Case report: intermittent fasting and probiotic yogurt consumption are associated with reduction of serum alpha-N-acetylgalactosaminidase and increased urinary excretion of lipophilic toxicants

Jerry Blythe1 and Stefania Pacini2

1Retired Medical Doctor, Indianapolis, IN, USA 2Silver Spring Sagl, Arzo-Mendrisio, Switzerland

Corresponding author

Running title: Key words:

Stefania Pacini, MD, PhD. Silver Spring Sagl. Via Raimondo Rossi 24, Arzo-Mendrisio 6864. Switzerland.
E-mail: info@bravo-europe.com

Intermittent fasting and lipophilic toxicants

intermittent fasting; probiotic yogurt; nagalase; detoxification; thyroid

Abbreviations: N-acetyl(2-hydroxypropyl)cysteine (NAHP); 3-Phenoxybenzoic acid (3PBA), N- acetyl phenyl cysteine (NAP), Phenylglycoxylic acid (PGO), Monoethylphthalate (MEP); 2- Hydroxyisobutyric Acid (2HIB); Thyroid Stimulating Hormone (TSH); Dr. Jerry Blythe (Dr. JB); chronic lymphocytic leukemia (CLL); Institutional Review Board (IRB); Gc protein-derived Macrophage Activating Factor (GcMAF).

Abstract

In this study, we describe the changes associated with three months of intermittent fasting and probiotic yogurt consumption in a 72-year-old marathon runner with chronic lymphocytic leukemia for a number of years. Serum alpha-N-acetylgalactosaminidase (nagalase), a marker of inflammation and cancer cell proliferation, was significantly decreased at the end of a three- month observation. These results are consistent with immune modulating properties of certain probiotics based on the fermentation of milk and colostrum. Urinary excretion of non-metal toxicants that accumulate in adipose tissue such as Perchlorate, N-acetyl(2- hydroxypropyl)cysteine (NAHP), 2,4-Dichlorophenoxyacetic acid, 3-Phenoxybenzoic acid (3PBA), N-acetyl phenyl cysteine (NAP), Phenylglycoxylic acid (PGO), Monoethylphthalate (MEP) and 2- Hydroxyisobutyric Acid (2HIB) was significantly increased. These results are consistent with the weight loss (5 Kg) associated with intermittent fasting and with the known features of probiotics as detoxification tools. Consistent with certain toxicants acting as endocrine disruptors, we observed an increased elimination of toxicants and a 33% decrease of serum Thyroid Stimulating Hormone (TSH), suggesting a trend toward normalization of thyroid function. These results support the hypothesis that a combination of intermittent fasting with the consumption of specific probiotic yogurts may lead to immune modulation, detoxification and other improvements.

Introduction

A recent review of the metabolic effects of intermittent fasting published in the July 2017 Annual Review of Nutrition suggests that nutritional regimens may “offer promising nonpharmacological approaches to improving health at the population level, with multiple public health benefits”.(1) This statement is consistent with the conclusions of a study published in 2013 describing how “Intermittent fasting is reported to improve the lipid profile; to decrease inflammatory responses, reflected by changes in serum adipokine levels; and to change the expression of genes related to inflammatory response and other factors”.(2) The interest in intermittent fasting as a nutritional approach to weight loss and a number of health issues, is demonstrated by the number of peer-reviewed papers on this topic. A PubMed query for “intermittent fasting” performed in July 2017, yielded, yielded 755 results with a quasi- exponential increase in the number of papers in the past 5 years.

Intermittent fasting appears particularly efficient in achieving sustained weight loss and enhancing body composition by reducing fat mass.(3) And, a reduction of fat mass is often further associated with a release of lipophilic toxicants that may have accumulated in adipocytes(3); such a release may pose a health danger if it is not accompanied by appropriate detoxification procedures. Among the different detoxification approaches, interventions involving modulation of the microbiota appear particularly promising as they target a system, in this instance, the microbiota that is involved in all aspects of human physiology. Such interventions have received a great deal of attention in the past few years.(4,5)

Thus, it has been known for more than 10 years that probiotic microbial strains have properties that enable them to bind metals and toxins from food and water; interestingly, binding of toxicants by probiotics appears to be instantaneous, thus leading to the proposal of exploiting these features for decontamination in food and intestinal models.(6)

Based on these premises, Dr. JB, a retired Medical Doctor, embarked on a three-month experience of intermittent fasting and probiotic yogurt consumption with the goal of weight loss and detoxification. In this study, we describe the changes associated with such a nutritional approach.

Subject and methods

Dr. JB was born through Cesarean section in 1945, was fed with formulas, and suffered several early childhood infections, including pneumonia at the age of 5, treated with chloromycetin (one of the first commercial uses of chloramphenicol was chloromycetin in 1949). The drug is seldom used today because of potential toxicity and bone marrow depression. At the age of 6, he developed a passion for running that continues to this day. He kept logs of his mileage and calculates that he has run approximately 112,000 miles (180,246 Km) including a number of marathons; his remarkable achievements have recently been described in the journal, The Socionomist.(7)

He has been healthy and energetic throughout his adult life without illness other than Typhoid Fever acquired outside the United States in 1970 and treated with chloramphenicol. His bone marrow suffered a residual leukopenia and lymphocytosis which appear to have not affected his daily activities. In 2012, he was diagnosed with chronic lymphocytic leukemia (CLL) at an Indianapolis Cancer Center, and found to have a 13q14.3 deletion. Retrospective study of blood analyses, however, suggests that the condition had been evolving over a number of years and diagnosable as CLL since 2008 when for the first time he developed a leukocytosis along with his lymphocytosis. After careful consideration, Dr. JB opted for complementary/alternative approaches for his condition with particular focus on healthy nutrition and exercise, avoiding refined sugars, alcohol, carbonated drinks (sodas), cookies, pies, candy or cakes; he maintained his consistent training program that he is still implementing, running on average 6 miles per day at a comfortable pace.

In March 2017, JB embarked on an intermittent fasting program patterned on the weight loss program described by He et al.(3) Shortly thereafter, he introduced daily consumption of 100 ml of probiotic yogurt aimed at supporting the immune system and helping detoxification, choosing a product whose properties have been previously described.(8,9)

Blood and urine analyses were performed immediately before and after the three-month experience. Analysis of serum alpha-N-acetylgalactosaminidase (nagalase) was performed at the European Laboratory of Nutrients, (Bunnik, The Netherlands); the results are expressed as Units = nmol/min/mg. Blood analyses were performed at Lab Corp, Laboratory Corporation of America. Analyses of toxicants in early morning urine samples were performed at The Great

Plains Laboratory Inc. (Lenexa, KS, USA) and are expressed as μg/g creatinine. Copies of the original records are conserved at the office of Dr. JB.

Since this is a single case report that does not produce generalizable knowledge, nor is it an investigation of an FDA regulated product, Institutional Review Board (IRB) review is not required for this activity.(10) Likewise, since this case report described the experience of one of the Authors, Dr. JB, MD, the concept of “informed consent” is implicit in the authorship of the study.

Results

After three months of intermittent fasting and probiotic yogurt consumption, Dr. JB’s body weight decreased from 70.7 to 66.2 Kg (6.4%). Serum nagalase values significantly decreased from 1.92 to 1.06 U (44.8%), a value that is very close to the upper normal value set by the laboratory (0.95). Serum TSH values also significantly decreased from 7.83 to 5.2 μIU/ml (33.6%). Leucocyte count decreased from 58 to 54.9 (cells x 103/μL) (5.3%) and the percentage of lymphocytes decreased from 94 to 90% (4.4%). The number of platelets favorably increased from 93 to 125 (cells x 103/μL) (34.4%).

Concomitant with these changes, urinary excretion of a number of lipophilic toxicants significantly increased (Table 1).

______________________________________________________________________________

Table 1
Non-metal toxicants in urine______________________________________________________________________________

Compound Before After

______________________________________________________________________________

Monoethylphthalate (MEP) 2-Hydroxyisobutyric acid (2HIB) Phenylglycoxylic acid (PGO) Perchlorate

1,925 3,353 4,364 7,349 36 184 3.5 13 N.D. 0.11 70 135 0.18 1.7 N.D. 0.27

N-acetyl phenyl cysteine (NAP)
N-acetyl(2-hydroxypropyl)cysteine (NAHP)
2,4-Dichlorophenoxyacetic acid
3-Phenoxybenzoic acid (3PBA) ______________________________________________________________________________ Values are expressed as μg/g creatinine; the first term (before) refers to the value observed before intermittent fasting and probiotic yogurt consumption; the second term (after) refers to the value observed after the three-month experience.______________________________________________________________________________

It is worth noticing that the toxicants whose excretion was increased concomitantly with implementation of intermittent fasting and probiotic yogurt consumption, are among the most common environmental pollutants. It may be argued that elevated initial levels of phthalates were associated with Dr. JB’s consistent running and consumption of water from years of soft, plastic water bottles possibly containing phthalates. Interpretation of initial elevated level of 2HIB is more complex. Thus, 2HIB is formed endogenously as a product of branched-chain amino acid degradation in patients with ketoacidosis.(11) However, 2HIB is also a metabolite of gasoline additives(12), and elevated levels of 2HIB can indicate exposure to environmental pollution, exposure that could be associated with activities such as running in urban environments. Since Dr. JB had no clinical or laboratory signs of ketoacidosis, we interpret the elevated levels of 2HIB as consequence of environmental exposure.

Discussion

The changes observed during a three-month experience of intermittent fasting and probiotic yogurt consumption seem to support the hypothesis that such a nutritional approach provides health benefits ranging from weight loss to detoxification and immune system support.

Decrease of nagalase can be interpreted as the result of modulatory activities of the probiotic yogurt on the immune system. It is known that fermentation of milk leads to formation of immune-modulatory and anti-oxidant molecules, and as such, may play a role in decreasing the inflammatory status responsible for the elevated nagalase levels.(13) In addition, the probiotic yogurt used in this study contains bovine colostrum, a supplement known to have intrinsic immune-modulating properties(14) and to be rich in vitamin D-binding protein that is the precursor of Gc protein-derived Macrophage Activating Factor (GcMAF).(15) It has been proposed that nagalase is a marker of cancer cell proliferation (16) as well as of systemic inflammation.(17) Therefore, the observed decrease of serum nagalase activity, together with the decrease of lymphocytes accompanied by an increase of platelets, may reflect a positive impact of the nutritional approach described herein on the underlying pathologic condition of Dr. JB.

Such a positive impact may also be consistent with the observed decrease of serum TSH values. We interpret this change as a consequence of the increased excretion of toxicants known to act as endocrine disruptors with particular reference to thyroid function. For example, phthalates bind to thyroid hormone receptors altering the signaling of thyroid hormones (18), and perchlorate interferes with iodine uptake with resulting increased production of TSH as compensatory mechanism.(19) Thus, it can be hypothesized that increased elimination of phthalates, perchlorate and other endocrine disruptors may be responsible for the trend toward normalization of thyroid function.

The observed increased urinary excretion of lipophilic toxicants is consistent with recent report describing how such a nutritional plan followed by Dr. JB is associated with an increase of serum concentrations of lipophilic pollutants such as polychlorinated biphenyls, possibly mobilized from adipose tissue.(3) We attribute the increased urinary excretion of lipophilic

chemicals to the effects of the cleansing procedures associated with the plan described in He et al.(3) as well as to the known detoxifying effect of probiotics. For example, Lactobacillus acidophilus, a component of the probiotic yogurt used in this study, is endowed with antioxidant effects, decreasing malondialdehyde and increasing the levels of the antioxidants, glutathione reductase, superoxide dismutase, and glutathione peroxidase.(20) Other probiotic strains provide significant protection against metal toxicity by decreasing the level of toxicants in the liver and kidney, and by preventing alterations in the levels of glutathione peroxidase and superoxide dismutase.(21) Another mechanism of action responsible for the detoxifying effects attributable to probiotic yogurt consumption lays in the observation that lactic acid bacterial strains remove toxins from liquid media by physical binding(22) , and protect against toxins such as polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, amino acid pyrolysates and mycotoxins.(23) In addition, it has been recently demonstrated that probiotics such as those represented in the probiotic yogurt used in this study may improve kidney function thus contributing to the overall detoxifying effect. (24)

The authors wish to emphasize that the data in this case report are from one individual, and not part of a larger study. Like Aronson who encourages reports of single cases in medicine(25) , the authors believe the history and observations may benefit those addressing the impact of environment on health.

Conclusions

The experience of Dr. JB suggests that intermittent fasting associated with probiotic yogurt consumption may lead to body weight reduction, increased excretion of toxicants and modulation of the immune system with overall positive effects on health.

Acknowledgements

The Authors wish to thank Mr. Peter Greenlaw, author of books on healthy nutrition, who introduced Dr. Blythe to the nutritional plan described in this study, and connected him with Dr. Pacini after completion of the three-month experience.

References

1. Patterson, R.E., Sears, D.D. Metabolic effects of intermittent fasting.
Annu. Rev. Nutr., Jul 17. doi: 10.1146/annurev-nutr-071816-064634. [Epub ahead of print].

2. Azevedo, F.R., Ikeoka, D. and Caramelli, B. Effects of intermittent fasting on metabolism in men. Rev. Assoc. Med. Bras. (1992). 2013; 59:167-173.
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3. He, F., Zuo, L., Ward, E., and Arciero, P.J. Serum polychlorinated biphenyls increase and oxidative stress decreases with a protein-pacing caloric restriction diet in obese men and women. Int. J. Environ. Res. Public Health; 2017 14: 59. doi:10.3390/ijerph14010059.

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8. Pacini, S., Punzi, T, Morucci, G. and Ruggiero, M. Macrophages of the mucosa-associated lymphoid tissue (malt) as key elements of the immune response to vitamin d binding protein- macrophage activating factor. It. J. Anat. Embryol. 2011; 116:136.

9. Schwalb, M., Taubmann, M., Hines, S., Reinwald, H., and Ruggiero, M.
Clinical observation of a novel, complementary, immunotherapeutic approach based on ketogenic diet, chondroitin sulfate, vitamin D3, oleic acid and a fermented milk and colostrum product. Am. J. Immunol. 2016; 12:91-98. doi:10.3844/ajisp.2016.91.98.

10. John Hopkins Medicine. 102.3 Organization Policy on Single Case Reports and Case Series. http://www.hopkinsmedicine.org/institutional_review_board/guidelines_policies/organization _policies/102_3.html
Accessed August 16, 2017.

11. Konkoľová, J., Chandoga, J., Kováčik, J., et al. Severe child form of primary hyperoxaluria type 2 – a case report revealing consequence of GRHPR deficiency on metabolism. BMC Med. Genet. 2017; 18: 59. doi:10.1186/s12881-017-0421-8.

12. Benson, J.M., Tibbetts, B.M. and Barr, E.B. The uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether inhaled alone and in combination with gasoline vapor. J. Toxicol. Environ. Health A. 2003; 66:1029-1052.

13. Ebner, J., Aşçı Arslan, A., Fedorova, M., Hoffmann, R., Küçükçetin, A. and Pischetsrieder, M. Peptide profiling of bovine kefir reveals 236 unique peptides released from caseins during its production by starter culture or kefir grains.
J. Proteomics. 2015; 117:41-57. doi: 10.1016/j.jprot.2015.01.005.

14. Shing, C.M., Hunter, D.C. and Stevenson, L.M. Bovine colostrum supplementation and exercise performance: potential mechanisms. Sports Med. 2009; 39:1033-1054. doi:10.2165/11317860-000000000-00000.

15. Senda, A., Fukuda, K., Ishii, T. and Urashima, T. Changes in the bovine whey proteome during the early lactation period. Anim. Sci J. 2011; 82:698-706. doi:10.1111/j.1740-0929.2011.00886.x.

16. Thyer, L., Ward, E., Smith, R., et al. GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients. Oncoimmunology. 2013. 2:e25769. doi: 10.4161/onci.25769.

17. Suh, M.J., Tovchigrechko, A., Thovarai, V. et al. Quantitative differences in the urinary proteome of siblings discordant for type 1 diabetes include lysosomal enzymes.
J. Proteome Res. 2015; 14:3123-3135. doi: 10.1021/acs.jproteome.5b00052.

18. Zoeller, R.T., Environmental chemicals as thyroid hormone analogues: new studies indicate that thyroid hormone receptors are targets of industrial chemicals?
Mol. Cell. Endocrinol. 2005; 242:10-15.

19. Leung, A.M., Pearce, E.N. and Braverman, L.E. Perchlorate, iodine and the thyroid. Best Pract. Res. Clin. Endocrinol. Metab. 2010; 24:133-141. doi:10.1016/j.beem.2009.08.009.

20. Bagherpour Shamloo, H., Golkari, S., Faghfoori, Z. et al. Lactobacillus Casei decreases organophosphorus pesticide diazinon cytotoxicity in human HUVEC cell line.
Adv. Pharm. Bull. 2016; 6: 201–210. doi:10.15171/apb.2016.028.

21. Majlesi, M., Shekarforoush, S.S., Ghaisari, H.R., Nazifi, S., Sajedianfard, J. and Eskandari, M.H. Effect of probiotic Bacillus coagulans and Lactobacillus plantarum on alleviation of mercury toxicity in rat. Probiotics Antimicrob. Proteins. 2017; Jan 13. doi:10.1007/s12602-016- 9250-x.

22. Haskard, C.A., El Nezami, H. S., Kankaanpää, P. E., Salminen, S. and Ahokas, J.T.
Surface binding of Aflatoxin B1 by lactic acid bacteria. Appl. Environ. Microbiol. 2001; 67:3086– 3091. doi: 10.1128/AEM.67.7.3086–3091.2001.

23. Zoghi, A., Khosravi-Darani, K. and Sohrabvandi, S. Surface binding of toxins and heavy metals by probiotics. Mini Reviews in Med. Chem. 2014; 14:84-98.

24. Abbasi, B., Ghiasvand, R. and Mirlohi, M. Kidney Function Improvement by Soy Milk Containing Lactobacillus plantarum A7 in Type 2 Diabetic Patients With Nephropathy: a Double- Blinded Randomized Controlled Trial. Iran J. Kidney Dis. 2017; 11:36-43.

25. Aronson, J. K. Unity from diversity: the evidential use of anecdotal reports of adverse drug reactions and interactions. J. Eval. Clin. Pract. 2005; 11:195–208.

Authors’ Contributions

Jerry Blythe, MD: performed the experience described in this study, provided critical input and assisted in revising and improving the paper.

Stefania Pacini, MD, PhD: wrote the first draft of this paper, provided critical input and assisted in revising and improving the paper.

Disclosures

Jerry Blythe discloses no conflict of interest. He bought all the foods and supplements used during his experience and paid for the analyses reported in this study.

Stefania Pacini works as external consultant for Silver Spring Sagl, the company producing the probiotic yogurt used in this experience. She had no prior knowledge of the nutritional plan followed by Dr. Blythe nor of the results of the analyses that were communicated only after completion of the experience.

Sulfate and Sulfation

Mimi says:

R.H. Waring, School of Biosciences, University of Birmingham, Birmingham. B15 2TT U.K.

https://www.epsomsaltcouncil.org/wp-content/uploads/2015/10/sulfation_benefits.pdf

1.  Sulfate is essential for many biological processes.
2.  Sulfate is needed for formation of proteins in joints; low levels of sulfate are found in   plasma and synovial fluid from patients with rheumatoid arthritis.
3.  Sulfate is needed to start the cascade of digestive enzymes released from the pancreas. Without proteases, lipases and amylases, food is not digested efficiently.
4. Sulfate is essential in forming the mucin proteins which line the gut walls. These have main functions- they stop the gut contents from ‘sticking’ and they block transport of toxins from the gut to the bloodstream. Low plasma sulfate has been found in patients with irritable bowel disease.
5. Sulfate is necessary for formation of brain tissue. Before birth, the functional units of the brain, ‘neurons’, are laid down on a scaffolding network of sulfated carbohydrate chains. Reduced sulfation can lead to faulty neuronal connections and later dysfunction.
6. Sulfation is a major pathway in detoxifying drugs and environmental contaminants.
7. Sulfate is not easily absorbed across the gut wall. Recent research has shown that it can be absorbed across the skin. It is also formed in the body by oxidation of the aminoacids cysteine and methionine. However, this pathway is often sub-optimal and many people benefit from sulfate supplementation.

Background

The process of sulfation involves adding on a sulfate residue, which has two negative charges, to a biological molecule. This leads to a number of ‘knock-on’ effects because the addition of a highly charged polar group can fundamentally alter the molecular configuration and properties. Sulfation can activate or inactivate a wide range of biological compounds and any aberration in the supply of sulfate can have potentially serious consequences.

Principally, sulfation is a major inactivation pathway for catecholamines such as the neurotransmitter dopamine, about 80% of which is sulfated in man (1). Usually, when chemical neurotransmitters are released in the central nervous system, they act at receptor proteins and are then inactivated by sulfation or by FAD-linked mono-oxygenases or alternatively are carried by transporter proteins back into the initiating neurone. Failure of a major pathway such as sulfation will lead to a neurotransmitter imbalance and this can have effects on behavior, mood and function, as can be seen in migraine where many patients have reduced sulfation capacity. The process of sulfation also affects the functioning of peptides and proteins. Mucin proteins, which line the gastrointestinal tract, are sulfated glyco-proteins which control adhesion/lubrication of gut contents and absorption of nutrients (2). They have long peptide backbones with repeating sub-units and also peptide side-chains, rather similar to a ‘bottle-brush’. These amino acid sequences are glycosylated with a range of sugars and both these carbohydrate residues and the peptides themselves are sulfated. As the addition of sulfate residues imparts net negative charges, the proteins adopt an extended configuration to minimise their interactions, since the negative charges repel each other . Removal of the sulfate residues leads to a protein which has a more globular structure and provides less protection for the tissues from the intestinal contents. Reduced sulfation has been linked with gut dysfunction in irritable bowel disease (3) and lower levels of sulfation of the ileal mucins have recently been shown to occur in children with autism (4) and in irritable bowel. This correlates with the finding that gut permeability is increased in a large percentage of autistic children (5). The effects of mucin modification can also be seen in the colonisation of the gut by microflora as sulfation increases resistance to colonisation by pathogenic bacteria (and viruses). It is interesting that Helicobacter pylori, which can colonise the stomach, only does so when it has produced a sulfatase enzyme to de-sulphate the gastric mucins (6). This reduced sulfation of mucin proteins may underlie the relatively common finding of Candida infections in the gut, since the slight negative charges on Candida cells would lead to their repulsion by the negatively charged sulfate groups on normal mucins. The reduction in mucin sulfation is probably an indirect cause of the alterations in gut flora which are often found in gastrointestinal inflammation.

Peptides can also be sulfated, usually on tyrosine residues,by the enzyme tyrosyl protein sulfotransferase (TPST); the gastric hormones gastrin and cholecystokinin are good examples of this pathway. Both are involved in the digestive process and both are activated by sulfation. In a complex cascade, gastrin is sulfated and, with hydrochloric acid from the stomach, causes release of cholecystokinin, which also requires sulfation. Together with peptide fragments released from gastric proteolysis (mediated by the hydrochloric acid), this acts with the peptide hormone secretin on pancreatic tissue to induce the secretion of a range of proteolytic enzymes and also amylase and lipases. Lower levels of pancreatic amylase alter the digestibility of starch-based foods and allow increased fermentation of pathogenic bacteria while the decreased pancreatic lipase activity promotes formation of foul-smelling fatty stools which contain undigested triglycerides. Without the sulfation process to trigger the release of pancreatic proteases such as trypsin and chymotrypsin, the complete digestion of proteins to their amino acid building blocks (proteolysis) cannot take place, so that peptides, rather than amino acids, are found in the gastrointestinal tract. As reduced sulphation of mucins may have made the gut more permeable, the stage is set to allow peptides to penetrate into the blood stream. Some peptides, particularly those derived from casein and gluten, have been found to be neuroactive with effects on the brain where they can act at opioid receptors, affecting behaviour, mood and responses to physical stimuli such as pain. This ‘leaky gut’ hypothesis therefore links with the opioid theory to explain why peptides from casein and gluten in particular seem to cause problems for some people. Although the blood-brain barrier is usually seen as being non-permeable to many compounds it may, like the gut, be ‘leaky’ in individuals with previous head injuries or who are living in stressful conditions. Several studies have reported the presence of brain-derived proteins and antibodies, such as those from myelin, within the peripheral circulation. If relatively large proteins can cross from the brain, it seems possible that peptides and proteins could potentially be transported into the brain, although the mechanisms involved are not known. Simple diffusion across ‘leaky’ gap junctions may be all that is necessary.

The supply of sulfate in man is rarely sufficient to allow synthesis of all the required sulfated biocomponents. There are two main sources of sulfate in vivo. Unlike other anions, such as chloride (Cl-), which seem to be readily and easily absorbed, sulfate is carried into the body from the g.i. tract via a sodium ion-linked sulfate transporter (7). This is easily saturated, so that ingestion of many small divided doses of sulfate over a period of time leads to higher blood sulfate concentrations than when a single large dose is taken. Anaerobic bacteria can convert sulfate (SO42-) into sulfide (S2-); individuals with bowel disease may have a range of pathogenic species of bacteria in the gastrointestinal tract which readily carry out this reduction so any ingested sulfate may be reduced to sulfide and therefore become unavailable. Magnesium sulfate is readily absorbed across the skin so topical applications or bathing may be more successful in raising plasma sulfate levels. Sulfate is also produced in vivo by oxidation of methionine or cysteine, both sulfur – containing amino acids which are provided from dietary proteins, and this pathway probably provides ∼80% of the sulfate required in man. The first stage in this process involves the enzyme cysteine dioxygenase (CDO); cysteine sulfinic acid is formed and undergoes fission to provide sulfite (SO32-) ions which are then further oxidised to sulfate (SO42-) ions by the enzyme sulfite oxidase (SOX). As can be seen, if CDO or SOX have reduced activity, the provision of sulfate will also be decreased. The human CDO gene has 5 exons and 4 introns and is localised to chromosome 5 (5q22-23); it is similar to the rat gene and proteins from the 2 genes have about 90% homology. The gene has a Ptx-3 response element, with a range of cis-acting elements (expressed in the CNS exclusively in dopaminergic neurones). The CDO protein is found in heart, thyroid and kidney, as well as brain and the liver. There seem to be variations in the properties of CDO enzymes from different tissues which may reflect differences in degree of glycosylation. CDO is known to be polymorphic in human populations and there are sub-sets with lower activity (~ 30% of the population) or nul activity (~ 3% of the population) (8). The nul S-oxidisers are heavily over-represented in chronic disease states with an auto-immune component such as rheumatoid arthritis and primary biliary cirrhosis (9,10). Even within ‘normal’ populations there is a wide variation in the extent of conversion of dietary sulfur amino acids to sulfate. In experiments carried out over decades in this department in practical classes with medical students, it was found that increased levels of protein in the diet produced proportional increases in urinary sulfate excretion, as would be expected. However, about 30% of the students reached a plateau in sulfate excretion, despite increasing ingestion of protein, suggesting that some pathway or pathways had become saturated. Provision of sulfate for in vivo metabolism is therefore a rate-limiting step for many people.

In some conditions, large amounts of sulfate are lost from the body in urine; the kidney (as well as the gut) may be ‘leaky’, so that sulfate is not resorbed as would be expected. Sometimes this is linked with an increased excretion of protein/peptides in urine , suggesting that kidney function is impaired. There may, of course, be dysregulation of the sulfate excretion system. On the apical or brush border side of the cells of the renal tubules, the sodium-dependent NaSi-1 sulfate co- transporter removes sulfate from the filtered fluid and this is exchanged for other anions, usually bicarbonate so that sulfate is transported into the blood from the baso-lateral side (11). Another renal sulfate transporter, Sat-1, is a sulfate/bicarbonate/oxalate exchanger and has been found in the brain and is highly expressed in the cerebellum and hippocampus (12). This complicated process suggests that conservation of sulfate anions is critical for human function. It has however been suggested that the kidney transporters respond to the levels of sulfate in the gastrointestinal tract so that sulfated glycosaminoglycans(GAGs) lost from the intestine during chronic infection could release sulfate and so provide a signal to the renal transporter to excrete the anion (13). The sulfate transporters NaSi-1 and Sat-1 are both subject to regulation by a member of hormonal and dietary changes (14). Renal re-absorption of inorganic sulfate is increased in growth and development (15) and regulated by vitamin D, dietary sulfate, glucocorticoids and thyroid hormones (16), also metabolic acidosis (17) and non-steroidal anti-inflammatory drugs (NSAIDS) (18) and chronic potassium depletion (19). To add to a complex interaction, the renal sulfate transporter proteins are themselves sulfated and the brush border contains a matrix of sulfated GAGs, so reduced levels of sulfate in vivo could have the effect of altering the structure of the systems required to retain sulfate in the body. This would of course result in a spiral of increasing dysfunction and it is unclear which are the controlling factors. Further, most of the work on sulfate metabolism has been done with the rat and should be invoked with caution since this species uses different pathways from those seen in human beings.

Not only is there an impaired level of sulfate in many disease states, there is also often a corresponding lack of sulfotransferase activity. These are the enzymes which carry out sulfation of a wide range of substrates. They belong to a super-family which uses PAPS (3′-phosphoadenosine – 5′-phosphosulfate) as co-factor and are widely distributed throughout the body (20). This process is critical for life as a defect in PAPS synthesis is lethal in humans (21) reflecting the wide range of biosubstrates which are sulfated. Sulfotransferases with endogenous substrates are important in tissue development and mice with defects in the Golgi-membrane enzyme which sulfates glycosaminoglycans are non-viable as these polysaccharides cannot be converted to the unique binding sites which are recognised as signals for growth (22, 23). Other mutant mice without the heparan sulphate 20-sulfotransferase gene die from defective kidney development (24) suggesting that the sulfate/sulfation axis is vital in kidney development and function. These enzymes with endogenous substrates are membrane-bound while those which sulfate signal molecules such as steroids, thyroid hormones and neurotransmitters are cytosolic (25). The same enzymes also carry out the second stage in metabolism of drugs and related compounds to give water-soluble derivatives which are more readily excreted. All sulfotransferases have common motifs, as they contain a five-stranded parallel β-sheet flanked by α-helices which contains both the PAPS binding site and also the centre of the catalytic site, structures which are highly conserved throughout evolution. The substrate-binding sites, however, are totally different in the membrane-bound and cytosolic enzymes (26).

The major enzymes responsible for the sulfation of phenols and catecholamines (SULT1A1 and 1A3 respectively) have been mainly studied in blood platelets where activity is approximately co-regulated with other tissues in the body, such as the gut and brain. SULT1A1 and 1A3 have 93% amino acid sequence identity while a third more recently discovered isoform, SULT1A2, differs by only 3 residues from SULT1A1. This is, however, sufficient to give it slightly different properties with regard to substrate repertoire, inhibitor sensitivity and thermal stability. All three genes are located close together on Chromosome 16. SULT1A1 is present in high concentration in the liver, brain, blood platelets and many other tissues while SULT1A3 is also found in platelets and brain but tends to be concentrated in the gastrointestinal tract rather than the liver. Only limited information is available on the distribution of SULT1A2 but it has been detected in the liver (by RNA) and some bladder tumours. There is a wide variation in SULT1A1 activity in human populations, as much as 50-fold having been reported by one group of workers (27). The level of enzyme activity in platelets correlates with the level of protein as detected by immunoblotting. A number of different alleles for SULT1A1 have been found and an alloenzyme with His 213 is less active than one with arginine at the same site. A range of different SULT1A2 alleles are also known and are in a linkage disequilibrium with the SULT1A1 alleles. Polymorphisms do not appear to have been reported for SULT1A3 although investigations on the platelet enzyme activity in families suggest that they may exist, since there is a wide range of activity which is highly heritable (28).

Sulfotransferase activity is known to be altered in some dysfunctional states, for example most patients with migraine have low SULT1A1 and sometimes low SULT1A3 activity (29). They are therefore less able to sulfate dietary phenols and catecholamines in the g.i. tract and in the bloodstream via the platelets as the SULT enzyme is co-regulated in both tissues. Sulfation results in inactivation of amines and these individuals are susceptible to foods which contain substrates for the enzymes (cheese/tyramine, chocolate/phenylethylamine, bananas/serotonin). The increased blood levels of compounds with neurotransmitter activity are thought to ‘trigger’ migraine headaches in those who are already susceptible. It has been shown that individuals who are susceptible to migraine are metabolically unstable (with raised excitotoxic amino acid levels) so that very small changes in blood and brain catecholamine levels are sufficient to provoke a migraine attack. SULT1A1 and 1A3 can also be inhibited by flavonoids (30) and by foods containing these compounds which typically occur in fruit and vegetables (31). The incubation of cytosolic preparations from 35 different fruits and vegetables with platelet sulfotransferases produced a wide range of results . Some cytosols inhibited the sulfation of both test substrates although most appeared to more potently inhibit 4-nitrophenol sulfation than dopamine. Ingestion of citrus fruit, especially oranges, and red wine is often reported as being a migraine ‘trigger’ and the component flavonoids (naringin and resveratrol) are inhibitors of both SULT1A1 and SULT1A3. Further studies with purified flavonoids have shown that some can virtually abolish SULT1A1 activity at very low (less than 100nM) concentrations but at higher concentrations (over 1μM) they appear to act as substrates for SULT1A3. The inhibitory effect can be partially overcome by the presence of magnesium ions. These not only appear to enhance enzyme activity, but also to decrease enzyme sensitivity to flavonoids like quercetin by a factor of about five.

Other sulfotransferases can also be affected. The enzyme tyrosylproteinsulfotransferase (TPST) is membrane-bound and found in most tissues of the body, including the platelet and the gastrointestinal tract, where it is co-regulated (32). The TPST enzyme is a 50-54 kDa integral membrane glycoprotein of the trans-Golgi network. At least 2 forms are known, TPST-1 and TPST-2. The latter is a type II transmembrane protein of 377 amino acid residues, encoded by m- RNA originating from seven exons of a gene located on chromosome 22. A 304- residue segment in the terminal domain of TPST-2 has 75% amino acid homology to the corresponding segment of TPST-1 including conservation of the residues involved in binding PAPS. The enzyme and requires the presence of acidic residues on the amino-terminal side of the target tyrosine (33). The TPST family is highly conserved from the evolutionary standpoint, presumably because the post- translation modification of tyrosine residues in peptides and proteins has been required for millennia. A number of substrates are known and these include the leukocyte adhesion molecule P- Selectin glycoprotein ligand 1 (PSGL-1) which is required for binding to P-selectin on activated endothelium and IgA, IgM, IgCT and complement C4 (34). TPST is the enzyme responsible for sulfation of gastrin and cholecystokinin as well as the sulfation of proto-mucin monomers. Sulfated cholecystokinin (CS) has receptors in the brain as well as the gut and is required for release of the peptide hormone oxytocin. Vitamin B6 (pyridoxal phosphate) has been reported to inhibit both SULT1A1 and TPST. However, studies in this laboratory have shown that, as with quercetin, this inhibition can be blocked by magnesium ions and that a ratio of at least 1:1 magnesium:B6 will restore enzyme activity.

Carbohydrates are also substrates for sulfation, particularly the polysaccharide chains attached to glycosaminoglycans (GAGs). Here, there are specific patterns of sulfation which control a range of functions and vary by cell type. These sulfated GAGs are involved in G-protein signalling, calcium signalling, gap junctions (cell-cell signalling) and in differentiation and growth (35). As the enzymes involved in carbohydrate sulfation have relatively high Michaelis constants (compared with SULT 1A1/2, for example), low levels of sulfate will affect this pathway preferentially. Because sulfated polysaccharides and GAGs are so important in the development of the foetal and neonatal brain, any alteration in their structure may have deleterious effects on their function. Sulfate transport across the placenta increases dramatically around the time of birth when most of the glial cells are being formed and these increased levels of sulfate are associated with formation of astrocytes and oligodendrocytes from progenitor cells (36). It is of interest that children have higher levels of plasma sulfate than adults (0.47 nmol/l at birth decreasing to 0.33 nmol/l at 36 months; adults levels are around 0.27 nmol/l although there can be a wide range). This relatively high level of sulfate, as compared with the adult state and with, for instance, laboratory rats, may be associated with a requirement for sulfate patterning of carbohydrates in neuronal development. Recent studies on perineuronal nets have found these structures coating the proximal dendrites of certain neurons including those in 5 areas of the brain involved in autism (37). It seems probable that the nets play a role in signal modulation of the GABA-ergic neurons; they contain chondroitin sulfate and so are distinctive from other structures. In rat studies, these perineural nets have been found in the thalamus, which controls sensory integration and emotions and also in the hypothalamus, controlling hormonal and autonomic regulation neurons. Neurones in the human cerebral cortex which are ensheathed by perineuronal nets rarely undergo cytoskeletal changes in Alzheimer’s disease, suggesting a neuro- protective effect of extracellular matrix components (38). Other proteoglycans with sulphated GAG chains also have a role in brain development, particularly the syndecan family which regulates the maturation of dendritic spines and also glypican which is expressed during axon growth. Neurocan is a chondroitin sulfate proteoglycan of the lectican family, mainly expressed during modeling and remodeling stages in the CNS. It binds to a wide range of cell surface molecules and to structural extracellular matrix components and has been suggested as being responsible for the ‘fine-tuning’ of neural networks as it modulates cell-binding and neurite outgrowth (39).

Sulfation of proteins and carbohydrate chains is also important for the function of joints in the body. A special protein/carbohydrate complex exists to allow the surfaces of the different bones in eg wrist, knee and ankle to slide over one another as the joint is used. This slippery lining to the joints is called the ‘synovium’; the surfaces of the bones are held slightly apart by the lubrication of synovial fluid. Rather like the mucin proteins in the gut walls, the synovium is heavily sulfated on proteins and associated GAGs to give a structure which is both very resistant to breakdown in use and also able to protect the bones in the joint from rubbing against each other and causing damage. Patients with rheumatoid arthritis have been shown to have very low levels of sulfate in plasma, synovial fluid and also in the synovium proteins (40) and this loss of sulfate is linked with the disease progression, showing that sulfation is vital for joint function.

Conclusion

As more research is done, the importance of the sulfate supply becomes more apparent, since sulfation plays such a major part in so many biochemical processes while sulfate generation in the body is often sub-optimal.

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