mammalian tissue needs [30].
vegetable items contain suboptimal concentrations of both SAAs [33,34,39] below the customary RDA guidelines.
This dietary handicap may be further deteriorated by
unsuitable food processing [40] and by
the presence in plant products of naturally occurring anti-nutritional factors
such as tannins in cereal grains and
anti-trypsin or anti-chymotrypsin inhibitors in soybeans and kidney beans [41].
LBM loss
LBM shrinking may be the result of either
dysmaturation of body protein tissues as an effect of protracted dietary SAA deprivation
or of cytokine-induced depletion of body stores.
Although causally unrelated and evolving along dissimilar adaptive processes,
both physiopathologic entities lead to comparable LBM downsizing best
identified by declining plasma TTR ( measured alone or within combined formulas )
and subsequently rising Hcy values.
All parameters are downregulated with the sole exception of RM flux rates, indicating that
maintenance of Met homeostasis remains a high metabolic priority in protein-depleted states.
Stressful disorders are characterized by
overstimulation of all
TM
RM
TS flux rates.
The severity and duration of initial impact determine the magnitude of protein tissue breakdown,
rendering an account of N : S urinary losses,
fluctuations of albuminuria and of
insulin resistance striving to contain LBM integrity.
Both physiopathologic entities are compromized in reducing the oxidative burden imposed by HHcy states owing to
defective synthesis and/or
enhanced overconsumption of Cys-GSH-H2S reducing molecules,
a condition still worsened by its co-existence with elemental S-deficiency.
IMPAIRMENT OF THE TRANSSULFURATION PATHWAY
The hypothesis that subclinical protein malnutrition might be involved in the occurrence of HHcy states via inhibition of cystathionine-b-synthase (CbS) activity
first arose in Senegal in 1986 [42] and was later corroborated in Central Africa [43]. The concept was clearly counterintuitive in that it was unexpected that
high Hcy plasma values might result from low intake of its precursor Met molecule.
Despite the low SAA intake of our vegetarian patients [27], plasma Met concentrations disclosed noticeable stability permitting
maintenance of the synthesis and functioning of myriads of Met dependent molecular, structural and metabolic compounds
These clinical investigations have received strong support from recent mouse [45] and rat [46] experiments submitted to Met-restricted regimens.
At the end of the Met-deprivation period, both animal species did manifest meaningful HHcy states (p<0.001) contrasting with
significantly lower BW (p<0.001) reduced by 33 % [45] and 44 % [46] of control, respectively.
the uniqueness of Met behavior stands in accordance with balance studies performed on large mammalian species showing
that the complete withdrawal of Met from otherwise normal diets causes the greatest rate of body loss,
nearly equal to that generated by protein-free regimens [47,48].
This efficient Met homeostatic mechanism is classically ascribed to a PLP-like inhibition of CbS activity exerted through
allosteric binding of S-adenosylmethionine (SAM) to the C-terminal regulatory domain of the enzyme [49,50].
The loss of CbS activity may develop via a (post)translational defect
independently from intrahepatic SAM concentrations [45].
We have postulated the existence of an independent sensor mechanism set in motion by TBS pool shrinkage and
reduced bioavailability of Met – its main building block – working as an inhibitory feedback loop of CbS activity [30].
Such Met-bodystat, likely to be centrally mediated, is to maintain unaltered Met disposal in conditions of
decreased dietary provision implies the fulfillment
of high metabolic priorities of survival value [30,44].
Whereas HHcy may be regarded as the dark side of a beneficial adaptive machinery [43],
impairment of the TS pathway also depresses the production of compounds situated downstream to the CbS blockade level,
notably Cys and GSH, keeping in mind that Cys may undergo reversible GSH conversion (Fig. 1).
The plasma concentration of both Cys and GSH reductants is indeed significantly decreased in our vegetarian subjects
by 33 % and 67 % of control, displaying negative correlations (r = –0.67 and –0.37, respectively) with HHcy values [27].
Reduced dietary intake of the preformed Cys molecule [27] and diminished Cys release from protein breakdown in malnourished states [51]
may contribute to the lowering effect.
The significantly decreased GSH blood levels may similarly be attributed to dietary composition since the tripeptide is mainly found in meat products
but is virtually absent from cereals, roots, milk and dairy items [52] and
because regimens lacking SAAs may lessen the production of blood GSH and its intrahepatic sequestration [53].
BIOGENESIS OF HYDROGEN SULFIDE
The TS degradation pathway schematically proceeds along two main PLP-dependent enzymatic reactions working in succession (Fig. 1).
The first is catalyzed by CbS (EC 4.2.1.22) governing the replacement of the hydroxyl group of serine with Hcy to generate Cysta plus H2O.
Cys may however substitute for serine and the replacement of its sulfhydryl group with Hcy releases Cysta and H2S instead of water [54].
The second is regulated by cystathionine-g-lyase (CgL, EC 4.4.1.1.) hydrolyzing Cysta to release Cys and alpha-ketobutyrate plus ammonia as side-products [55].
Cys may also undergo nonoxidative desulfuration pathways leading to H2S or sulfanesulfur production [56] under the control of CbS or CgL enzymes.
Cys may otherwise undergo oxidative conversion regulated by cysteine-dioxygenase (CDO, EC 1.13.11.20) which
catalyzes the replacement of the SH- group of Cys by SO3 – to yield cysteine-sulfinate [56].
This last compound may be further decarboxylated to hypotaurine that is finally oxidized to Tau (67 %) and SO4 2- oxyanions (33 %) [56]. CbS and CgL, both cytosolic enzymes,
their relative contribution to the generation of H2S may vary according to
animal strains,
tissue specificities and
nutritional or physiopathological circumstances [23,24].
CbS and CgL are expressed in most organs such as liver, kidneys, brain, heart, large vessels, ileum and pancreas [57,58] potentially
subjected to HHcy-induced ROS injury while keeping the capacity to desulfurate Cys and to
locally produce H2S as cytoprotectant signaling agent.
CbS is the principal TS enzyme found in
cerebral glial cells and astrocytes [59].
CgL predominates in the
vascular system [60] whereas
both enzymes are present in the renal proximal tubules [61].
H2S is the third gaseous substrate found in the biosphere [18] after NO and CO. All three gases are characterized by
severe toxicity when inhaled at high concentrations.
In particular, H2S produced by anaerobic fermentation is
capable of causing respiratory death by
inhibition of mitochondrial cytochrome C oxidase [62].
NO, CO and H2S are synthesized from arginine, glycine and Cys, respectively, exerting at low concentrations major biological functions in living organisms.
Most of our knowledge on these atypical signal messengers [63] are derived from animal experiments and tissue cultures. These transmitter molecules may
share some properties in common such as penetration of cellular membranes independently from specific receptors [64].
They are also manifesting dissimilar activities: whereas NO and CO activate guanylyl cyclase to generate biological responses via cGMP-dependent kinases,
H2S induces Ca2+-dependent effects through ATP-sensitive K+ channels [65].
Some of these potentialities may work in concert while others operate antagonistically. For instance,
NO and H2S express vasorelaxant tone on endogenous smooth muscle [66]
but reveal different effects on large artery vessels [67].
These gaseous substances maintain whole body homeostasis through complex interactions and multifaceted crosstalks between signaling pathways.
Elemental S (32.064 as atomic mass) is a primordial constituent of lava flows in areas of volcanic or sedimental origin usually presenting as crown-shaped
stable octamolecules – hence its S8 symbolic denomination – which may conglomerate to form brimstone rocks. The vegetable kingdom is
unable to assimilate S8 and requires as prior step its natural or bacterial oxidation to SO4 2- derivatives before launching
the synthesis of SAA molecules along narrowly regulated metabolic pathways [30,44].
Distinct anabolic processes are identified in mammalian tissues which lack the enzymatic equipment required to organize sulfate oxyanions
but possess the capacity of direct S8 conversion into H2S.
S8 is poorly soluble in tap waters [68] may be taken up and transported to mammalian tissues loosely fastened to serum albumin (SA) [69].
S may also be covalently bound to intracellular S-atoms taking the form of sulfane-sulfur compounds [70] either
firmly attached to cytosolic organelles or in
untied form to mitochondria [57,58,71,72] to undergo
later release in response to specific endogenous requirements [71].
Sulfane-sulfur compounds are somewhat unstable and may decompose in the presence of reducing agents allowing the restitution of S [70,71].
S may either endorse the role of stimulatory factor of several mammalian apoenzyme activities as shown for
succinic dehydrogenase [73] and NADH dehydrogenase [74] or
operate as inhibitory agent of other mammalian apoenzymes such as
adenylate kinase [75] and liver tyrosine aminotransferase [76].
Elemental S resulting from dietary supply or from sulfane-sulfur decay may be subjected to
non-enzymatic reduction in the presence of Cys and GSH [25,26] and/or reducing equivalents obtained from
glucose oxidation [25], hence yielding at physiological pH additional provision of H2S.
The gaseous mediator is a weakly acidic molecule endowed with strong lipophilic affinities. In experimental models, the blockade of the TS cascade
at CbS or CgL levels significantly depresses or even
abolishes the vitally required production of Cys
operating at the crossroad of multiple converting processes (Fig. 1).
Addition of Cys to the incubation milieu
resumes the generation of H2S [19] in a Cys concentration-dependent manner [77].
The compounds situated downstream both cystathionases in the context of SAA deprivation
keep their functional potentialities
but are unable to express their converting Cys – H2S capacities
in the absence of precursor substrate.
Summing up
inhibition of CbS activity contributes to
promote efficient RM processes and
maintenance of Met homeostasis
but entails as side-effects
upstream sequestration of Hcy molecules in biological fluids
while decreasing the bioavailability of Cys and GSH
working as limiting factors for H2S production.
These last adverse effects thus constitute the Achilles heel of a remarkable adaptive machinery.
ROLES PLAYED BY HYDROGEN SULFIDE
The first demonstration that human tissues may reduce S to H2S was incidentally provided in 1924 when a man given colloid sulfur
for the treatment of polyarthritis did rapidly exhale the typical rotten egg malodor [78].
H2S may be produced by the intestinal flora [79] and serves as a metabolic fuel for colonocytes [80].
Prevention of endogenous poisoning by excessive enteral production is insured by the detoxifying activities of mucosal cells [81],
hindering any systemic effect of the gaseous substrate.
The normal H2S concentration measured in mammalian plasmas usually ranges from 10 to 100 μM with a mean average turning around 40-50 μM [19,21,82,83].
This H2S plasma level, appearing as the net product of organs possessing CbS and CgL enzymes and supplemented by the non-enzymatic conversion of S,
flows transiently into the vasculature and freely penetrates into all body cells.
Supposing that the gaseous reductant is evenly distributed in total body water (45 L in a 70 kg reference man) allows an estimate of
bioavailable H2S pool turning around 2 mM which represents, in terms of S participation, largely less than 1 / 1,000 of TBS.
The peculiar adaptive physiology of vegetarian subjects renders very unlikely that their TBS pool might be solicited to release
S-substrates prone to undergo conversion to nascent H2S molecules since
they adapt to declining energy and nutrient intakes
by switching overall body economy toward downregulated steady state activities.
The release from TBS of substantial amounts of S-compounds occurs
only during the onset of hypercatabolic states as documented in trauma patients [31]
and in infectious diseases [84], exacting as preliminary step
cytokine-induced breakdown of tissue proteins, a selective hallmark of stressful disorders [85].
H2S in fulfilling ROS Scavenger Tasks
The limited disposal of H2S endogenously produced might be readily exhausted in fulfilling ROS scavenging tasks at the site of oxidative lesions.
All body organs generating H2S from TS enzymes are
simultaneously producers and consumers of the gaseous substrate whose actual concentration
reflects the balance between synthetic and catabolic rates [86].
Clinical investigations show that H2S concentrations found in cerebral homogenates from Alzheimer’s disease (AD) patients are
very much lower than expected from values measured in healthy brains [87], suggesting that
the gaseous messenger is locally submitted to enhanced consumption rates reflecting disease severity.
The concept is strongly supported by studies pointing to the
negative correlation linking the severity of AD to H2S plasma values [88].
in pediatric [89] and elderly [90] hypertensive patients as well
more severe HHcy-dependent oxidative burden is
associated with more intense H2S uptake rates.
These H2S cleansing properties are mainly exerted by mitochondrial organelles
known to be centrally involved in oxidative disorders [20,91].
Malnourished subjects deprived of Cys and GSH disposal thus incur the risk of H2S-deficiency
rendering them unable to properly overcome HHcy-imposed oxidative lesions.
The rapid exhaustion of H2S stores have detrimental consequences as shown disclosing
the beneficial effects of exogenous administration of commonly used sulfide salt donors (Na2S and NaHS)
generating H2S gas once in solution.
Such supply significantly augments
H2S plasma concentrations allowing to counteract ROS damages.
H2S was primarily recognized as a physiological substrate working as
neuromodulator [92] and soon later as
vasorelaxant factor [65].
H2S is now regarded as endowed with a broader spectrum of biological properties [18],
operating as a general protective mediator
against most degenerative organ injuries,
being capable of neutralizing or
abolishing most ROS harmful effects.
Table 1 collects findings displaying that H2S may promote the synthesis and activity of several
anti-oxidative enzymes (catalases, Cu- and Mn-superoxide dismutases, GSH-peroxidases) and
stimulate the production of anti-inflammatory reactants (interleukin-10) or
conversely downregulate
pro-oxidative enzymes (collagenases, elastases),
pro-inflammatory cytokines (interleukine-1b, tumor-necrosis factor a) and
It has been calculated that 81.5% of H2S undergoes catabolic disintegration in the form of hydrosulfide anion (HS-) or sulfide anion (S2-) [117].
Since S is the main element in the diprotonated H2S molecule (34.08 as molecular mass), it may be considered that
partial or complete repair of HHcy-induced lesions constitutes the therapeutic proof that
S-deficiency is causally involved in the development of ROS damages.
The concept is sustained by the observation that all synthetic drugs (diclofenac, indomethacine, sildenafil) utilized as surrogate providers of H2S [64,118] are
characterized by a large diversity of molecular conformations but
share in common the presence of Satom(s) mimicking, once released,
H2S-like pharmacological properties.
It remains to be clarified whether the beneficial effects of S-fortification to S-deficient subjects are mediated, among other possible mechanisms, via
stimulation [73,74] of anti-oxidative enzymes or inhibition [75,76] of pro-oxidative enzymes.
It is only very recently that the essentiality of S has been recognized, causing Hcy elevation in deficient individuals [119]. It is worth reminding that the
gaseous NO substrate may work in concert or antagonistically [66,83] to fine-tuning the helpful properties exerted by H2S on body tissues.
Preliminary studies suggest for instance that NO operates, in combination with H2S, as a potential modulator of endothelial remodeling since
NO-synthase isoforms contribute to the activation of metalloproteinases involved in the regulation of the collagen/elastin balance defining vascular elastance [83,120].
SUBCLINICAL MALNUTRITION AS WORLDWIDE SCOURGE
A growing body of data collected along the last decades indicates that
large proportions of mankind still suffer varying degrees of protein and energy deficiency that is associated with
increased morbidity and mortality rates.
The determinants of malnutrition are complex and interrelated, comprising
socioeconomic and political conditions,
insufficient dietary intakes,
inadequate caring practices and
superimposed inflammatory burden.
Children living in developing countries are paying a heavy toll to chronic malnutrition [121,122] whereas adult populations are handicapped by
feeble physical and working capacities,
increased vulnerability to infectious complications and
reduced life expectancy [123,124].
Cross-sectional studies collected in the eighties indicate that chronic malnutrition remains a worldwide scourge with
top prevalence recorded in Asia, whereas
sub-Saharan Africa endures medium nutritional distress and
Latin America appears as the least affected [125,126].
Along the last decades, significant progresses have been achieved in some countries such as Vietnam [127] and Bangladesh [128]
owing to appropriate education programs and improved economic development.
Inequalities however persist between middle class population groups mainly located in affluent urban areas and
underprivileged rural communities remaining stagnant on the sidelines of household income growth.
Representative models of these socio-economic disparities in global nutrition and health are illustrated in the two most populated countries in the world, China and India.
Large surveys undertaken in 105 counties of China and recently published have concluded that the rural communities haven’t yet reached the stage of overall welfare [129].
In India, similar investigations have documented that extreme poverty still prevails in the northern mountainous states of the subcontinent [130]. Taken together, southern
Asian countries fail to overcome malnutrition burden [131]. In some African countries, there exists even upward trends suggesting nutritional
deterioration over the years [132] still aggravated by a severe drought. The assessment of malnutrition in children usually rely on anthropometric criteria such as height-for-age, weight for-height, mid upper arm circumference and skinfold thickness allowing to draw the degree of stunting and wasting from these estimates. In adult subjects, BW and BMI are currently selected parameters to which some biochemical measurements are frequently added, notably SA, classical marker of protein nutritional status, and creatininuria (u-Cr), held as indicator of sarcopenia. The former biometric approaches are very useful in that they correctly provide a static picture of the declared stages of malnutrition but fail to recognize the dynamic mechanisms occurring during the preceding months and the adaptive alterations running behind.
Table 1. Reversal of HHcy-Induced Oxidative Damages by Administration of Exogenous H2S
BRAIN EFFECTS
H2S is overproduced in response to neuronal excitation [93], and
increases the sensitivity of N-methyl-D-aspartate (NMDA) reactions to glutamate in hippocampal neurons [23,94].
improves long-term potentiation, a synaptic model of memory [92,93]
stimulates the inhibitory effects of catalase and superoxide dismutase (SOD) in oxidative stress of endothelial cells [95].
regulates Ca 2+ homeostasis in microglial cells [96]and it inhibits TNFa expression in microglial cultures [97].
protects brain cells from neurotoxicity by preventing the rise of ROS in mitochondria [98].
CARDIOVASCULAR EFFECTS
H2S releases vascular smooth muscle,
inhibits platelet aggregation and
reduces the force output of the left ventricule of the heart [18].
maintains vascular smooth muscle tone [66] and
insures protection against arterial hypertension [99].
modifies leucocyte-vascular epithelium interactions in vivo by
modulating leucocyte adhesion and
diapedesis at the site of inflammation [100].
attenuates myocardial ischemia-reperfusion injury by
depressing IL-1b and mitochondrial function [20].
upregulates the expression of depressed anti-oxidative enzymes in heart infarction and
inhibits myocardial injury [21].
alleviates smooth muscle pain by
stimulating K+ ATP channels [101].
prevents apoptosis of human neutrophil cells
by inhibiting p38 MAP kinase and caspase 3 [102].
potentiates angiogenesis and wound healing [103].
RENAL EFFECTS
H2S downregulates the increased activity of metalloproteinases 2 and 9 involved in extracellular matrix degradation (elastases, collagenases) [19].
Prevents apoptotic cell death in renal cortical tissues [19].
Improves the expression of desmin (marker of podocyte injury) and
restores the drop of nephrin (component of normal slit diaphragm) in the cortical tissues
resulting in reduced proteinuria [19].
Induces hypometabolism revealing protective effects on renal function and survival [104].
Normalizes GSH status and production of ROS in renal diseases [19].
Controls renal ischemia-reperfusion injury and dysfunction [105].
Depresses the expression of inflammatory molecules involved in glomerulosclerosis [106].
Prevents lung oxidative stress in hypoxic pulmonary hypertension caused by low GSH content [113].
Promotes SOD and catalase activities and reduces the production of malondialdehyde in oxidative lung injury [114].
Reduces lung inflammation and remodeling in asthmatic animals [115] and in pulmonary hypertension [116]. ..(see OCCJ 2011;4:34-44)
Assessing Protein-Depleted States
SA is an insensitive marker of protein-depleted states compared to TTR [134]
SA is an indicator of population than of individual protein status in subclinical PEM.
u-Cr is likewise a meagerly informative tool as 10 % loss of muscle mass is required before it reaches significantly decreased urinary concentrations [135].
The data imply that the magnitude of subclinical malnutrition is largely
underscored when classical biometric and laboratory investigations are performed.
Moreover, ruling out the protein component involved in HHcy epidemiology and confining solely attention to the B-vitamin triad led to unachieved conclusions.
surveys undertaken in Taiwan [136] and in India [137] established HHcy variance turning around 30 %, indicating that
a sizeable percentage of subjects do not come within the vitamin shortage concept.
only one recent review recommending the use of TTR in vegetarian subjects [138].
The main reason for making the choice of TTR is grounded on the striking similar plasma profile disclosed by this marker with both LBM and Hcy [29].
Under healthy conditions, the 3 parameters –
TTR,
LBM,
Hcy –
indeed show low concentrations at birth,
linear increase without sexual difference in preadolescent children,
gender dimorphism in teenagers with higher values recorded in adolescent male subjects
thereafter maintenance of distinct plateau levels during adulthood [29,139,140].
Under morbid circumstances, the plasma concentrations of
Hcy manifest gradual elevation
negatively correlated with LBM downsizing and
TTR decline.
In vegetarian subjects and subclinically malnourished patients,
rising Hcy and
diminished TTR plasma concentrations look as mirror image of each other,
revealing divergent distortion from normal and
allowing early detection of preclinical steps
at the very same time both SA and u-Cr markers still remain silent.
Any disease process characterized by quantitative or qualitative dietary protein restriction or intestinal malabsorption
may cause LBM shrinking,
downregulation of TTR concentrations and
subsequent HHcy upsurge.
These conditions are documented in frank kwashiorkor [141], subclinical protein restriction [27,43] and anorexia nervosa [142].
In patients submitted to weight-reducing programs,
LBM was found the sole independent variable
negatively correlated with rising Hcy values [143].
Morbid obesity may be alleviated by medical treatment [143] or surgical gastroplasty [144,145],
conditions frequently associated with secondary malabsorptive syndromes and malnutrition [146],
How does this account play out in the typical patient with excessive body fat, lipoprotein disoreder, and perhaps diabetes and disordered sleep – an account of acquired HHcy?
Have the studies been done? Would you expect to see a clear benefit from reduced HHcy_emia based on a 30 min daily walk, and
eating of well fat trimmed meats, fruits and vegetables, and fish, flax seed, or krill oil?
In westernized countries, subclinical protein-depleted states are illustrated in immigrants originating from
developing regions but keeping alive their traditional feeding practices [147] or
by communities having adopted, for socio-cultural reasons, strict vegan dietary lifestyles [148].
THE ADDITIONAL BURDEN OF S-DEFICIENCY
After N, K and P, elemental S is recognized as the fourth most important macronutrient required for plant development. The essentiality of S in the vegetable kingdom
arose from observations made many decades ago by pedologists and agronomists [149,150] revealing that the withdrawal of sulfate salts from nutrient sources produces
rapid growth retardation,
depressed chlorophyllous synthesis,
yellowing of leaves and
reduction in fertility and crop yields.
A large number of field studies, mainly initiated for economical reasons, has provided continuing gain in fundamental and applied knowledge and led to the overall consensus
that SO4 2- -deficiency is a major wordwide problem [151,152].
Field investigations have shown that the concentration of SO4 2- oxyanions in soils and drinking waters
may reveal considerable variations ranging from less than 2 mg/L to more than 1 g/L,
meaning a ratio exceeding 1 / 500 under extreme circumstances [30].
The main causal factors responsible for unequal distribution of SO4 2- oxyanions are geographical distance from eruptive sites and
intensity of soil weathering in rainy countries.
SO4 2- -dependent nutritional deficiencies entail detrimental effects to most African and Latin American crops [151]
reaching nevertheless top incidence in southeastern Asia [151,153].
and the Indo-Gangetic plain extending from Pakistan to Bangladesh and covering the North of India and Nepal [154].
Intensive agricultural production,
lack of animal manure and
use of fertilizers providing N, K and P substrates
but devoid of sulfate salts may further aggravate that imbalanced situation.
As global population increases steadily and the production of staple plants predicted to escalate considerably,
SO4 2- deficient disorders are expected to become more pregnant along the coming years [155] with significant harmful impact for mankind.
Nevertheless, effective preventive efforts are developed in some countries aiming at fortification of soils mainly
by ammonium sulfate or calcium sulfate (gypsum) salts,
resulting in meaningful improvements in crop yield,
SAAs content and biological value and
opening more optimistic perspectives for livestock and human consumption [152,155-158].
Contrasting with the tremendously high amounts of data accumulated over decades by pedologists and agronomists on sulfate requirements and metabolism,
the available knowledge on elemental sulfur in human nutrition looks like a black hole. Despite the fact that S8 follows H, C, O, N, Ca and P as the seven most
abundant element in mammalian tissues, it appears as a forgotten item. Not the slightest attention is dedicated to S8 in the authoritative “Present Knowledge
in Nutrition” series of monographs even though they go over most oligo- and trace-elements in minute detail.
The geographical distribution of S8 throughout the earth’s crust is not well-known
as extreme paucity of measurements in soils and tap waters prevents reaching a comprehensive overview.
Nevertheless, and because S8 is the obligatory precursor substrate for the oxidative production of sulfate salts,
a decremental dispersion pattern paralleling those of SO4 2- oxyanions is likely to occur with
highest values recorded in the vicinity of volcano sources
and lowest values found in remote and washed-out areas.
Obviously, a great deal of research on elemental S remains to be completed by clinical biochemists before rejoining the status of plant agronomy.
Taken together, these data imply that subclinically malnourished subjects living in areas recognized as
SO4 2- -deficient for the vegetable kingdom also
incur increased risks to become S8-depleted.
This clinical entity most probably prevails in all regions, notably Northern India, where protein malnutrition [130] and sulfur-deficiency [154] coexist.
Combination of both nutritional deprivations explains why the bulk of local dwellers, including young subjects [159,160], may develop HHcy states and CVD disorders
characterized by strong refractoriness to vitamin-B supplementation [160] or
high incidence of stroke [161] unrelated to the classical Framingham criteria.
The current consensus is that “the problem of CVD in South Asia is different in etiology and magnitude from other parts of the world” [162]. These disquieting findings are
confirmed in several Asian countries [163] and have prompted local cardiologists to exhort their governments to focus more attention on CVD epidemiology [164].
CONCLUDING REMARKS
vegetarian subjects are not protected against the risk of CVD and stroke which should no longer be regarded as solely affecting populations living in westernized societies
whose morbidity and mortality risks are stratified by classical Framingham criteria.
Likewise hypercholesterolemia, hyperhomocysteinemia should be incriminated as
emblematic risk factor for a panoply of CVD and related disorders.
Whereas the causality of cholesterol and lipid fractions largely prevails in affluent societies consuming high amounts of animal-based items,
that of homocysteine predominates in population groups whose dietary lifestyle gives more importance to plant products.
MAIN PHYSICO-CHEMICAL AND METABOLIC CHARACTERISTICS* OF 3 CARRIER-PROTEINS INVOLVED IN THE STRESS RESPONSE
CBG
TTR
RBP
Molecular mass (Da.)
42,650
54,980
21,200
Conformation
monomeric
tetrameric
monomeric
Amino acid sequence
383
4 x 127
182
Carbohydrate load
18 % glycosylated
unglycosylated
unglycosylated
Hormonal binding sites
one for cortisol
two for TH
one for retinol
Association constant (M-1)
3 x 107
7 x 107 (T4)
1.9 x 107
Normal plasma concentration
30 mg/L.
300 mg/L.
50 mg/L.
Biological half-life
5 days
2 days
14 hrs
Bound ligand concentration
120 µg/L.
80 µg TT4/L.
500 µg/L.
Free ligand concentration
5 µg/L.
20 ng FT4/L.
1 µg/L.
Ratio free : bound ligands
4 %
0.034 %
0.14 %
Distribution volume of free moieties
18 L.
12 L.
18 L.
STIMULATORY AND INHIBITORY EFFECTS MODULATED
BY GLUCOCORTICOIDS
TARGET SYSTEMS
INDUCED EFFECTS
REF.
Thymidine kinase
_
transcription of induced DNA into RNA
112
Alkaline phosphodiesterase I
_
cleavage of phosphodiester bonds
113
Tyrosine transaminase
_
transfer of tyrosine amino group
114
Tryptophane oxygenase
_
formylkynurenine and Trp catabolites
115
Alkaline phosphatase
_
release of P from phosphoric esters
116
Phosphoenolpyruvate carboxykinase (liver)
_
glycolysis from pyruvate and ATP production
117
Mannolsyltransferases
_
dolichol-linked glycosylation of APRs
118
Haptoglobin
_
APR combining with hemoglobin
119
α1-Anti (chymo) trypsin (α1 AT, α1 ACT)
_
serpin molecules allowing N-sparing effects
120
α1-Acid glycoprotein (AGP)
_
glycosylated APR with antibody-like actions
121
Serum amyloid protein (SAA)
_
defense systems against oxidative burst
122
γ-Fibrinogen
_
clotting processes and tissue repair
123
C-Reactive Protein (CRP)
_
complement processes and opsonization
124
Corticosteroid-binding globulin (CBG)
_
CBG levels, favoring free hypercortisolemia
100
Phosphoenolpyruvate carboxykinase (adipocytes)
_
ATP turnover and glycolysis
113
THE DUAL MORBID ENTITIES CAUSING LBM DOWNSIZING AND SUBSEQUENT Hcy UPSURGE
TTR coupled with CRP or other inflammatory indices (31,177,178,284,285)
Insulin resistance status
Normal or low (286)
Increased in proportion of tissue breakdown (177,178,181-183)
status of Cys-GSH-H2S reducing molecules
Decreased enzymatic and non-enzymatic production (39,161,162,287)
Increased production cancelled out by tissue overconsumption (78,171)
Urinary SO42- and S-compounds
Decreased kidney output (76,78,79)
Variable depending on exogenous SAA supply and
extent of tissue breakdown (78,163,168,173)
Transmethylation pathway
Depressed (48,93)
Overstimulated (169)
Remethylation pathway
Stimulated (76,83,153)
Overstimulated (169)
Transsulfuration pathway
Inhibited (49,76,83)
Overstimulated (170,173)
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