A. Appendix and Nomenclature

ZPD zero path difference

This appendix presents the definition of terms/notation used throughout the chapter.

References

1621S-1625S

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Sciences. 1993;694:161-176

DOI: 10.1097/00001648-199505000-00005

10.1001/jama.262.20.2847

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[8] Wilcox AJ, Lie RT, Solvoll K, Taylor J, McConnaughey DR, Abyholm F, Vindenes H, Vollset SE, Drevon CA. Folic acid supplements and risk of facial clefts: National population based

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### Author details

Rahaman Raziya Sultana1 \*, Sheik Nizamuddin Zafarullah<sup>2</sup> and Navamani Hephzibah Kirubamani<sup>3</sup>

\*Address all correspondence to: saalimraziya@gmail.com


### References

IR infrared spectroscopy LMP last menstrual period

100 B Group Vitamins - Current Uses and Perspectives

ZPD zero path difference

Notation Definition

or gas

R1 Intensity ratio parameter 1

value for the group.

Author details

Std. Dev1

Rahaman Raziya Sultana1

Navamani Hephzibah Kirubamani<sup>3</sup>

\*Address all correspondence to: saalimraziya@gmail.com

variance of two normal distribution are not known.

3 Saveetha Medical College, Kanchipuram, India

2 Department of Physics, Easwari Engineering College, Chennai, India

MSAF maternal serum alpha fetoprotein

A. Appendix and Nomenclature

indicated by the first day of bleeding

SGOT serum glutamic oxaloacetic transaminase

This appendix presents the definition of terms/notation used throughout the chapter.

FTIR Fourier-transform infrared spectrometer simultaneously collects high-spectral-resolution data over a wide

LMP The last menstrual period (LMP) refers to the start date of a woman's most recent menstruation, or period as

DTGS Deuterated triglycine sulfate detector (DTGS) is a very sensitive room-temperature detector for mid-infrared

t-test A t-test is an investigation of two population means which implies using statistical examination, a t-test with two samples is commonly utilized with size of a small sample, testing the contrast or difference between the

F-test An F-test is any statistical test in which the statistic test has an F-distribution under the null hypothesis.

spectral range. It is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid

range measurements that employs temperature-sensitive ferroelectric crystals of deuterated triglycine sulfate

Standard deviation 1 is a quantity expressing by how much the members of a group differ from the mean

\*, Sheik Nizamuddin Zafarullah<sup>2</sup> and

1 Department of Physics, Justice Basheer Ahmed Sayeed College for Women, Chennai, India

SGPT serum glutamic pyruvic transaminase


[14] Beard CM, Panser LA, Katusic SK. Is excess folic acid supplementation a risk factor for autism? Medical Hypotheses. 2011;77(1):15-17. DOI: 10.1016/j.mehy.2011.03.013

[29] Silverstein RM, Webster FX, Kiemle DJ. Spectrometric Identification of Organic Compounds. 7th ed. State Universtiy of New York, College of Environmental Science & Forestry, John Wiley & sons. Inc. 2005. ISBN-10: 0471393622; ISBN-13: 978-0471393627 [30] Huleihel M, Salman A, Erukhimovich V, Ramesh J, Hammody Z, Mordechai S. Novel optical method for study of viral carcinogenesis in vitro. Journal of Biochemical and

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[31] Gunasekaran S, Sankari G. FTIR and UV–visible spectral study on normal and diseased

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[33] Silverstein RM, Bassler GC, Morrill TC. Spectrometric Identification of Organic Com-

[34] Ragamathunnisa M, Jasmine Vasantha Rani E, Padmavathy R, Radha N. Spectroscopic study on thiourea and thiosemicarbazide in non-aqueous media. NIOSR Journal of Applied Physics (IOSR-JAP). 2013;4(1):05-08. e-ISSN: 2278-4861. www.iosrjournals.org

[35] Ke J, Laskar DD, Chen SL. Biodegradation of hardwood lignocellulosics by the western poplar clearwing borer, Paranthrene robiniae (Hy. Edwards). Biomacromolecules. 2011;12:

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[14] Beard CM, Panser LA, Katusic SK. Is excess folic acid supplementation a risk factor for autism? Medical Hypotheses. 2011;77(1):15-17. DOI: 10.1016/j.mehy.2011.03.013

[15] Hamilton J. Folic Acid for Pregnant Mothers Cuts Kids' Autism Risk. US: National Public

[16] Sultana RR, Zafarullah SN, Hephzibah Kirubamani N. Utility of FTIR spectroscopic analysis of saliva of diabetic pregnant women in each trimester. Indian Journal of Science

[17] Zhang J, Rana S, Srivastava R, Misra R. On the chemical synthesis and drug delivery response of folate receptor-activated, polyethylene glycol-functionalized magnetite nanoparticles. Acta

[18] Wiswell TE, Tuttle DJ, Northam RS, Simonds GR. Major congenital neurologic malformations. A 17-year survey. American Journal of Diseases of Children. 1990;144(1):61-67 [19] Bower C, Miller M, Payne J, Serna P. Promotion of folate for the prevention of neural tube defects: Who benefits? Paediatric and Perinatal Epidemiology. 2005;19(6):435-444

[20] Danielsson K. What's the Big Deal with Folic Acid and Pregnancy?; 2009. About.com.

[21] Callaway L, Colditz PB, Fisk NM. Folic acid supplementation and spontaneous preterm

[22] Facts about Folic Acid CDC [Internet]. 2009. Available form: http://www.cdc.gov/ncbddd/

[23] Haberg SE, London SJ, Stigum H, Nafstad P, Nystad W. Folic acid supplements in pregnancy and early childhood respiratory health. Archives of Disease in Childhood.

[24] Sherwood KL, Houghton LA, Tarasuk V, O'Connor DL. One-third of pregnant and lactating women may not be meeting their folate requirements from diet alone based on mandated levels of folic acid fortification. The Journal of Nutrition. 2006;136:

[25] Beauchaine JP, Peterman JW, Rosenthal RJ. Applications of FT-IR/microscopy in forensic

[26] Prati S, Joseph E, Sciutto G, Mazzeo R. New advances in the application of FTIR microscopy and spectroscopy for the characterization of artistic materials. Accounts of Chemical

[27] Gaft M, Reisfeld R, Panczer G. Luminescence Spectroscopy of Minerals and Materials. Berlin, Heidelberg, NewYork: Springer; 2005. p. 263. ISBN: 3540219188. http://books.

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analysis. Microchimica Acta. 1988;94(1–6):133-138. DOI: 10.1007/BF01205855

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Biomaterialia. 2008;4(1):40-48

102 B Group Vitamins - Current Uses and Perspectives


**Section 4**

**Other B vitamins**

**Section 4**

**Other B vitamins**

**Chapter 7**

**Provisional chapter**

**Niacin, Metabolic Stress and Insulin Resistance in Dairy**

**Niacin, Metabolic Stress and Insulin Resistance in Dairy** 

The periparturient period in cows is associated with metabolic stress and a state of negative energy balance, which are characterized by increased lipolysis, ketogenesis, hepatic steatosis, oxidative stress and insulin resistance. Such metabolic changes may exert adverse effects on the health and milk yield of lactating cows. The pharmacokinetics of niacin in ruminants is specific as rumen microorganisms facilitate both the synthesis of tryptophan and the degradation of niacin. Niacin administration to cows leads to an increase in the coenzyme activity, encompassing the activity of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These coenzymes are actively involved in the metabolism of lipids and carbohydrates, whereas NAD protects the organism from oxidative stress. In periparturient cows, the supplementation of niacin has been found to induce depressed lipolysis and a limited impact of nonesterified fatty acids on all metabolic processes. It also results in decreased lipid peroxidation regardless of the magnitude of lipolysis in the periparturient period. Furthermore, niacin reduces the concentration of ketone bodies, thus preventing the development of fatty lever disease and ketosis in cows. The anti-inflammatory effect of niacin is manifested in stimulating the secretion of adiponectin and inhibiting immune

**Keywords:** transition period, nonesterified fatty acid, insulin, glucose, oxidative stress

Metabolic stress in dairy cows occurs around the time of parturition as a consequence of heightened milk production requirements, accompanied by depressed feed intake and

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.77268

**Cows**

**Cows**

Branislava Belić

Branislava Belić

**Abstract**

cells.

**1. Introduction**

Marko Cincović, Talija Hristovska and

Marko Cincović, Talija Hristovska and

http://dx.doi.org/10.5772/intechopen.77268

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

### **Niacin, Metabolic Stress and Insulin Resistance in Dairy Cows Niacin, Metabolic Stress and Insulin Resistance in Dairy Cows**

DOI: 10.5772/intechopen.77268

Marko Cincović, Talija Hristovska and Branislava Belić Marko Cincović, Talija Hristovska and Branislava Belić

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77268

### **Abstract**

The periparturient period in cows is associated with metabolic stress and a state of negative energy balance, which are characterized by increased lipolysis, ketogenesis, hepatic steatosis, oxidative stress and insulin resistance. Such metabolic changes may exert adverse effects on the health and milk yield of lactating cows. The pharmacokinetics of niacin in ruminants is specific as rumen microorganisms facilitate both the synthesis of tryptophan and the degradation of niacin. Niacin administration to cows leads to an increase in the coenzyme activity, encompassing the activity of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These coenzymes are actively involved in the metabolism of lipids and carbohydrates, whereas NAD protects the organism from oxidative stress. In periparturient cows, the supplementation of niacin has been found to induce depressed lipolysis and a limited impact of nonesterified fatty acids on all metabolic processes. It also results in decreased lipid peroxidation regardless of the magnitude of lipolysis in the periparturient period. Furthermore, niacin reduces the concentration of ketone bodies, thus preventing the development of fatty lever disease and ketosis in cows. The anti-inflammatory effect of niacin is manifested in stimulating the secretion of adiponectin and inhibiting immune cells.

**Keywords:** transition period, nonesterified fatty acid, insulin, glucose, oxidative stress

### **1. Introduction**

Metabolic stress in dairy cows occurs around the time of parturition as a consequence of heightened milk production requirements, accompanied by depressed feed intake and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

a negative energy balance. Accordingly, the organism enters a state of increased lipolysis, oxidative stress and insulin resistance. Niacin is a well-known antilipolytic vitamin which enhances gluconeogenesis and insulin concentrations in the blood [1]. A review of the literature has revealed that the effect of niacin is dependent upon the dosage administered, pharmaceutical form, duration of administration and biological features of cows [2, 3]. There is a limited body of information on the relationship between niacin and insulin productions and efficiency in cows, as well as on the NAD and NADP response to niacin administration. This chapter will elucidate the mechanism of metabolic stress in cows, the pharmacokinetics of niacin, the physiology of the niacin-containing NAD and NADP coenzymes, as well as the biological effect of niacin on lipolysis, lipolysis-dependent metabolic adaptations and insulin resistance in dairy cows.

the mammary gland in order to maintain the persistence of lactation. This is achieved through a number of metabolic adaptations induced by the hormonal changes and associated tissue responses. Increased liver gluconeogenesis, liver glycogen depletion, increased lipolysis, protein catabolism and limited glucose utilization by all tissues other than the mammary gland represent some of the alternative means by which the organism meets the energy requirements of the mammary gland. Growth hormone levels increase around parturition, resulting in the increased responsiveness of adipose tissue to lipolytic signals such as norepinephrine. An increased release of NEFAs from adipose tissue subsequently ensues, which are converted by the liver to ketone bodies and used as alternate fuels for extramammary tissues. The ketones serve as alternate fuels which can replace glucose in many tissues, thus conserving glucose for milk synthesis. Elevated somatotropin levels also enhance gluconeogenesis [8]. An increase in corticosteroid concentrations around parturition enhances the responsiveness of adipocytes to the action of catecholamines and stimulates glycogenolysis as well as gluconeogenesis [9]. Depressed plasma insulin concentrations and decreased insulin sensitivity enable the insulinindependent uptake of nutrients by the mammary gland, whereas insulin-dependent tissues

Niacin, Metabolic Stress and Insulin Resistance in Dairy Cows

http://dx.doi.org/10.5772/intechopen.77268

109

Adipose tissue has a pivotal role in homeorhesis and metabolic stress in cows. In the dry period and late lactation, anabolic processes predominate as the cow's body stores triglycerides in adipose tissue, which is thereafter sensitive to insulin (the key antilipolytic hormone reducing the degradation of triglycerides in adipose tissue cells and facilitating the synthesis of fatty acids and glycerol). A negative energy balance in early lactation is associated with a number of metabolic changes, leading to increased lipid catabolism in adipose tissue and the mobilization of body fat stores. The degradation of triglycerides stored in adipocytes also ensues, accompanied by the release of NEFAs and glycerol. The mobilization of adipose tissue fat is mediated by the following similarly functioning enzymes: monoglyceride lipase (MGL), hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) [10]. ATGL initiates lipolysis followed by the actions of HSL on diacylglycerol and MGL on monoacylglycerol. An increase in the action of triglyceride lipase is recorded at low insulin levels in the blood. The name of hormone-sensitive lipase itself suggests that hormones such as catecholamines, adrenocorticotropic hormone (ACTH) and glucagon stimulate the action of this intracellular neutral lipase [11]. The mobilization of fatty acids from body stores is induced by both energy deficits and changes in neuroendocrine regulation. Hormonal changes such as low insulin and glucagon concentrations significantly contribute to initiating and maintaining the mobilization of depot fat, whereas reduced insulin resistance, as an indicator of decreased insulin functional capacity, is of paramount importance [12]. Low plasma insulin concentrations enhance the action of triacylglycerol lipase and inhibit the entry of NEFAs, glycerol and glucose into adipocytes by reducing the action of lipoprotein lipase (the enzyme which hydrolyzes triacylglycerols in chylomicrons and very-low-density lipoproteins) as well as the expression/translocation of GLUT4 molecules. Lipolysis occurs in a state of reduced insulin sensitivity or low serum insulin concentrations (which is characteristic of early lactation), resulting in increased serum NEFA concentrations. In ruminants, acetate is a major substrate for the de novo synthesis of fatty acids, and adipose tissue is of overriding importance to the process. The degree of in vitro incorporation of acetate in the de novo synthesis of fatty acids

increase the oxidation of fatty acids and reduce the utilization of glucose.

### **2. Metabolic stress in cows**

In addition to a number of metabolic and physiological adaptations, the periparturient period in cows is associated with a dramatic increase in the nutritional requirements essential for foetal growth and milk synthesis. The nutritional requirements of the placenta and foetus are highest in the last 3 weeks of pregnancy, whereas the dry matter intake (DMI) is reduced by 10–30% relative to the DMI in the early dry period. As milk production surges from the onset of lactation to the yield required to sustain the calf, the ongoing adaptations occur rapidly, resulting in a marked discrepancy between the varying nutritional requirements and concomitant adaptations. The peak of lactation is expected to be reached in weeks 4–8 postpartum, whereas the highest dry matter intake is achieved in weeks 10–22 after parturition [1, 2]. The greatest negative energy balance in dairy cows is recorded around day 14 of lactation, continuing even to day 72 of lactation (as reported by the same author) [4]. During early lactation, the energy requirements for milk production and proper tissue function exceed the amount of energy ingested. To compensate for a negative energy balance, energy and protein body reserves are mobilized, expending to approximately 600 g/d of fat and 40 g/d of protein in the first 8 weeks after parturition [5]. The failure of these adaptive mechanisms has been implicated in the occurrence of common metabolic disorders in early lactation. Postpartum metabolic disorders are interrelated and concurrent, greatly influencing the fertility of cows. Fatty liver syndrome and degeneration, ketosis, parturient paresis, mastitis, hypomagnesaemia, rumen acidosis, displaced abomasum, laminitis, postpartum infections and fertility problems are the predominant diseases of dairy cows in the periparturient period [6]. A glucose mass of 72 g is required to produce 1 kg of milk [7]. In ruminants, the largest amount of carbohydrates ingested is fermented in the rumen, whereas very little glucose is absorbed from the digestive tract. Consequently, glucose requirements of dairy cows are, for the most part, met by gluconeogenesis, i.e. the synthesis of glucose from propionates, amino acids, glycerol and liver lactates. A postpartum increase in the expression of key enzymes, i.e. pyruvate carboxylase and phosphoenolpyruvate carboxylase, enhances the magnitude of gluconeogenesis in the liver. Substantial discrepancies between the depressed feed intake and elevated energy requirements of the mammary gland in this period incite the organism to provide sufficient energy to the mammary gland in order to maintain the persistence of lactation. This is achieved through a number of metabolic adaptations induced by the hormonal changes and associated tissue responses. Increased liver gluconeogenesis, liver glycogen depletion, increased lipolysis, protein catabolism and limited glucose utilization by all tissues other than the mammary gland represent some of the alternative means by which the organism meets the energy requirements of the mammary gland. Growth hormone levels increase around parturition, resulting in the increased responsiveness of adipose tissue to lipolytic signals such as norepinephrine. An increased release of NEFAs from adipose tissue subsequently ensues, which are converted by the liver to ketone bodies and used as alternate fuels for extramammary tissues. The ketones serve as alternate fuels which can replace glucose in many tissues, thus conserving glucose for milk synthesis. Elevated somatotropin levels also enhance gluconeogenesis [8]. An increase in corticosteroid concentrations around parturition enhances the responsiveness of adipocytes to the action of catecholamines and stimulates glycogenolysis as well as gluconeogenesis [9]. Depressed plasma insulin concentrations and decreased insulin sensitivity enable the insulinindependent uptake of nutrients by the mammary gland, whereas insulin-dependent tissues increase the oxidation of fatty acids and reduce the utilization of glucose.

a negative energy balance. Accordingly, the organism enters a state of increased lipolysis, oxidative stress and insulin resistance. Niacin is a well-known antilipolytic vitamin which enhances gluconeogenesis and insulin concentrations in the blood [1]. A review of the literature has revealed that the effect of niacin is dependent upon the dosage administered, pharmaceutical form, duration of administration and biological features of cows [2, 3]. There is a limited body of information on the relationship between niacin and insulin productions and efficiency in cows, as well as on the NAD and NADP response to niacin administration. This chapter will elucidate the mechanism of metabolic stress in cows, the pharmacokinetics of niacin, the physiology of the niacin-containing NAD and NADP coenzymes, as well as the biological effect of niacin on lipolysis, lipolysis-dependent metabolic adaptations and insulin

In addition to a number of metabolic and physiological adaptations, the periparturient period in cows is associated with a dramatic increase in the nutritional requirements essential for foetal growth and milk synthesis. The nutritional requirements of the placenta and foetus are highest in the last 3 weeks of pregnancy, whereas the dry matter intake (DMI) is reduced by 10–30% relative to the DMI in the early dry period. As milk production surges from the onset of lactation to the yield required to sustain the calf, the ongoing adaptations occur rapidly, resulting in a marked discrepancy between the varying nutritional requirements and concomitant adaptations. The peak of lactation is expected to be reached in weeks 4–8 postpartum, whereas the highest dry matter intake is achieved in weeks 10–22 after parturition [1, 2]. The greatest negative energy balance in dairy cows is recorded around day 14 of lactation, continuing even to day 72 of lactation (as reported by the same author) [4]. During early lactation, the energy requirements for milk production and proper tissue function exceed the amount of energy ingested. To compensate for a negative energy balance, energy and protein body reserves are mobilized, expending to approximately 600 g/d of fat and 40 g/d of protein in the first 8 weeks after parturition [5]. The failure of these adaptive mechanisms has been implicated in the occurrence of common metabolic disorders in early lactation. Postpartum metabolic disorders are interrelated and concurrent, greatly influencing the fertility of cows. Fatty liver syndrome and degeneration, ketosis, parturient paresis, mastitis, hypomagnesaemia, rumen acidosis, displaced abomasum, laminitis, postpartum infections and fertility problems are the predominant diseases of dairy cows in the periparturient period [6]. A glucose mass of 72 g is required to produce 1 kg of milk [7]. In ruminants, the largest amount of carbohydrates ingested is fermented in the rumen, whereas very little glucose is absorbed from the digestive tract. Consequently, glucose requirements of dairy cows are, for the most part, met by gluconeogenesis, i.e. the synthesis of glucose from propionates, amino acids, glycerol and liver lactates. A postpartum increase in the expression of key enzymes, i.e. pyruvate carboxylase and phosphoenolpyruvate carboxylase, enhances the magnitude of gluconeogenesis in the liver. Substantial discrepancies between the depressed feed intake and elevated energy requirements of the mammary gland in this period incite the organism to provide sufficient energy to

resistance in dairy cows.

**2. Metabolic stress in cows**

108 B Group Vitamins - Current Uses and Perspectives

Adipose tissue has a pivotal role in homeorhesis and metabolic stress in cows. In the dry period and late lactation, anabolic processes predominate as the cow's body stores triglycerides in adipose tissue, which is thereafter sensitive to insulin (the key antilipolytic hormone reducing the degradation of triglycerides in adipose tissue cells and facilitating the synthesis of fatty acids and glycerol). A negative energy balance in early lactation is associated with a number of metabolic changes, leading to increased lipid catabolism in adipose tissue and the mobilization of body fat stores. The degradation of triglycerides stored in adipocytes also ensues, accompanied by the release of NEFAs and glycerol. The mobilization of adipose tissue fat is mediated by the following similarly functioning enzymes: monoglyceride lipase (MGL), hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) [10]. ATGL initiates lipolysis followed by the actions of HSL on diacylglycerol and MGL on monoacylglycerol. An increase in the action of triglyceride lipase is recorded at low insulin levels in the blood. The name of hormone-sensitive lipase itself suggests that hormones such as catecholamines, adrenocorticotropic hormone (ACTH) and glucagon stimulate the action of this intracellular neutral lipase [11]. The mobilization of fatty acids from body stores is induced by both energy deficits and changes in neuroendocrine regulation. Hormonal changes such as low insulin and glucagon concentrations significantly contribute to initiating and maintaining the mobilization of depot fat, whereas reduced insulin resistance, as an indicator of decreased insulin functional capacity, is of paramount importance [12]. Low plasma insulin concentrations enhance the action of triacylglycerol lipase and inhibit the entry of NEFAs, glycerol and glucose into adipocytes by reducing the action of lipoprotein lipase (the enzyme which hydrolyzes triacylglycerols in chylomicrons and very-low-density lipoproteins) as well as the expression/translocation of GLUT4 molecules. Lipolysis occurs in a state of reduced insulin sensitivity or low serum insulin concentrations (which is characteristic of early lactation), resulting in increased serum NEFA concentrations. In ruminants, acetate is a major substrate for the de novo synthesis of fatty acids, and adipose tissue is of overriding importance to the process. The degree of in vitro incorporation of acetate in the de novo synthesis of fatty acids in adipose tissue is significantly reduced in late pregnancy (15 days antepartum), compared to days 120 and 240 of lactation, and completely impeded in early lactation [13]. Depressed lipogenesis is mainly attributable to hypoinsulinemia and decreased insulin sensitivity of adipose tissue, i.e. increased insulin resistance.

of niacin in the small intestine, the lipid layers in the pallet are relatively undegradable in the

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111

The pharmacokinetics of orally administered medications in ruminants depends on the form of the medication (rumen-protected vs. not rumen-protected), whereas the pharmacokinetics of niacin is further influenced by two niacin vitamers: nicotinic acid and nicotinamide. The metabolism of nicotinic acid and nicotinamide, which are involved in the biosynthesis of NAD, may differ markedly. Unlike differences found in the concentration of each niacin vitamers [20], there is no difference in the total amount of niacin in the rumen between roughage and concentrate rations in a 40:60 to 60:40 ratio, respectively. Rumen microorganisms also synthesize niacin. The ruminal synthesis of niacin exceeds 2.2 g/d [21]. Increased amounts of nonfibrous carbohydrates facilitate the synthesis of niacin, whereas the roughage-to-concentrate ratio of the diet exerts no effect [22]. The duration of niacin administration greatly affects nicotinic acid concentrations in ruminal and intestinal fluids. One hour after administering niacin which is not rumen-protected, nicotinic acid concentrations in the rumen reach the peak by the conversion of nicotinamide to nicotinic acid or other forms (Campebell et al. [23] failed to detect nicotinamide in the ruminal fluid). The results of Santschi et al. [20] suggest that a considerable portion of both niacin vitamers are synthesized in the rumen, whereas the largest portion of nicotinic acid and the entire portion of nicotinamide are bound within the microbes. Although direct absorption from the rumen is possible, the absorption of niacin from the small intestine appears to be the main route by which niacin is made available to the host. Only 17% of niacin administered is found in the duodenum as free nicotinic acid [23]. According to Santschi et al. [21], as much as 98.5% of niacin is degraded in the rumen of dairy cows. Nicotinic acid concentrations were found to be elevated in the duodenum of nicotinamide-supplemented cows (12 g/d) compared to cows supplemented with nicotinic acid [23]. However, this research focused solely on the analysis of duodenum fluids although niacin can also be found in solid intestinal contents. The niacin administered was not ruminally protected. Owing to the extensive ruminal degradation of niacin, considerably higher doses of niacin were administered to cows (12–36 g/d) in a number of studies [18]. When higher doses of niacin are administered, a surplus of undegraded niacin is more likely to reach the lower parts of the digestive tract. Increased niacin concentrations have been found in the duodenum of niacin-supplemented cows [23, 24]. A loss of niacin occurs even after abomasal infusion, to a lesser extent (approximately 85%), which corroborates the presence of both abomasal and

Duodenal niacin concentrations are essentially dependent on the pharmaceutical form of niacin (rumen-protected vs. not rumen-protected), the amount of niacin available and the roughage-to-concentrate ratio of the diet. According to Niehoff et al. [25], the total amount of niacin (nicotinic acid + nicotinamide) reaching the duodenum increases with an increase in the dietary share of concentrates and nicotinic acid supplements, whereas the amount of nicotinamide is solely dependent on nicotinic acid supplementation. Unsaturated nicotinic acid is of low rumen stability. Santschi et al. [21] reported that unsaturated nicotinic acid has a bioavailability of 5%. The administration of rumen-protected niacin in dairy cows leads to

rumen and thereby prevent the degradation of niacin by rumen microorganisms [19].

duodenal niacin absorptions [21].

### **3. Pharmacokinetics of niacin, NAD and NADP**

Niacin is a vitamin essential to energy metabolism. Physiologically, niacin is incorporated into the coenzyme nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are involved in numerous metabolic processes: (1) anabolic pathways (NADPH/NADP) such as the syntheses of lipids and nucleic acids which require reducing equivalents provided by NADPH and (2) catabolic pathways (NADH/NAD). NAD is involved in a great many oxidation-reduction reactions as an electron carrier capable of accepting and donating electrons. NAD+, the oxidized form of NAD, can accept electrons in the reduction of NAD+ to NADH, whereas it can donate electrons in the oxidation of NADH to NAD+. Moreover, NAD is a source of adenosine diphosphate ribose (ADP-ribose) for protein modification. It is also a precursor of two second messenger molecules (cyclic ADP-ribose and nicotinamide adenine dinucleotide phosphate), which augment intracellular calcium concentrations and have a central role in a number of metabolic pathways. Another physiological effect of nicotinic acid is the potential to suppress lipolysis when administered in higher doses [14, 15]. Niacin is a generic descriptor for two vitamers: nicotinic acid and nicotinamide. Both forms of niacin are nutritionally equivalent and can be used for the synthesis of NAD. However, their biological proportions vary, and only nicotinamide can act as a reactive component [16].

In addition to ingested feed as a niacin source, niacin can also be synthesized in animals by the enzymatic conversion of tryptophan and quinolinic acid to niacin. Furthermore, rumen microorganisms synthesize niacin as well, using aspartates and dihydroxyacetone phosphate [17]. Previous research suggested that dairy cows did not require an exogenous supplementation of vitamin B due to a sufficient supply of this vitamin from the feed ingested and the synthesis of niacin in the rumen. However, milk production in high-yielding dairy cows has significantly increased, accompanied by increased vitamin B requirements. Depressed feed intake in the periparturient period, often continuing long after the onset of lactation, impedes the inflow of feed precursors (which are of immense importance to the ruminal synthesis of niacin), thus further increasing the need for niacin supplementation.

As an oral supplement, niacin can be rumen-protected or not rumen-protected. Niacin supplements which are not ruminally protected are less stable in the rumen and are readily degraded and, thus, should be administered in higher pharmacological doses [18]. The rumen-protected form of niacin is often found encapsulated and is commonly referred to as encapsulated niacin. These products are practically small pallets with niacin placed in the centre and covered by several layers of lipids. As encapsulation enhances the bioavailability of niacin in the small intestine, the lipid layers in the pallet are relatively undegradable in the rumen and thereby prevent the degradation of niacin by rumen microorganisms [19].

in adipose tissue is significantly reduced in late pregnancy (15 days antepartum), compared to days 120 and 240 of lactation, and completely impeded in early lactation [13]. Depressed lipogenesis is mainly attributable to hypoinsulinemia and decreased insulin sensitivity of adi-

Niacin is a vitamin essential to energy metabolism. Physiologically, niacin is incorporated into the coenzyme nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are involved in numerous metabolic processes: (1) anabolic pathways (NADPH/NADP) such as the syntheses of lipids and nucleic acids which require reducing equivalents provided by NADPH and (2) catabolic pathways (NADH/NAD). NAD is involved in a great many oxidation-reduction reactions as an electron carrier capable of accepting and donating electrons. NAD+, the oxidized form of NAD, can accept electrons in the reduction of NAD+ to NADH, whereas it can donate electrons in the oxidation of NADH to NAD+. Moreover, NAD is a source of adenosine diphosphate ribose (ADP-ribose) for protein modification. It is also a precursor of two second messenger molecules (cyclic ADP-ribose and nicotinamide adenine dinucleotide phosphate), which augment intracellular calcium concentrations and have a central role in a number of metabolic pathways. Another physiological effect of nicotinic acid is the potential to suppress lipolysis when administered in higher doses [14, 15]. Niacin is a generic descriptor for two vitamers: nicotinic acid and nicotinamide. Both forms of niacin are nutritionally equivalent and can be used for the synthesis of NAD. However, their biological proportions vary, and only nicotinamide can

In addition to ingested feed as a niacin source, niacin can also be synthesized in animals by the enzymatic conversion of tryptophan and quinolinic acid to niacin. Furthermore, rumen microorganisms synthesize niacin as well, using aspartates and dihydroxyacetone phosphate [17]. Previous research suggested that dairy cows did not require an exogenous supplementation of vitamin B due to a sufficient supply of this vitamin from the feed ingested and the synthesis of niacin in the rumen. However, milk production in high-yielding dairy cows has significantly increased, accompanied by increased vitamin B requirements. Depressed feed intake in the periparturient period, often continuing long after the onset of lactation, impedes the inflow of feed precursors (which are of immense importance to the ruminal synthesis of

As an oral supplement, niacin can be rumen-protected or not rumen-protected. Niacin supplements which are not ruminally protected are less stable in the rumen and are readily degraded and, thus, should be administered in higher pharmacological doses [18]. The rumen-protected form of niacin is often found encapsulated and is commonly referred to as encapsulated niacin. These products are practically small pallets with niacin placed in the centre and covered by several layers of lipids. As encapsulation enhances the bioavailability

niacin), thus further increasing the need for niacin supplementation.

pose tissue, i.e. increased insulin resistance.

110 B Group Vitamins - Current Uses and Perspectives

act as a reactive component [16].

**3. Pharmacokinetics of niacin, NAD and NADP**

The pharmacokinetics of orally administered medications in ruminants depends on the form of the medication (rumen-protected vs. not rumen-protected), whereas the pharmacokinetics of niacin is further influenced by two niacin vitamers: nicotinic acid and nicotinamide. The metabolism of nicotinic acid and nicotinamide, which are involved in the biosynthesis of NAD, may differ markedly. Unlike differences found in the concentration of each niacin vitamers [20], there is no difference in the total amount of niacin in the rumen between roughage and concentrate rations in a 40:60 to 60:40 ratio, respectively. Rumen microorganisms also synthesize niacin. The ruminal synthesis of niacin exceeds 2.2 g/d [21]. Increased amounts of nonfibrous carbohydrates facilitate the synthesis of niacin, whereas the roughage-to-concentrate ratio of the diet exerts no effect [22]. The duration of niacin administration greatly affects nicotinic acid concentrations in ruminal and intestinal fluids. One hour after administering niacin which is not rumen-protected, nicotinic acid concentrations in the rumen reach the peak by the conversion of nicotinamide to nicotinic acid or other forms (Campebell et al. [23] failed to detect nicotinamide in the ruminal fluid). The results of Santschi et al. [20] suggest that a considerable portion of both niacin vitamers are synthesized in the rumen, whereas the largest portion of nicotinic acid and the entire portion of nicotinamide are bound within the microbes.

Although direct absorption from the rumen is possible, the absorption of niacin from the small intestine appears to be the main route by which niacin is made available to the host. Only 17% of niacin administered is found in the duodenum as free nicotinic acid [23]. According to Santschi et al. [21], as much as 98.5% of niacin is degraded in the rumen of dairy cows. Nicotinic acid concentrations were found to be elevated in the duodenum of nicotinamide-supplemented cows (12 g/d) compared to cows supplemented with nicotinic acid [23]. However, this research focused solely on the analysis of duodenum fluids although niacin can also be found in solid intestinal contents. The niacin administered was not ruminally protected. Owing to the extensive ruminal degradation of niacin, considerably higher doses of niacin were administered to cows (12–36 g/d) in a number of studies [18]. When higher doses of niacin are administered, a surplus of undegraded niacin is more likely to reach the lower parts of the digestive tract. Increased niacin concentrations have been found in the duodenum of niacin-supplemented cows [23, 24]. A loss of niacin occurs even after abomasal infusion, to a lesser extent (approximately 85%), which corroborates the presence of both abomasal and duodenal niacin absorptions [21].

Duodenal niacin concentrations are essentially dependent on the pharmaceutical form of niacin (rumen-protected vs. not rumen-protected), the amount of niacin available and the roughage-to-concentrate ratio of the diet. According to Niehoff et al. [25], the total amount of niacin (nicotinic acid + nicotinamide) reaching the duodenum increases with an increase in the dietary share of concentrates and nicotinic acid supplements, whereas the amount of nicotinamide is solely dependent on nicotinic acid supplementation. Unsaturated nicotinic acid is of low rumen stability. Santschi et al. [21] reported that unsaturated nicotinic acid has a bioavailability of 5%. The administration of rumen-protected niacin in dairy cows leads to augmented free niacin concentrations in the blood [19]. Morey et al. argue that encapsulated niacin treatments increase plasma nicotinamide concentrations (24 g/d of encapsulated niacin provides 9.6 g/d of bioavailable niacin).

Our results suggest [28] that blood NAD and NADP concentrations are a sensitive indicator of the niacin status of cows. The NAD concentrations obtained ranged from 860 to 895 pmol/mL in the control group in the weeks before and after calving. In niacin-supplemented cows, the following NAD concentrations were obtained: 1724.6 pmol/L in the week of calving (week 0), 1968.6 pmol/mL in the first week after calving and 1771.8 pmol/L in the second week after calving. The NADP concentrations obtained in the control group ranged from 385.09 to 425.62 pmol/ mL during the entire period under consideration. In niacin-supplemented cows, the following NADP concentrations were obtained: 704.45 pmol/L in the week of calving (week 0), 778.36 pmol/L in the first week after calving and 796.18 pmol/L in the second week after calving.

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**4. Effects of niacin administration on lipolysis, ketogenesis and** 

cles. Depressed feed intake and metabolic changes subsequently ensue.

acid can suppress the release of fat from adipose tissue [14].

NEFAs are the major component of triglycerides (the fat stores in the body), which consist of three fatty acids linked to glycerol. The hydrolysis of stored triglycerides (fat) in adipose tissue by hormone-sensitive lipase liberates NEFAs and glycerol. Plasma NEFA concentrations are elevated in periparturient dairy cows. Accordingly, cows mobilize fatty acids from adipose tissue as a means of adapting to a number of metabolic changes and a negative energy balance in the periparturient period. The large influx of NEFAs into the liver exceeds its fatty acid oxidation capacity and results in storing NEFAs as triglycerides in hepatocytes and mus-

In their review paper on the administration of niacin which is not rumen-protected, Niehoff et al. [3] argue that nicotinic acid can decrease NEFA concentrations under certain conditions, whereas nicotinamide does not exert the same effect. When the effect of nicotinic acid is minimized, a rebound of NEFAs above basal values occurs, followed by a return to normal concentrations. To induce these effects, the amount of niacin reaching the duodenum should be large, which can be achieved by high-dose niacin supplementation. High doses of nicotinic

GPR109A (HM74A in humans and PUMA-G in mice) is a G-protein-coupled receptor for nicotinic acid, which has been shown to mediate the nicotinic acid-induced antilipolytic effects [15, 29]. The high-affinity receptor for nicotinic acid HM74A enhances the therapeutic effect of nicotinic acid by inhibiting adenylyl cyclase and reducing the intracellular level of cAMP in adipocytes. In vivo studies suggest that administering pharmacological doses of nicotinic acid decrease plasma NEFA concentrations by inhibiting lipolysis in cattle [14, 26, 30]. This antilipolytic potential of nicotinic acid is most likely realized by the activation of GPR109A [31–35]. The GPR109A antilipolytic pathway, already described in other mammal species, has only recently been shown to exist in a functioning form in bovine tissues under in vitro conditions. Conversely, nicotinamide has a low affinity for binding to GPR109A. The activation of GPR109A by nicotinic acid results in decreased cellular cAMP concentrations and the inhibition of adenyl cyclase. Decreased cAMP concentrations in adipocytes lead to the inactivation of protein kinase A and decreased phosphorylation of hormone-sensitive lipase, thus inducing

**oxidative stress**

The intestinal absorption of nicotinic acid and nicotinamide approximates to 73% and 94%, respectively, with an overall average niacin absorption of 84% from the duodenum [21]. Nicotinic acid is mostly absorbed from the duodenum. The intestinal mucosa is rich in niacin conversion enzymes such as NAD glycohydrolase. It is highly unlikely that nicotinic acid is directly converted to nicotinamide. However, nicotinic acid is readily converted to NAD in the intestinal mucosa, and excessive amounts of NAD are subsequently hydrolyzed to nicotinamide by NAD+ glycohydrolase [17]. NAD+ glycohydrolase is an enzyme that catalyzes the hydrolysis of NAD+ to produce ADP-ribose and nicotinamide [26]. Morey et al. [26] found that plasma nicotinamide concentrations decreased in niacin-treated cows 50 h after niacin administration but still exceeded those of control animals. Nicotinamide is the primary circulating form of niacin and is converted into its coenzyme forms (NAD and NADP) in the tissues. The transport of niacin in the blood is mainly associated with erythrocytes. Niacin rapidly leaves the blood stream and enters the kidney, liver and adipose tissue. There is a considerable dispute over the presence of nicotinic acid in the blood. Therefore, nicotinamide is considered the primary circulating form of niacin [17]. Nicotinic acid, which is not metabolized by the liver, can be transported to different tissues in the body by administering higher pharmacological doses of niacin.

Nicotinamide is a reactive part of NAD and NADP, which are involved in numerous oxidation-reduction reactions as coenzymes, i.e. cofactors. Enzymes containing NAD and NADP are important links in a series of reactions associated with carbohydrate, protein and lipid metabolism [27]. NAD and NADP act as an intermediate in most of the H+ transfers in metabolism, including more than 200 reactions in the metabolism of carbohydrates, fatty acids and amino acids. The most important metabolic reactions catalyzed by NAD and NADP are summarized as follows: carbohydrate metabolism (glycolysis, i.e. the anaerobic and aerobic oxidation of glucose, and the TCA (Krebs) cycle), lipid metabolism (the synthesis and breakdown of glycerol, the oxidation and synthesis of fatty acids and the synthesis of steroids) and protein metabolism (the degradation and synthesis of amino acids and the oxidation of carbon chains via the TCA cycle). The NAD and NADP coenzymes can be synthesized from niacin vitamers, tryptophan and quinolinic acid. The primary function of the liver is to synthesize NADP from tryptophan by hydrolysis in order to release niacin for its use in extrahepatic tissues. The brain, muscles and, to a lesser extent, testicles can take up nicotinamide from the bloodstream and utilize it without the previous deaminization. The nicotinamide nucleotide coenzymes are catabolized from four enzymes: NAD pyrophosphatase, NAD glycohydrolase, ADPribosyltransferase and poly (ADP-ribose) polymerase. Under normal conditions, there is little or no urinary excretion of either nicotinamide or nicotinic acid as both vitamers are actively reabsorbed from the glomerular filtrate. Such excretion only occurs when nicotinamide and/ or nicotinic acid concentrations are so high that the transport mechanism is saturated. N1 - Methylnicotinamide and N-methyl-2-pyridone-5-carboxamide are the two principal urinary metabolites of nicotinamide in humans, rats and pigs. In herbivores, niacin is seemingly not metabolized by methylation but is mostly excreted unchanged.

Our results suggest [28] that blood NAD and NADP concentrations are a sensitive indicator of the niacin status of cows. The NAD concentrations obtained ranged from 860 to 895 pmol/mL in the control group in the weeks before and after calving. In niacin-supplemented cows, the following NAD concentrations were obtained: 1724.6 pmol/L in the week of calving (week 0), 1968.6 pmol/mL in the first week after calving and 1771.8 pmol/L in the second week after calving. The NADP concentrations obtained in the control group ranged from 385.09 to 425.62 pmol/ mL during the entire period under consideration. In niacin-supplemented cows, the following NADP concentrations were obtained: 704.45 pmol/L in the week of calving (week 0), 778.36 pmol/L in the first week after calving and 796.18 pmol/L in the second week after calving.

### **4. Effects of niacin administration on lipolysis, ketogenesis and oxidative stress**

augmented free niacin concentrations in the blood [19]. Morey et al. argue that encapsulated niacin treatments increase plasma nicotinamide concentrations (24 g/d of encapsulated niacin

The intestinal absorption of nicotinic acid and nicotinamide approximates to 73% and 94%, respectively, with an overall average niacin absorption of 84% from the duodenum [21]. Nicotinic acid is mostly absorbed from the duodenum. The intestinal mucosa is rich in niacin conversion enzymes such as NAD glycohydrolase. It is highly unlikely that nicotinic acid is directly converted to nicotinamide. However, nicotinic acid is readily converted to NAD in the intestinal mucosa, and excessive amounts of NAD are subsequently hydrolyzed to nicotinamide by NAD+ glycohydrolase [17]. NAD+ glycohydrolase is an enzyme that catalyzes the hydrolysis of NAD+ to produce ADP-ribose and nicotinamide [26]. Morey et al. [26] found that plasma nicotinamide concentrations decreased in niacin-treated cows 50 h after niacin administration but still exceeded those of control animals. Nicotinamide is the primary circulating form of niacin and is converted into its coenzyme forms (NAD and NADP) in the tissues. The transport of niacin in the blood is mainly associated with erythrocytes. Niacin rapidly leaves the blood stream and enters the kidney, liver and adipose tissue. There is a considerable dispute over the presence of nicotinic acid in the blood. Therefore, nicotinamide is considered the primary circulating form of niacin [17]. Nicotinic acid, which is not metabolized by the liver, can be transported to different tissues in the body by administering higher

Nicotinamide is a reactive part of NAD and NADP, which are involved in numerous oxidation-reduction reactions as coenzymes, i.e. cofactors. Enzymes containing NAD and NADP are important links in a series of reactions associated with carbohydrate, protein and lipid metabolism [27]. NAD and NADP act as an intermediate in most of the H+ transfers in metabolism, including more than 200 reactions in the metabolism of carbohydrates, fatty acids and amino acids. The most important metabolic reactions catalyzed by NAD and NADP are summarized as follows: carbohydrate metabolism (glycolysis, i.e. the anaerobic and aerobic oxidation of glucose, and the TCA (Krebs) cycle), lipid metabolism (the synthesis and breakdown of glycerol, the oxidation and synthesis of fatty acids and the synthesis of steroids) and protein metabolism (the degradation and synthesis of amino acids and the oxidation of carbon chains via the TCA cycle). The NAD and NADP coenzymes can be synthesized from niacin vitamers, tryptophan and quinolinic acid. The primary function of the liver is to synthesize NADP from tryptophan by hydrolysis in order to release niacin for its use in extrahepatic tissues. The brain, muscles and, to a lesser extent, testicles can take up nicotinamide from the bloodstream and utilize it without the previous deaminization. The nicotinamide nucleotide coenzymes are catabolized from four enzymes: NAD pyrophosphatase, NAD glycohydrolase, ADPribosyltransferase and poly (ADP-ribose) polymerase. Under normal conditions, there is little or no urinary excretion of either nicotinamide or nicotinic acid as both vitamers are actively reabsorbed from the glomerular filtrate. Such excretion only occurs when nicotinamide and/ or nicotinic acid concentrations are so high that the transport mechanism is saturated. N1

Methylnicotinamide and N-methyl-2-pyridone-5-carboxamide are the two principal urinary metabolites of nicotinamide in humans, rats and pigs. In herbivores, niacin is seemingly not

metabolized by methylation but is mostly excreted unchanged.


provides 9.6 g/d of bioavailable niacin).

112 B Group Vitamins - Current Uses and Perspectives

pharmacological doses of niacin.

NEFAs are the major component of triglycerides (the fat stores in the body), which consist of three fatty acids linked to glycerol. The hydrolysis of stored triglycerides (fat) in adipose tissue by hormone-sensitive lipase liberates NEFAs and glycerol. Plasma NEFA concentrations are elevated in periparturient dairy cows. Accordingly, cows mobilize fatty acids from adipose tissue as a means of adapting to a number of metabolic changes and a negative energy balance in the periparturient period. The large influx of NEFAs into the liver exceeds its fatty acid oxidation capacity and results in storing NEFAs as triglycerides in hepatocytes and muscles. Depressed feed intake and metabolic changes subsequently ensue.

In their review paper on the administration of niacin which is not rumen-protected, Niehoff et al. [3] argue that nicotinic acid can decrease NEFA concentrations under certain conditions, whereas nicotinamide does not exert the same effect. When the effect of nicotinic acid is minimized, a rebound of NEFAs above basal values occurs, followed by a return to normal concentrations. To induce these effects, the amount of niacin reaching the duodenum should be large, which can be achieved by high-dose niacin supplementation. High doses of nicotinic acid can suppress the release of fat from adipose tissue [14].

GPR109A (HM74A in humans and PUMA-G in mice) is a G-protein-coupled receptor for nicotinic acid, which has been shown to mediate the nicotinic acid-induced antilipolytic effects [15, 29]. The high-affinity receptor for nicotinic acid HM74A enhances the therapeutic effect of nicotinic acid by inhibiting adenylyl cyclase and reducing the intracellular level of cAMP in adipocytes. In vivo studies suggest that administering pharmacological doses of nicotinic acid decrease plasma NEFA concentrations by inhibiting lipolysis in cattle [14, 26, 30]. This antilipolytic potential of nicotinic acid is most likely realized by the activation of GPR109A [31–35]. The GPR109A antilipolytic pathway, already described in other mammal species, has only recently been shown to exist in a functioning form in bovine tissues under in vitro conditions. Conversely, nicotinamide has a low affinity for binding to GPR109A. The activation of GPR109A by nicotinic acid results in decreased cellular cAMP concentrations and the inhibition of adenyl cyclase. Decreased cAMP concentrations in adipocytes lead to the inactivation of protein kinase A and decreased phosphorylation of hormone-sensitive lipase, thus inducing a reduction of lypolysis. The GPR109A receptors are found primarily in adipose tissue and immune cells, as well as in the brain, liver and muscles of cattle. BHB is the endogenous ligand of the human GPR109A, whereas nicotinamide acts as a very weak agonist at GPR109A producing no alterations in plasma lipoprotein profiles. Nicotinic acid, nicotinamide and BHB, as the ligands of the cattle GPR109A, exhibit different levels of efficiency in the induced antilipolysis under in vitro conditions. Nicotinic acid decreases the phosphorylation of hormonesensitive lipase, thereby reducing the lipolytic response. However, nicotinamide does not exert a suppressing impact on the lipolytic activity in bovine tissues under in vitro conditions, whereas only extremely high BHB concentrations can significantly reduce the release of glycerol and phosphorylation of hormone-sensitive lipase.

marked decrease in plasma BHB concentrations was recorded in cows supplemented with 12 g/d of niacin (in a crystal powder form), whereas a slighter decrease in plasma BHB con-

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In addition to lipolysis and ketogenesis, niacin exerts a major effect on lipid peroxidation and oxidative stress. Oxidative stress occurs when excess prooxidants (free radicals) overwhelm the antioxidant capacity of the organism. Such a state is associated with metabolic stress in periparturient cows [43]. It most commonly occurs when there is an imbalance between the increased production of free radicals and the decreased ability of the organism to neutralize them. Oxidation is part of the biochemical regulatory processes of the organism responsible for generating the energy required to sustain life. During these processes, free radicals are formed, having positive physiological functions. However, a physiological imbalance between excess free radicals and the ability of the organism to neutralize them changes the oxidative status of the organism, which thereafter enters a state of real oxidative stress (conducive to a number of various disorders and diseases). The degree of oxidative stress is determined by measuring the concentration and activity of prooxidants and antioxidants. Prooxidants are reactive oxygen metabolites containing an unpaired electron in the outermost electron shell, thereby participating in oxidation-reduction reactions. These reactive molecules can integrate into genetic and/or

anatomical cell structures, inducing significant changes in cellular function [44, 45].

and TBARS concentrations and NEFA and BHB concentrations [46, 47].

The reactive molecules most essential to periparturient cows are formed in the process of increased lipid mobilization. Nonesterified fatty acids are fairly reactive molecules susceptible to oxidation and free radical reactions. Fats are considered the best indicator of oxidative stress. Malondialdehyde (MDA) results from the reaction between free radicals and polyunsaturated fatty acids. It readily reacts with thiobarbituric acid to form thiobarbituric acid reactive substances (TBARS). MDA and/or TBARS concentrations are significantly increased in dairy cows after parturition. Moreover, a positive correlation has been found between MDA

In ruminants, antioxidants are divided into three major categories: (1) intracellular antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), (2) nonenzymatic protein antioxidants in plasma such as protein thiol groups in albumin and (3) nonenzymatic low-molecular-weight antioxidants such as glutathione, alpha-tocopherol, beta-carotene, etc. The antioxidant capacity of the cow's body is greatly dependent on the energy balance of the body. Therefore, ketotic cows, due to a negative energy balance, have been found to exhibit decreased antioxidant activity and increased concentrations of reactive lipid molecules. Antioxidant protection is also influenced by the diet and milk yield of cows. Calving has a significant impact on the antioxidant system of the cow, leading to a decrease in antioxidant concentrations in early lactation [15, 17, 18]. Niacin has been shown to exert the following antioxidant effects: decreasing lipolysis and lipid peroxidation, participating in the conversion of oxidized glutathione (GSSG) to the reduced form (GSH) by glutathione reductase (GR), decreasing the NADH+H+/NADP+ ratio and increasing the NAD+ content. As previously mentioned herein, niacin administration increases NAD concentrations [27].

In the study of Hristovska et al. [48], niacin-supplemented cows were found to exhibit lower NEFA concentrations in the periparturient period. NEFA concentrations remained invariant

centrations was found in cows receiving 6 g/d of niacin [42].

Pires and Grummer [14] administered abomasal infusions of nicotinic acid (0, 6, 30 or 60 mg of NA/kg of body weight (BW)) to feed-restricted Holstein cows as a single bolus 48 h after the initiation of feed restriction. Plasma NEFA concentrations decreased from 546 to 208 ± 141 μEq/L at 1 h after the infusion of 6 mg of NA/kg of BW and to less than 100 ± 148 μEq/L at 3 h after the abomasal infusion of the two highest doses of NA. Upon the termination of NA infusions, a rebound occurred following the initial decrease of plasma NEFA concentrations. The rebound lasted up to 9 h for the 30 mg dose of NA and up to 6 h for the 6 mg dose. On balance, nicotinic acid was shown to be a potent antilipolytic agent in feed-restricted cattle with a negative energy balance. Sustained reductions in plasma NEFA concentrations are achieved as long as there is a constant supply of nicotinic acid to the lower parts of the digestive tract. The antilipolytic effect of nicotinic acid may be favourable to dairy cows provided that niacin is administered in optimal doses and forms, accompanied by a postruminal source of nicotinic acid. Nevertheless, the optimal dose of nicotinic acid should be determined, exerting a moderately inhibiting effect on lipolysis and NEFAs (adipose tissue NEFAs are an important energy source and precursors for the synthesis of fatty acids at the onset of lactation). In their study on the administration of rumen-protected niacin, Morey et al. [26] found that 24 g/d of encapsulated niacin (providing 9.6 g/d of bioavailable niacin) inhibited lipolysis in postpartum cows by decreasing postpartum NEFA concentrations. The treatment protocols used in this study are unequivocally associated with suppressing lipolysis in cattle, causing no rebound lipolysis. A total of 24 g/d of encapsulated niacin provides a source of bioavailable niacin which modifies lipid metabolism [36].

Notwithstanding the large influx of NEFAs into hepatocytes of early-lactation cows, decreased triglyceride concentrations were found in the liver of cows supplemented with rumen-protected niacin. As the accumulation of hepatic triglycerides is directly related to blood NEFA concentrations, reductions in blood NEFA concentrations lead to decreased triglyceride accumulation in postpartum niacin-supplemented cows [36]. In addition to fatty liver, the occurrence of ketosis is another negative consequence of elevated NEFA concentrations, i.e. the incomplete metabolism of NEFAs which are converted to ketone bodies. To prevent ketosis, cows should be supplemented with niacin alongside glycogen precursors such as propylene glycol and sodium propionate [37, 38]. The previous research suggests that niacin supplementation decreases plasma BHBA and NEFA concentrations with an increase in serum glucose [39, 40]. Erickson et al. [41] report significant effects of niacin on plasma BHB concentrations in niacin-supplemented cows compared to control animals. Relative to the control group, a marked decrease in plasma BHB concentrations was recorded in cows supplemented with 12 g/d of niacin (in a crystal powder form), whereas a slighter decrease in plasma BHB concentrations was found in cows receiving 6 g/d of niacin [42].

a reduction of lypolysis. The GPR109A receptors are found primarily in adipose tissue and immune cells, as well as in the brain, liver and muscles of cattle. BHB is the endogenous ligand of the human GPR109A, whereas nicotinamide acts as a very weak agonist at GPR109A producing no alterations in plasma lipoprotein profiles. Nicotinic acid, nicotinamide and BHB, as the ligands of the cattle GPR109A, exhibit different levels of efficiency in the induced antilipolysis under in vitro conditions. Nicotinic acid decreases the phosphorylation of hormonesensitive lipase, thereby reducing the lipolytic response. However, nicotinamide does not exert a suppressing impact on the lipolytic activity in bovine tissues under in vitro conditions, whereas only extremely high BHB concentrations can significantly reduce the release of glyc-

Pires and Grummer [14] administered abomasal infusions of nicotinic acid (0, 6, 30 or 60 mg of NA/kg of body weight (BW)) to feed-restricted Holstein cows as a single bolus 48 h after the initiation of feed restriction. Plasma NEFA concentrations decreased from 546 to 208 ± 141 μEq/L at 1 h after the infusion of 6 mg of NA/kg of BW and to less than 100 ± 148 μEq/L at 3 h after the abomasal infusion of the two highest doses of NA. Upon the termination of NA infusions, a rebound occurred following the initial decrease of plasma NEFA concentrations. The rebound lasted up to 9 h for the 30 mg dose of NA and up to 6 h for the 6 mg dose. On balance, nicotinic acid was shown to be a potent antilipolytic agent in feed-restricted cattle with a negative energy balance. Sustained reductions in plasma NEFA concentrations are achieved as long as there is a constant supply of nicotinic acid to the lower parts of the digestive tract. The antilipolytic effect of nicotinic acid may be favourable to dairy cows provided that niacin is administered in optimal doses and forms, accompanied by a postruminal source of nicotinic acid. Nevertheless, the optimal dose of nicotinic acid should be determined, exerting a moderately inhibiting effect on lipolysis and NEFAs (adipose tissue NEFAs are an important energy source and precursors for the synthesis of fatty acids at the onset of lactation). In their study on the administration of rumen-protected niacin, Morey et al. [26] found that 24 g/d of encapsulated niacin (providing 9.6 g/d of bioavailable niacin) inhibited lipolysis in postpartum cows by decreasing postpartum NEFA concentrations. The treatment protocols used in this study are unequivocally associated with suppressing lipolysis in cattle, causing no rebound lipolysis. A total of 24 g/d of encapsulated niacin provides a source of bioavailable

Notwithstanding the large influx of NEFAs into hepatocytes of early-lactation cows, decreased triglyceride concentrations were found in the liver of cows supplemented with rumen-protected niacin. As the accumulation of hepatic triglycerides is directly related to blood NEFA concentrations, reductions in blood NEFA concentrations lead to decreased triglyceride accumulation in postpartum niacin-supplemented cows [36]. In addition to fatty liver, the occurrence of ketosis is another negative consequence of elevated NEFA concentrations, i.e. the incomplete metabolism of NEFAs which are converted to ketone bodies. To prevent ketosis, cows should be supplemented with niacin alongside glycogen precursors such as propylene glycol and sodium propionate [37, 38]. The previous research suggests that niacin supplementation decreases plasma BHBA and NEFA concentrations with an increase in serum glucose [39, 40]. Erickson et al. [41] report significant effects of niacin on plasma BHB concentrations in niacin-supplemented cows compared to control animals. Relative to the control group, a

erol and phosphorylation of hormone-sensitive lipase.

114 B Group Vitamins - Current Uses and Perspectives

niacin which modifies lipid metabolism [36].

In addition to lipolysis and ketogenesis, niacin exerts a major effect on lipid peroxidation and oxidative stress. Oxidative stress occurs when excess prooxidants (free radicals) overwhelm the antioxidant capacity of the organism. Such a state is associated with metabolic stress in periparturient cows [43]. It most commonly occurs when there is an imbalance between the increased production of free radicals and the decreased ability of the organism to neutralize them. Oxidation is part of the biochemical regulatory processes of the organism responsible for generating the energy required to sustain life. During these processes, free radicals are formed, having positive physiological functions. However, a physiological imbalance between excess free radicals and the ability of the organism to neutralize them changes the oxidative status of the organism, which thereafter enters a state of real oxidative stress (conducive to a number of various disorders and diseases). The degree of oxidative stress is determined by measuring the concentration and activity of prooxidants and antioxidants. Prooxidants are reactive oxygen metabolites containing an unpaired electron in the outermost electron shell, thereby participating in oxidation-reduction reactions. These reactive molecules can integrate into genetic and/or anatomical cell structures, inducing significant changes in cellular function [44, 45].

The reactive molecules most essential to periparturient cows are formed in the process of increased lipid mobilization. Nonesterified fatty acids are fairly reactive molecules susceptible to oxidation and free radical reactions. Fats are considered the best indicator of oxidative stress. Malondialdehyde (MDA) results from the reaction between free radicals and polyunsaturated fatty acids. It readily reacts with thiobarbituric acid to form thiobarbituric acid reactive substances (TBARS). MDA and/or TBARS concentrations are significantly increased in dairy cows after parturition. Moreover, a positive correlation has been found between MDA and TBARS concentrations and NEFA and BHB concentrations [46, 47].

In ruminants, antioxidants are divided into three major categories: (1) intracellular antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), (2) nonenzymatic protein antioxidants in plasma such as protein thiol groups in albumin and (3) nonenzymatic low-molecular-weight antioxidants such as glutathione, alpha-tocopherol, beta-carotene, etc. The antioxidant capacity of the cow's body is greatly dependent on the energy balance of the body. Therefore, ketotic cows, due to a negative energy balance, have been found to exhibit decreased antioxidant activity and increased concentrations of reactive lipid molecules. Antioxidant protection is also influenced by the diet and milk yield of cows. Calving has a significant impact on the antioxidant system of the cow, leading to a decrease in antioxidant concentrations in early lactation [15, 17, 18]. Niacin has been shown to exert the following antioxidant effects: decreasing lipolysis and lipid peroxidation, participating in the conversion of oxidized glutathione (GSSG) to the reduced form (GSH) by glutathione reductase (GR), decreasing the NADH+H+/NADP+ ratio and increasing the NAD+ content. As previously mentioned herein, niacin administration increases NAD concentrations [27].

In the study of Hristovska et al. [48], niacin-supplemented cows were found to exhibit lower NEFA concentrations in the periparturient period. NEFA concentrations remained invariant in the niacin group within the first 2 weeks after calving, whereas a significant increase in NEFA concentrations was recorded in the control group in the same period. Niacin administration exerted a significant effect on metabolic adaptations in early-lactation cows. Cows supplemented with niacin exhibited considerably lower BHB concentrations, higher cholesterol and triglyceride concentrations, lower MDA concentrations, higher glucose concentrations, lower total bilirubin concentrations, lower liver enzyme activity (AST, ALP and GGT), higher albumin concentrations and lower urea and phosphorus concentrations. As the magnitude of lipolysis increases, niacin administration greatly reduces ketogenesis and lipid peroxidation. Niacin also exerts a substantial impact on the relationship between NEFA concentrations and other metabolic parameters; thus, a weak regression relationship was found between NEFA values and glucose, cholesterol, triglycerides, total bilirubin, AST, albumin, urea and phosphorus values. Niacin reduces the dependence of metabolic adaptations in early-lactation cows on the degree of lipid mobilization. Furthermore, niacin administration to periparturient cows positively affects lipid metabolism in early lactation, i.e. decreased lipid mobilization (decreased NEFA concentrations), ketogenesis (decreased BHB concentrations) and liver lipidosis (higher triglyceride and cholesterol concentrations in the blood and higher cholesterol concentrations per unit NEFA) [49].

Increased glucose concentrations were recorded during a rebound of plasma NEFA concentrations (upon the initial decrease), whereas insulin concentrations followed a similar pattern to that of the NEFA rebound [14]. Pires et al. [14] argue that decreased NEFA concentrations in feed-restricted Holstein cows infused with nicotinic acid enhance the insulin response and glucose uptake with an increase in insulin sensitivity (suggesting that blood NEFA concentrations are a relevant factor in the occurrence of insulin resistance in dairy cows with a negative energy balance). These results are consistent with the results obtained in a study involving human subjects infused with acipimox (a long-acting nicotinic acid analogue). Acipimox was shown to decrease blood NEFA concentrations, increase the response to the oral glucose tolerance test and enhance the insulin-stimulated glucose uptake in peripheral tissue (using the

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Niacin has been shown to greatly affect glucose concentrations. An increase in glucose concentrations is dependent upon the niacin dose administered and treatment duration. Pescara et al. [55] claim that the mechanism by which nicotinic acid increases plasma glucose concentrations is unelucidated, but it may be attributable to the increased hepatic production of glucose or reduced blood glucose clearance or both. Blood insulin concentrations followed a similar dynamic pattern to that of blood glucose concentrations. An increase in glucose concentrations was recorded on days 10 and 12 of nicotinic acid infusions, continuing 1 day after treatment termination, whereas blood insulin concentrations increased during the entire treatment process [30]. According to Titgemeyer et al. [30], it is inconclusive whether an increase in glucose concentrations leads to an increase in insulin concentrations or insulin resistance causes elevated glucose concentrations. Their model is at variance with those stating that increased NEFA concentrations are associated with insulin resistance during nicotinic acid treatments. Differences in the results obtained can partially be accounted for by different energy supplies and degrees of lipolysis, indicating that both insulin and glucose concentrations in the blood are affected by niacin. Titgemeyer et al. [30] also found that glucagon concentrations were not significantly altered, inferring that glucagon was of little or no sig-

One of our studies has hypothesized that niacin administration to dairy cows in the transition period can influence insulin responsiveness and resistance in adipose tissue by virtue of niacin-induced changes in NEFA, glucose and insulin concentrations [56]. A total of 30 clinically healthy, multiparous Holstein-Friesian cows in late gestation were enrolled in the study. Insulin resistance was calculated on the basis of the following insulin resistance indicators: the glucose-to-insulin (G:I) ratio and the Revised Quantitative Insulin Sensitivity Check Index (RQUICKI). The formula for the glucose-to-insulin ratio is as follows: G:I = glucose (mg/dL)/insulin (μU/ml). The RQUICKI is calculated on the basis of plasma concentrations of glucose (mg/dl), insulin (μU/ml) and free fatty acids (mmol/l), using the following formula: RQUICKI = 1/[log (glucose mg/dL) + log (insulin μU/ml) + log (NEFA mmol/l)]. The RQUICKI is a good indicator of insulin resistance in dairy cows. Although lipolysis-dependent, the RQUICKI correlates with numerous metabolic parameters [57, 58]. The influence of niacin supplementation, in the week of calving and the first week after parturition, on glucose, insulin and NEFA concentrations, as well as RQUICKI values, was analyzed using the analysis of variance (ANOVA). According to the RQUICKI values obtained, niacin-supplemented and

hyperinsulinemic-euglycemic clamp technique) [53, 54].

nificance to the effect of niacin on blood glucose concentrations.

### **5. Effects of niacin administration on insulin resistance**

Insulin resistance is a state of reduced biological effect of insulin, leading to a compensatory increase in insulin concentrations [12]. It is associated with a diminished insulin response to glucose, i.e. insulin hyporesponsiveness (reduced beta cell function) and/or insulin sensitivity (depressed insulin-regulated glucose uptake in tissues). From the receptor's perspective, insulin resistance is referred to as pre-receptor (decreased insulin secretion and/or increased insulin degradation), receptor (a decreased number of receptors and/or their affinity for binding insulin) and post-receptor (defects in cell signalling and translocating glucose transporters. Insulin resistance in periparturient cows is attributed to the primary glucose requirements essential for foetal growth, udder development and lactation. In Holstein cows, insulin resistance is further influenced by plasma NEFA concentrations.

As niacin decreases lipolysis and increases glycaemia, it can facilitate insulin production and efficiency, as well as reduce insulin resistance. Some studies have failed to show significant effects of either niacin treatments or niacin treatment duration on blood glucose in cows receiving either rumen-protected or not rumen-protected niacin [26, 36, 50, 51]. Тhornton and Schultz [52] reported the following changes in the metabolism of glucose in ruminants upon administering pharmacological doses of nicotinic acid: increased plasma glucose and insulin concentrations, reduced tolerance to glucose and reduced insulin resistance. Di Costanzo et al. [18] found a significant increase in blood glucose concentrations in cows supplemented with 36 g/d of nicotinic acid. Such an increase in blood glucose concentrations can enhance the cellular gluconeogenic activity, induced by the partial suppression of lipogenesis. Feedrestricted cows, abomasally infused with pharmacological doses of nicotinic acid, were found to exhibit elevated insulin concentrations 4–8 h after the termination of NA infusions. Increased glucose concentrations were recorded during a rebound of plasma NEFA concentrations (upon the initial decrease), whereas insulin concentrations followed a similar pattern to that of the NEFA rebound [14]. Pires et al. [14] argue that decreased NEFA concentrations in feed-restricted Holstein cows infused with nicotinic acid enhance the insulin response and glucose uptake with an increase in insulin sensitivity (suggesting that blood NEFA concentrations are a relevant factor in the occurrence of insulin resistance in dairy cows with a negative energy balance). These results are consistent with the results obtained in a study involving human subjects infused with acipimox (a long-acting nicotinic acid analogue). Acipimox was shown to decrease blood NEFA concentrations, increase the response to the oral glucose tolerance test and enhance the insulin-stimulated glucose uptake in peripheral tissue (using the hyperinsulinemic-euglycemic clamp technique) [53, 54].

in the niacin group within the first 2 weeks after calving, whereas a significant increase in NEFA concentrations was recorded in the control group in the same period. Niacin administration exerted a significant effect on metabolic adaptations in early-lactation cows. Cows supplemented with niacin exhibited considerably lower BHB concentrations, higher cholesterol and triglyceride concentrations, lower MDA concentrations, higher glucose concentrations, lower total bilirubin concentrations, lower liver enzyme activity (AST, ALP and GGT), higher albumin concentrations and lower urea and phosphorus concentrations. As the magnitude of lipolysis increases, niacin administration greatly reduces ketogenesis and lipid peroxidation. Niacin also exerts a substantial impact on the relationship between NEFA concentrations and other metabolic parameters; thus, a weak regression relationship was found between NEFA values and glucose, cholesterol, triglycerides, total bilirubin, AST, albumin, urea and phosphorus values. Niacin reduces the dependence of metabolic adaptations in early-lactation cows on the degree of lipid mobilization. Furthermore, niacin administration to periparturient cows positively affects lipid metabolism in early lactation, i.e. decreased lipid mobilization (decreased NEFA concentrations), ketogenesis (decreased BHB concentrations) and liver lipidosis (higher triglyceride and cholesterol concentrations in the blood and higher choles-

Insulin resistance is a state of reduced biological effect of insulin, leading to a compensatory increase in insulin concentrations [12]. It is associated with a diminished insulin response to glucose, i.e. insulin hyporesponsiveness (reduced beta cell function) and/or insulin sensitivity (depressed insulin-regulated glucose uptake in tissues). From the receptor's perspective, insulin resistance is referred to as pre-receptor (decreased insulin secretion and/or increased insulin degradation), receptor (a decreased number of receptors and/or their affinity for binding insulin) and post-receptor (defects in cell signalling and translocating glucose transporters. Insulin resistance in periparturient cows is attributed to the primary glucose requirements essential for foetal growth, udder development and lactation. In Holstein cows, insulin

As niacin decreases lipolysis and increases glycaemia, it can facilitate insulin production and efficiency, as well as reduce insulin resistance. Some studies have failed to show significant effects of either niacin treatments or niacin treatment duration on blood glucose in cows receiving either rumen-protected or not rumen-protected niacin [26, 36, 50, 51]. Тhornton and Schultz [52] reported the following changes in the metabolism of glucose in ruminants upon administering pharmacological doses of nicotinic acid: increased plasma glucose and insulin concentrations, reduced tolerance to glucose and reduced insulin resistance. Di Costanzo et al. [18] found a significant increase in blood glucose concentrations in cows supplemented with 36 g/d of nicotinic acid. Such an increase in blood glucose concentrations can enhance the cellular gluconeogenic activity, induced by the partial suppression of lipogenesis. Feedrestricted cows, abomasally infused with pharmacological doses of nicotinic acid, were found to exhibit elevated insulin concentrations 4–8 h after the termination of NA infusions.

terol concentrations per unit NEFA) [49].

116 B Group Vitamins - Current Uses and Perspectives

**5. Effects of niacin administration on insulin resistance**

resistance is further influenced by plasma NEFA concentrations.

Niacin has been shown to greatly affect glucose concentrations. An increase in glucose concentrations is dependent upon the niacin dose administered and treatment duration. Pescara et al. [55] claim that the mechanism by which nicotinic acid increases plasma glucose concentrations is unelucidated, but it may be attributable to the increased hepatic production of glucose or reduced blood glucose clearance or both. Blood insulin concentrations followed a similar dynamic pattern to that of blood glucose concentrations. An increase in glucose concentrations was recorded on days 10 and 12 of nicotinic acid infusions, continuing 1 day after treatment termination, whereas blood insulin concentrations increased during the entire treatment process [30]. According to Titgemeyer et al. [30], it is inconclusive whether an increase in glucose concentrations leads to an increase in insulin concentrations or insulin resistance causes elevated glucose concentrations. Their model is at variance with those stating that increased NEFA concentrations are associated with insulin resistance during nicotinic acid treatments. Differences in the results obtained can partially be accounted for by different energy supplies and degrees of lipolysis, indicating that both insulin and glucose concentrations in the blood are affected by niacin. Titgemeyer et al. [30] also found that glucagon concentrations were not significantly altered, inferring that glucagon was of little or no significance to the effect of niacin on blood glucose concentrations.

One of our studies has hypothesized that niacin administration to dairy cows in the transition period can influence insulin responsiveness and resistance in adipose tissue by virtue of niacin-induced changes in NEFA, glucose and insulin concentrations [56]. A total of 30 clinically healthy, multiparous Holstein-Friesian cows in late gestation were enrolled in the study. Insulin resistance was calculated on the basis of the following insulin resistance indicators: the glucose-to-insulin (G:I) ratio and the Revised Quantitative Insulin Sensitivity Check Index (RQUICKI). The formula for the glucose-to-insulin ratio is as follows: G:I = glucose (mg/dL)/insulin (μU/ml). The RQUICKI is calculated on the basis of plasma concentrations of glucose (mg/dl), insulin (μU/ml) and free fatty acids (mmol/l), using the following formula: RQUICKI = 1/[log (glucose mg/dL) + log (insulin μU/ml) + log (NEFA mmol/l)]. The RQUICKI is a good indicator of insulin resistance in dairy cows. Although lipolysis-dependent, the RQUICKI correlates with numerous metabolic parameters [57, 58]. The influence of niacin supplementation, in the week of calving and the first week after parturition, on glucose, insulin and NEFA concentrations, as well as RQUICKI values, was analyzed using the analysis of variance (ANOVA). According to the RQUICKI values obtained, niacin-supplemented and control cows (n = 15 cow × 3 week = 45) were allocated to two groups: a more resistant group (RQUICKI < 0.5) and a less resistant group (RQUICKI ≥ 0.5). Differences in glucose, insulin and NEFA concentrations between the two groups were determined using paired t-tests. Moreover, a linear regression analysis (Y = bXi + a) was performed on the basis of all the parameter values obtained in the niacin-supplemented and control groups in order to determine differences in the slope of regression lines (differences in the b parameters). Cows in the niacin group, which were more resistant to insulin (RQUICKI < 0.5), exhibited higher concentrations of nonesterified fatty acids compared to more sensitive cows in the same group but still lower than those recorded in control animals. The regression analyses performed suggest the following characteristics of niacin-supplemented cows relative to the control group: increased insulin response to glucose, decreased antilipolytic effect of insulin and increased insulin efficiency (expressed as the glucose-to-insulin ratio) with a decrease in NEFA concentrations. Niacin was found to exert a dual influence on insulin resistance in early-lactation dairy cows: decreased NEFA concentrations led to a decrease in insulin resistance (due to an increase in insulin efficiency and the insulin sensitivity index), whereas elevated insulin and glucose concentrations most likely caused an increase in insulin resistance in dairy cows (due to the lower insulin sensitivity index and antilipolytic effect of insulin).

ascending regulation of proinflammatory cytokines, the activation of TLR4 can lead to the inflammatory response [64–67]. The activation of the innate immune response is incited by the activation of TLR receptors present on immune and non-immune cells (able to identify pathogens). TLR4 identifies lipopolysaccharides (endotoxins), which are the major component of the outer membrane of Gram-negative bacteria [68]. The typical proinflammatory response to lipopolysaccharides entails the expression of several acute-phase cytokines (TNF, IL-1 and IL-8) and leukocyte-endothelial adhesion molecules, as well as the influx and activation of neutrophils in inflamed tissues. Furthermore, increased lipid hydroperoxide concentrations, associated with oxidative stress, have been found to induce an increase in the proinflammatory phenotype of endothelial cells [69, 70]. TNF-α, IL-1, IL-6 and IL-8 have been implicated in the occurrence of coliform mastitis in periparturient cows in a state

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Niacin reduces adipose tissue inflammation by increasing adiponectin concentrations, thereby regulating the metabolism of carbohydrates and insulin sensitivity of adipose tissue. Such results have been obtained in cows and laboratory mice [72, 73]. Nicotinic acid increases

Niacin administration reduces TNF-α and IL-6, as well as the activation of NF-κB in the lungs and kidneys of rats [74, 75]. In monocytes, niacin suppresses the NF-κB signalling pathway, thus reducing proinflammatory mediators (namely, TNF-α, IL-6 and MCP-1) [76, 77] and inhibiting monocyte chemotaxis [78]. It also decreases C-reactive protein (CRP) concentrations, as well as macrophage accumulation in the liver and hepatocyte inflammation, which results in reducing acute-phase protein production [79, 80]. The anti-inflammatory effect of

Niacin should be administered to ruminants in adequate pharmacological doses and forms on account of their complex stomach. The antilipolytic effect of niacin reduces metabolic stress in periparturient cows. Moreover, metabolic adaptations in the periparturient period are significantly less dependent on the magnitude of lipolysis provided niacin is administered. Niacin reduces lipid peroxidation and the degree of oxidative stress in cows by the NAD and NADP coenzymes. The antilipolytic effect of niacin decreases insulin resistance in cows. However, its potential to elevate glucose and insulin concentrations may attenuate the antilipolytic effect of insulin due to increased insulin resistance in a state of metabolic stress. Niacin exerts its anti-inflammatory effect by stimulating the secretion of adiponectin and

adiponectin secretion through G-protein-coupled receptor signalling in cattle.

niacin is associated with the activation of niacin receptors [76, 77].

of oxidative stress [71].

**7. Conclusion**

inhibiting immune cells.

**Conflict of interest**

The authors declare no conflict of interest.

### **6. Effects of niacin administration on the inflammatory response following metabolic stress**

Inflammation is the common denominator of a number of processes occurring in cows during the periparturient period. Therefore, increased lipolysis may precipitate a substantial release of proinflammatory cytokines within adipose tissue, i.e. adipokines, the most important of which is tumour necrosis factor alpha (TNF-α) [59]. Ohtuska et al. reported increased serum TNF-α activity in cows with moderate-to-severe fatty liver syndrome [60]. The organism protects itself from inflammation by secreting acute-phase proteins. Plasma haptoglobin and serum amyloid A concentrations have been found to be elevated in cows with fatty liver [61]. In addition to decreased albumin and cholesterol concentrations, Bertoni et al. recorded increased bilirubin, AST and GGT concentrations in cows with a high inflammatory index, which is indicative of the biochemical profile of fatty liver [62]. Inflammatory mediators were directly implicated in metabolic changes by Trevisi et al. upon the peroral administration of interferon-α during the last 2 weeks of gestation, which led to liver inflammation and the release of acute-phase proteins [63]. Relative to the control group, cows treated with interferon-α were found to exhibit significantly higher plasma ketone concentrations during the first 2 weeks after parturition. A number of experimental studies have shown a direct impact of NEFAs on inflammatory processes such as the regulation of peroxisome proliferator-activated receptors (PPARs). PPARs modulate the inflammatory response in many cells such as adipocytes. In monocytes, PPARs activate certain polyunsaturated fatty acids such as α-linolenic acid and docosapentaenoic acid, which can suppress the inflammatory response. Another instance of the effect of lipids on receptor binding is the activation of Toll-like receptors (TLRs), especially TLR4. In addition to the ascending regulation of proinflammatory cytokines, the activation of TLR4 can lead to the inflammatory response [64–67]. The activation of the innate immune response is incited by the activation of TLR receptors present on immune and non-immune cells (able to identify pathogens). TLR4 identifies lipopolysaccharides (endotoxins), which are the major component of the outer membrane of Gram-negative bacteria [68]. The typical proinflammatory response to lipopolysaccharides entails the expression of several acute-phase cytokines (TNF, IL-1 and IL-8) and leukocyte-endothelial adhesion molecules, as well as the influx and activation of neutrophils in inflamed tissues. Furthermore, increased lipid hydroperoxide concentrations, associated with oxidative stress, have been found to induce an increase in the proinflammatory phenotype of endothelial cells [69, 70]. TNF-α, IL-1, IL-6 and IL-8 have been implicated in the occurrence of coliform mastitis in periparturient cows in a state of oxidative stress [71].

Niacin reduces adipose tissue inflammation by increasing adiponectin concentrations, thereby regulating the metabolism of carbohydrates and insulin sensitivity of adipose tissue. Such results have been obtained in cows and laboratory mice [72, 73]. Nicotinic acid increases adiponectin secretion through G-protein-coupled receptor signalling in cattle.

Niacin administration reduces TNF-α and IL-6, as well as the activation of NF-κB in the lungs and kidneys of rats [74, 75]. In monocytes, niacin suppresses the NF-κB signalling pathway, thus reducing proinflammatory mediators (namely, TNF-α, IL-6 and MCP-1) [76, 77] and inhibiting monocyte chemotaxis [78]. It also decreases C-reactive protein (CRP) concentrations, as well as macrophage accumulation in the liver and hepatocyte inflammation, which results in reducing acute-phase protein production [79, 80]. The anti-inflammatory effect of niacin is associated with the activation of niacin receptors [76, 77].

### **7. Conclusion**

control cows (n = 15 cow × 3 week = 45) were allocated to two groups: a more resistant group (RQUICKI < 0.5) and a less resistant group (RQUICKI ≥ 0.5). Differences in glucose, insulin and NEFA concentrations between the two groups were determined using paired t-tests. Moreover, a linear regression analysis (Y = bXi + a) was performed on the basis of all the parameter values obtained in the niacin-supplemented and control groups in order to determine differences in the slope of regression lines (differences in the b parameters). Cows in the niacin group, which were more resistant to insulin (RQUICKI < 0.5), exhibited higher concentrations of nonesterified fatty acids compared to more sensitive cows in the same group but still lower than those recorded in control animals. The regression analyses performed suggest the following characteristics of niacin-supplemented cows relative to the control group: increased insulin response to glucose, decreased antilipolytic effect of insulin and increased insulin efficiency (expressed as the glucose-to-insulin ratio) with a decrease in NEFA concentrations. Niacin was found to exert a dual influence on insulin resistance in early-lactation dairy cows: decreased NEFA concentrations led to a decrease in insulin resistance (due to an increase in insulin efficiency and the insulin sensitivity index), whereas elevated insulin and glucose concentrations most likely caused an increase in insulin resistance in dairy cows (due

to the lower insulin sensitivity index and antilipolytic effect of insulin).

**following metabolic stress**

118 B Group Vitamins - Current Uses and Perspectives

**6. Effects of niacin administration on the inflammatory response** 

Inflammation is the common denominator of a number of processes occurring in cows during the periparturient period. Therefore, increased lipolysis may precipitate a substantial release of proinflammatory cytokines within adipose tissue, i.e. adipokines, the most important of which is tumour necrosis factor alpha (TNF-α) [59]. Ohtuska et al. reported increased serum TNF-α activity in cows with moderate-to-severe fatty liver syndrome [60]. The organism protects itself from inflammation by secreting acute-phase proteins. Plasma haptoglobin and serum amyloid A concentrations have been found to be elevated in cows with fatty liver [61]. In addition to decreased albumin and cholesterol concentrations, Bertoni et al. recorded increased bilirubin, AST and GGT concentrations in cows with a high inflammatory index, which is indicative of the biochemical profile of fatty liver [62]. Inflammatory mediators were directly implicated in metabolic changes by Trevisi et al. upon the peroral administration of interferon-α during the last 2 weeks of gestation, which led to liver inflammation and the release of acute-phase proteins [63]. Relative to the control group, cows treated with interferon-α were found to exhibit significantly higher plasma ketone concentrations during the first 2 weeks after parturition. A number of experimental studies have shown a direct impact of NEFAs on inflammatory processes such as the regulation of peroxisome proliferator-activated receptors (PPARs). PPARs modulate the inflammatory response in many cells such as adipocytes. In monocytes, PPARs activate certain polyunsaturated fatty acids such as α-linolenic acid and docosapentaenoic acid, which can suppress the inflammatory response. Another instance of the effect of lipids on receptor binding is the activation of Toll-like receptors (TLRs), especially TLR4. In addition to the

Niacin should be administered to ruminants in adequate pharmacological doses and forms on account of their complex stomach. The antilipolytic effect of niacin reduces metabolic stress in periparturient cows. Moreover, metabolic adaptations in the periparturient period are significantly less dependent on the magnitude of lipolysis provided niacin is administered. Niacin reduces lipid peroxidation and the degree of oxidative stress in cows by the NAD and NADP coenzymes. The antilipolytic effect of niacin decreases insulin resistance in cows. However, its potential to elevate glucose and insulin concentrations may attenuate the antilipolytic effect of insulin due to increased insulin resistance in a state of metabolic stress. Niacin exerts its anti-inflammatory effect by stimulating the secretion of adiponectin and inhibiting immune cells.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Marko Cincović<sup>1</sup> \*, Talija Hristovska2 and Branislava Belić<sup>1</sup>

\*Address all correspondence to: marko.cincovic@polj.uns.ac.rs

1 Department of Veterinary Medicine, Faculty of Agriculture, Laboratory of Pathophysiology, University of Novi Sad, Serbia

2 Faculty of Veterinary Medicine, University of Bitola, Bitola, Macedonia

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**Author details**

120 B Group Vitamins - Current Uses and Perspectives

Marko Cincović<sup>1</sup>

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\*, Talija Hristovska2

Pathophysiology, University of Novi Sad, Serbia

Washington, DC: Natl. Acad. Press; 2001

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\*Address all correspondence to: marko.cincovic@polj.uns.ac.rs

1 Department of Veterinary Medicine, Faculty of Agriculture, Laboratory of

2 Faculty of Veterinary Medicine, University of Bitola, Bitola, Macedonia

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**Chapter 8**

**Provisional chapter**

**Vitamin B2 and Innovations in Improving Blood Safety**

Although transfusion of blood components is becoming increasingly safe, the risk of transmission of known and unknown pathogens persists. The application of vitamin B2 (riboflavin) and UV light to pathogen inactivation has several appealing factors. Riboflavin is a naturally occurring vitamin with a well-known and well-characterized safety profile. This photochemical-based method is effective against clinically relevant pathogens and inactivates leukocytes without significantly compromising the content and the efficacy of whole blood or blood component. This chapter gives an overview of the innovative technology for pathogen inactivation, the Mirasol® pathogen reduction technology (PRT) System, based on riboflavin and UV light, summarizing the mechanism of action, toxicology profile, pathogen reduction performance and clinical efficacy of the process. **Keywords:** riboflavin, pathogen reduction technology, blood transfusion, safety

The collection, processing, transfusion of whole blood, red blood cells, platelets, plasma, and infusion of fractionated plasma components are essential medical practices, often required for the preservation of life and for the treatment of disease. Although the transfusion/infusion of these components is a vital therapy, transfusions are still associated with some risk for

Worldwide measures to reduce the risk of transmission of diseases to recipients through blood have been continuously implemented and improved [2]. Blood safety improvements include donor's questionnaire, self-deferrals and donation screening methods designed to detect possible contaminating agents in blood. Serological testing and nucleic acid testing

**Vitamin B2 and Innovations in Improving Blood Safety**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.78260

Raymond P. Goodrich, Marcia Cardoso and

Raymond P. Goodrich, Marcia Cardoso and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78260

Susanne Marschner

Susanne Marschner

**Abstract**

**1. Introduction**

transmission of disease to the patient [1].

### **Vitamin B2 and Innovations in Improving Blood Safety Vitamin B2 and Innovations in Improving Blood Safety**

DOI: 10.5772/intechopen.78260

Raymond P. Goodrich, Marcia Cardoso and Susanne Marschner Raymond P. Goodrich, Marcia Cardoso and Susanne Marschner

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78260

### **Abstract**

Although transfusion of blood components is becoming increasingly safe, the risk of transmission of known and unknown pathogens persists. The application of vitamin B2 (riboflavin) and UV light to pathogen inactivation has several appealing factors. Riboflavin is a naturally occurring vitamin with a well-known and well-characterized safety profile. This photochemical-based method is effective against clinically relevant pathogens and inactivates leukocytes without significantly compromising the content and the efficacy of whole blood or blood component. This chapter gives an overview of the innovative technology for pathogen inactivation, the Mirasol® pathogen reduction technology (PRT) System, based on riboflavin and UV light, summarizing the mechanism of action, toxicology profile, pathogen reduction performance and clinical efficacy of the process.

**Keywords:** riboflavin, pathogen reduction technology, blood transfusion, safety

### **1. Introduction**

The collection, processing, transfusion of whole blood, red blood cells, platelets, plasma, and infusion of fractionated plasma components are essential medical practices, often required for the preservation of life and for the treatment of disease. Although the transfusion/infusion of these components is a vital therapy, transfusions are still associated with some risk for transmission of disease to the patient [1].

Worldwide measures to reduce the risk of transmission of diseases to recipients through blood have been continuously implemented and improved [2]. Blood safety improvements include donor's questionnaire, self-deferrals and donation screening methods designed to detect possible contaminating agents in blood. Serological testing and nucleic acid testing

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

have become staples of modern blood banking and have greatly reduced the risk of disease transmission by blood product transfusion. Yet, growing socio-political changes of contemporary society together with environmental changes challenge the practice of blood transfusion with a continuous source of unforeseeable threats with the emergence and re-emergence of blood-borne pathogens [2, 3].

**2. Vitamin B2 and UV light: the chemistry**

photochemistry are known [17, 19, 20].

tion process has been described in the literature.

**2.1. Action spectra and absorbance spectra**

**Figure 1.** Action spectra and absorbance spectra of riboflavin with lambda phage.

Riboflavin (RB) has absorption maxima at 220, 265, 375, and 446 nm in water and is yelloworange in color. When aqueous solutions containing RB are exposed to sunlight, RB is converted into lumichrome (LC) under neutral conditions, and into lumiflavin (LF) in alkaline solutions [17, 18]. LC is also a known metabolic breakdown product of RB in the human body [19]. Flavin systems are known to be photochemically active, and the products of flavin

Vitamin B2 and Innovations in Improving Blood Safety http://dx.doi.org/10.5772/intechopen.78260 129

The mechanism of pathogen reduction using RB likely involves three potential pathways: Type I Photochemistry [47, 48] Type II Photochemistry, [21] and the effects of UV light alone. The contribution of each of these three pathways to the Mirasol PRT System pathogen reduc-

The reported mode of action of RB in the reduction of pathogens is postulated to be based in part on the ability of RB to interact with nucleic acids and to undergo chemistry with those nucleic acids upon exposure to light. This chemistry is believed to involve both oxygen-dependent and oxygen-independent (electron transfer) processes. It has been described thoroughly in the chemical literature over the past several decades [22–30]. The use of UV light with platelets and plasma also affords a third contributor to pathogen kill via the direct action of light.

**Figure 1** depicts the action spectrum of RB and lambda phage minus light alone (yellow) over-laid with the absorbance profile of RB in PBS (pink) and absorbance of DNA in PBS (blue). At wavelengths lower than 300 nm, RB acts to shield the effects on DNA due to the

In the last two decades, several pathogen reduction/inactivation technologies (PRT) have been developed to allow treatment of blood products with the intent of reducing the levels of infectivity and eventually inactivating white blood cells that can cause immunological complications to blood recipients. PRT methods involve physicochemical disruption of pathogen structural elements, mostly applied to the production of plasma-derived fractionated products or photochemical modification of nucleic acids to prevent replication, applicable to labile blood components like platelet concentrates, therapeutic plasma and eventually red cell concentrates [4, 5].

One of these PRT technologies, the Mirasol PRT System, uses riboflavin or vitamin B2 as a photochemical sensitizer and relies on the association of riboflavin with nucleic acid and activation with UV-light to generate a photochemical reaction that modifies guanine residues and thus prevents replication processes. This method creates irreversible damage via electron transfer processes at the sites where riboflavin-guanine base chemistry occurs [6].

Flavins are present in all biologic fluids and tissues. The most common biologically important flavins are riboflavin and its nucleotides: riboflavin-5′-phosphate (flavin mono-nucleotide, FMN), and the intramolecular complex of FMN with adenosine-5′-monophosphate (flavin adenine dinucleotide, FAD) [7]. Riboflavin in its coenzyme form is a component of many oxidation-reduction reactions and of energy production. It is essential for growth and tissue repair in all animals from protozoa to man [8] unlike fat-soluble vitamins, which are stored in body fat, riboflavin is a water-soluble vitamin and excess amounts are rapidly excreted. Because there are no physiological stores of riboflavin and excretion is constant, frequent dietary intake is important to maintain sufficient concentration and in the case of excess, return to normal levels is commensurate with renal function [9].

The choice of riboflavin as photosensitizer in the Mirasol PRT System was reinforced by its well-documented safety profile, being widely used as food coloring in the United States, where it is "generally regarded as safe" by the FDA [10]. Neonates, including preterm and very low birth weight (VLBW) infants, requiring nutritional supplementation due to immature gastrointestinal and metabolic systems, commonly undergo parenteral nutrition with a multivitamin preparation which includes vitamin B12, thiamine, folate and riboflavin [11]. The FDA concluded in their review that the LD50 is orders of magnitude greater than the Recommended Daily Allowance (RDA); additionally, no reports on carcinogenicity, mutagenicity or teratogenicity associated with riboflavin have been reported to the agency [12]. In Europe, it has been approved by the Scientific Committee on Food [13]. Furthermore, an anti-neoplastic action of riboflavin photoproducts to hematological malignancies and solid tumors has been postulated, whereas high dose of riboflavin has been suggested for migraine prophylaxis [14–16].

### **2. Vitamin B2 and UV light: the chemistry**

have become staples of modern blood banking and have greatly reduced the risk of disease transmission by blood product transfusion. Yet, growing socio-political changes of contemporary society together with environmental changes challenge the practice of blood transfusion with a continuous source of unforeseeable threats with the emergence and re-emergence of

In the last two decades, several pathogen reduction/inactivation technologies (PRT) have been developed to allow treatment of blood products with the intent of reducing the levels of infectivity and eventually inactivating white blood cells that can cause immunological complications to blood recipients. PRT methods involve physicochemical disruption of pathogen structural elements, mostly applied to the production of plasma-derived fractionated products or photochemical modification of nucleic acids to prevent replication, applicable to labile blood components like platelet concentrates, therapeutic plasma and eventually red cell

One of these PRT technologies, the Mirasol PRT System, uses riboflavin or vitamin B2 as a photochemical sensitizer and relies on the association of riboflavin with nucleic acid and activation with UV-light to generate a photochemical reaction that modifies guanine residues and thus prevents replication processes. This method creates irreversible damage via electron

Flavins are present in all biologic fluids and tissues. The most common biologically important flavins are riboflavin and its nucleotides: riboflavin-5′-phosphate (flavin mono-nucleotide, FMN), and the intramolecular complex of FMN with adenosine-5′-monophosphate (flavin adenine dinucleotide, FAD) [7]. Riboflavin in its coenzyme form is a component of many oxidation-reduction reactions and of energy production. It is essential for growth and tissue repair in all animals from protozoa to man [8] unlike fat-soluble vitamins, which are stored in body fat, riboflavin is a water-soluble vitamin and excess amounts are rapidly excreted. Because there are no physiological stores of riboflavin and excretion is constant, frequent dietary intake is important to maintain sufficient concentration and in the case of excess,

The choice of riboflavin as photosensitizer in the Mirasol PRT System was reinforced by its well-documented safety profile, being widely used as food coloring in the United States, where it is "generally regarded as safe" by the FDA [10]. Neonates, including preterm and very low birth weight (VLBW) infants, requiring nutritional supplementation due to immature gastrointestinal and metabolic systems, commonly undergo parenteral nutrition with a multivitamin preparation which includes vitamin B12, thiamine, folate and riboflavin [11]. The FDA concluded in their review that the LD50 is orders of magnitude greater than the Recommended Daily Allowance (RDA); additionally, no reports on carcinogenicity, mutagenicity or teratogenicity associated with riboflavin have been reported to the agency [12]. In Europe, it has been approved by the Scientific Committee on Food [13]. Furthermore, an anti-neoplastic action of riboflavin photoproducts to hematological malignancies and solid tumors has been postulated, whereas high dose of riboflavin has been suggested for migraine

transfer processes at the sites where riboflavin-guanine base chemistry occurs [6].

return to normal levels is commensurate with renal function [9].

blood-borne pathogens [2, 3].

128 B Group Vitamins - Current Uses and Perspectives

concentrates [4, 5].

prophylaxis [14–16].

Riboflavin (RB) has absorption maxima at 220, 265, 375, and 446 nm in water and is yelloworange in color. When aqueous solutions containing RB are exposed to sunlight, RB is converted into lumichrome (LC) under neutral conditions, and into lumiflavin (LF) in alkaline solutions [17, 18]. LC is also a known metabolic breakdown product of RB in the human body [19]. Flavin systems are known to be photochemically active, and the products of flavin photochemistry are known [17, 19, 20].

The mechanism of pathogen reduction using RB likely involves three potential pathways: Type I Photochemistry [47, 48] Type II Photochemistry, [21] and the effects of UV light alone. The contribution of each of these three pathways to the Mirasol PRT System pathogen reduction process has been described in the literature.

The reported mode of action of RB in the reduction of pathogens is postulated to be based in part on the ability of RB to interact with nucleic acids and to undergo chemistry with those nucleic acids upon exposure to light. This chemistry is believed to involve both oxygen-dependent and oxygen-independent (electron transfer) processes. It has been described thoroughly in the chemical literature over the past several decades [22–30]. The use of UV light with platelets and plasma also affords a third contributor to pathogen kill via the direct action of light.

### **2.1. Action spectra and absorbance spectra**

**Figure 1** depicts the action spectrum of RB and lambda phage minus light alone (yellow) over-laid with the absorbance profile of RB in PBS (pink) and absorbance of DNA in PBS (blue). At wavelengths lower than 300 nm, RB acts to shield the effects on DNA due to the

**Figure 1.** Action spectra and absorbance spectra of riboflavin with lambda phage.

direct action of UV light. Greater levels of inactivation in the presence of RB occur at wavelengths between 300 and 350 nm compared to the prediction due to the absorbance profile of free RB in solution. This is also observed for wavelengths higher than 500 nm. In the region of 308–575 nm, in order to achieve the same magnitude of log reduction that was observed between 266 and 304 nm, the energy required for the experiments between 308 and 575 nm was increased 50-fold from 0.1 to 5.0 J/mL. There is no inactivation of lambda phage with light of wavelength ≥330 nm in the absence of RB.

These studies identify the precise site of the lesions induced in nucleic acids treated with monochromatic 266, 308, or 355 nm light from either an excimer or Yttrium Aluminum Garnet (YAG) laser in the presence and absence of RB. The results demonstrate that in the presence of RB, the predominant modifications occur to guanine bases, as evidenced by the formation of 8-oxodGuo. The extent of the oxidized guanines formed in the presence of RB is far in excess of those observed upon exposure to light alone. These results are consistent with the literature reports of Cadet and co-workers of the mechanism of action of RB with regard to nucleic acid chemistry [24]. The results were contrasted to those using UV light alone in the absence of RB, and suggest that the addition of RB to the system specifically enhances the damage to DNA

Vitamin B2 and Innovations in Improving Blood Safety http://dx.doi.org/10.5772/intechopen.78260 131

Virus reactivation is a phenomenon, which is known to occur through the use of host cell nucleic acid repair mechanisms. In the context of virus inactivation, the desired end target for these treatments is the prevention of virus replication. It is also desirable, in this context, to prevent repair of damaged virus particles because such repairs may render non-infectious agents capable of transmitting the disease when re-infused. This may be accomplished by generating either an extent of damage that the host system cannot repair or a type of damage

Studies of the inability of bacteriophage to repair the lesions (Weigle reactivation) induced by RB and light as contrasted to the observations with light exposure alone have been conducted [31]. These studies confirm that the rescue of DNA damaged phage does not occur to the same extent when RB is present in samples exposed to light. These observations are consistent with the data suggesting that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA or RNA. These data also suggest that this type and extent of damage to nucleic acids of virus in the presence of RB makes it less likely to be repaired by normal repair pathways available in host cells [36]. This result is essential for a system intended to assure the highest and

In summary, the Mirasol PRT process works through three independent mechanisms of action in rendering pathogens inactive. These include oxygen dependent chemistry induced by the combination of RB and light, electron transfer chemistry induced by the direct interaction of excited RB molecules with nucleic acid base pairs (primarily guanine bases) leading to oxidation products, and effects that are due to the action of UV light alone. In essence, the presence of RB in this system enhances the effects, which are due to UV light alone, creating a condition of greater sensitivity of the pathogen to the UV light to which the sample is exposed (photosensitization effect). The combination of these three modes affords broad and extended

Although the safety of RB has been extensively studied, there were no reports that directly supported its use in the Mirasol PRT System. Therefore a comprehensive preclinical safety

that the host system does not have the capability of repairing.

most complete levels of pathogen inactivation attainable.

levels of pathogen inactivation with this process.

**3. Toxicology and safety**

induced by UV light alone.

**2.3. Phage reactivation**

The action spectrum (AS) do not correlate perfectly with the absorption curve of either RB or LC in PBS over the entire wavelength regime. There appears to be essentially an identical amount of viral inactivation at 355 and 500 nm, although the optical densities (of solutions containing the same concentration of RB) differ by a factor of five at these two wavelengths. The phage reduction obtained at 320 nm and at 500 nm is greater than that expected based on the absorption spectrum of RB in PBS at these wavelengths. The effect is clearly seen in **Figure 1** which plots lambda phage inactivation achieved in the presence of RB at various concentrations minus that realized in its absence.

### **2.2. RB sensitized modification of nucleic acids**

Several studies have been conducted in order to examine the ability of RB sensitization to modify nucleic acids [31]. The DNA fragmentation studies in leukocytes and bacteria utilized chemical agents that bind to portions of the DNA strand, which have been severed or broken because of chemical modification. The fragments that are produced leave regions that can be chemically tagged with a fluorescence probe and subsequently measured to provide an estimate of the extent of fragmentation that has occurred. Single-strand breaks throughout the nucleic acid sequence can be identified in this way. More complete breaks leading to denaturation of the nucleic acid can also be monitored by gel electrophoresis. In the latter case, the complete denaturation of the nucleic acid can be followed by examining migration patterns on polyacrylamide gels [31, 32]. This assay looks for much more severe and complete nucleic acid degradation than single-strand breaks.

In one set of studies, the level of DNA fragmentation occurring in white cell DNA was determined using a flow cytometric assay (Trevigen Apoptotic Cell System (TACS) assay. The level of DNA fragmentation obtained was significantly increased in the presence of RB. Similar observations were made for samples of plasmid DNA and for DNA isolated from *Escherichia coli* following treatment in the presence and absence of RB [31]. These combined studies demonstrate a sensitizing effect, with respect to nucleic acid damage, which RB imparts to samples treated with UV light. These observations are consistent with literature reports for RB.

Cadet and co-workers have evaluated the chemistry involved in the formation of specific lesions induced in nucleic acids by RB and light. These lesions differ from those induced by exposure to light alone in that chemically distinct oxidized species of guanine where residues are formed. This chemistry DNA fragmentation in isolated white cell DNA following exposure to light in the presence and absence of RB was evaluated because of the fact that mammalian systems do not normally contain enzymatic systems capable of repairing these types of lesions. This is in stark contrast to the predominant lesion (thymine-thymine dimers formed) upon exposure of nucleic acids or agents containing nucleic acids to light alone [33–35].

These studies identify the precise site of the lesions induced in nucleic acids treated with monochromatic 266, 308, or 355 nm light from either an excimer or Yttrium Aluminum Garnet (YAG) laser in the presence and absence of RB. The results demonstrate that in the presence of RB, the predominant modifications occur to guanine bases, as evidenced by the formation of 8-oxodGuo. The extent of the oxidized guanines formed in the presence of RB is far in excess of those observed upon exposure to light alone. These results are consistent with the literature reports of Cadet and co-workers of the mechanism of action of RB with regard to nucleic acid chemistry [24]. The results were contrasted to those using UV light alone in the absence of RB, and suggest that the addition of RB to the system specifically enhances the damage to DNA induced by UV light alone.

### **2.3. Phage reactivation**

direct action of UV light. Greater levels of inactivation in the presence of RB occur at wavelengths between 300 and 350 nm compared to the prediction due to the absorbance profile of free RB in solution. This is also observed for wavelengths higher than 500 nm. In the region of 308–575 nm, in order to achieve the same magnitude of log reduction that was observed between 266 and 304 nm, the energy required for the experiments between 308 and 575 nm was increased 50-fold from 0.1 to 5.0 J/mL. There is no inactivation of lambda phage with light

The action spectrum (AS) do not correlate perfectly with the absorption curve of either RB or LC in PBS over the entire wavelength regime. There appears to be essentially an identical amount of viral inactivation at 355 and 500 nm, although the optical densities (of solutions containing the same concentration of RB) differ by a factor of five at these two wavelengths. The phage reduction obtained at 320 nm and at 500 nm is greater than that expected based on the absorption spectrum of RB in PBS at these wavelengths. The effect is clearly seen in **Figure 1** which plots lambda phage inactivation achieved in the presence of RB at various

Several studies have been conducted in order to examine the ability of RB sensitization to modify nucleic acids [31]. The DNA fragmentation studies in leukocytes and bacteria utilized chemical agents that bind to portions of the DNA strand, which have been severed or broken because of chemical modification. The fragments that are produced leave regions that can be chemically tagged with a fluorescence probe and subsequently measured to provide an estimate of the extent of fragmentation that has occurred. Single-strand breaks throughout the nucleic acid sequence can be identified in this way. More complete breaks leading to denaturation of the nucleic acid can also be monitored by gel electrophoresis. In the latter case, the complete denaturation of the nucleic acid can be followed by examining migration patterns on polyacrylamide gels [31, 32]. This assay looks for much more severe and complete

In one set of studies, the level of DNA fragmentation occurring in white cell DNA was determined using a flow cytometric assay (Trevigen Apoptotic Cell System (TACS) assay. The level of DNA fragmentation obtained was significantly increased in the presence of RB. Similar observations were made for samples of plasmid DNA and for DNA isolated from *Escherichia coli* following treatment in the presence and absence of RB [31]. These combined studies demonstrate a sensitizing effect, with respect to nucleic acid damage, which RB imparts to samples treated with UV light. These observations are consistent with literature reports for RB.

Cadet and co-workers have evaluated the chemistry involved in the formation of specific lesions induced in nucleic acids by RB and light. These lesions differ from those induced by exposure to light alone in that chemically distinct oxidized species of guanine where residues are formed. This chemistry DNA fragmentation in isolated white cell DNA following exposure to light in the presence and absence of RB was evaluated because of the fact that mammalian systems do not normally contain enzymatic systems capable of repairing these types of lesions. This is in stark contrast to the predominant lesion (thymine-thymine dimers formed) upon exposure of nucleic acids or agents containing nucleic acids to light alone [33–35].

of wavelength ≥330 nm in the absence of RB.

130 B Group Vitamins - Current Uses and Perspectives

concentrations minus that realized in its absence.

**2.2. RB sensitized modification of nucleic acids**

nucleic acid degradation than single-strand breaks.

Virus reactivation is a phenomenon, which is known to occur through the use of host cell nucleic acid repair mechanisms. In the context of virus inactivation, the desired end target for these treatments is the prevention of virus replication. It is also desirable, in this context, to prevent repair of damaged virus particles because such repairs may render non-infectious agents capable of transmitting the disease when re-infused. This may be accomplished by generating either an extent of damage that the host system cannot repair or a type of damage that the host system does not have the capability of repairing.

Studies of the inability of bacteriophage to repair the lesions (Weigle reactivation) induced by RB and light as contrasted to the observations with light exposure alone have been conducted [31]. These studies confirm that the rescue of DNA damaged phage does not occur to the same extent when RB is present in samples exposed to light. These observations are consistent with the data suggesting that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA or RNA. These data also suggest that this type and extent of damage to nucleic acids of virus in the presence of RB makes it less likely to be repaired by normal repair pathways available in host cells [36]. This result is essential for a system intended to assure the highest and most complete levels of pathogen inactivation attainable.

In summary, the Mirasol PRT process works through three independent mechanisms of action in rendering pathogens inactive. These include oxygen dependent chemistry induced by the combination of RB and light, electron transfer chemistry induced by the direct interaction of excited RB molecules with nucleic acid base pairs (primarily guanine bases) leading to oxidation products, and effects that are due to the action of UV light alone. In essence, the presence of RB in this system enhances the effects, which are due to UV light alone, creating a condition of greater sensitivity of the pathogen to the UV light to which the sample is exposed (photosensitization effect). The combination of these three modes affords broad and extended levels of pathogen inactivation with this process.

### **3. Toxicology and safety**

Although the safety of RB has been extensively studied, there were no reports that directly supported its use in the Mirasol PRT System. Therefore a comprehensive preclinical safety evaluation program in support of the Mirasol PRT System, designed to investigate all potential sources of concern, was conducted as part of the overall development program. In vivo animal and in vitro toxicity studies were performed using RB, lumichrome and photolyzed RB (see **Table 1**).

To obtain a consistent test article in as humane a fashion as possible for those studies, speciesspecific plasma was used rather than platelets. The photochemistry of RB yields equivalent photoproduct profiles in plasma products and in platelet products (which consist mainly of plasma). The absence of platelets eliminates the possibility of detecting toxic alterations to the platelet surface; however, that issue was addressed in the neoantigenicity and <sup>14</sup>C-RB binding studies.

### **3.1. Systemic toxicity**

No toxicologically significant findings were observed in any of the studies of acute toxicity. In the repeated-dose toxicity study, the levels of RB and lumichrome in blood samples from animals receiving Mirasol PRT-treated products were below the limits of quantification, as were the levels in blood samples from animals receiving untreated plasma. These results were consistent with the observed rapid clearance of RB after IV administration, both in the literature [9] and in the pharmacokinetic study with 14C-RB in Mirasol PRT-treated products. RB and its photoproducts naturally occur in human blood, see **Figure 2**. All photoproducts were found to be present in apheresis platelet products that had not undergone any photochemical treatment, although at a much lower concentration. The presence of these agents in human blood, the ubiquitous nature of RB exposure, its presence in human diets and the ability of humans to metabolize it and manage its inherent photochemistry suggests a low risk profile for this product.

observed in the Ames test for treated or control human platelets, or for lumichrome. The in vitro and in vivo tests for clastogenicity in mammalian cells (chromosomal aberration in cultured CHO cells and micronucleus test in mouse bone marrow cells, respectively) were also performed with Mirasol PRT-treated products. Human platelets treated with the Mirasol

**Figure 2.** Riboflavin and its photoproducts are naturally present in human blood; no new compounds are formed after

Vitamin B2 and Innovations in Improving Blood Safety http://dx.doi.org/10.5772/intechopen.78260 133

Results of studies using <sup>14</sup>C-labeled RB and exposure of platelets and plasma to UV light did not demonstrate any detectable binding of RB or its photoproducts to platelets or to plasma proteins. No evidence of neoantigenicity was observed with the Ouchterlony assay, indicating that no new antigens were formed during treatment with the Mirasol PRT System. Treatment with the Mirasol PRT System did not result in greater immunoglobulin G binding than what was observed in comparison with untreated controls, when assessed with the Capture-P assay. In the tests of lumichrome cytotoxicity, and of the cytotoxicity of Mirasol

In tests of hemocompatibility, no hemolysis was observed. In functional assessments, when mixed with thrombocytopenic whole blood, the function of Mirasol PRT-treated platelets was well preserved, in comparison with controls [37]. Treated platelets displayed no evidence of

After a single IV administration of Mirasol treated plasma containing photolyzed <sup>14</sup>C-RB, the radioactivity was well distributed from the whole blood to tissues selected for assay within

PRT System gave negative results in all genotoxicity experiments.

**3.3. Neoantigenicity and cytotoxicity**

hyperactivation or hypercoagulability.

**3.4. Hemocompatibility**

Mirasol treatment.

PRT-treated products, no cytotoxicity was observed.

**3.5. Pharmacokinetics of photolyzed 14C-RB in rats**

### **3.2. Developmental toxicity and genotoxicity**

No developmental toxicity was observed in the embryo-fetal development study. All fetuses were examined for malformations and developmental variations. No mutagenicity was


**Table 1.** In vivo\* and in vitro# toxicology.

**Figure 2.** Riboflavin and its photoproducts are naturally present in human blood; no new compounds are formed after Mirasol treatment.

observed in the Ames test for treated or control human platelets, or for lumichrome. The in vitro and in vivo tests for clastogenicity in mammalian cells (chromosomal aberration in cultured CHO cells and micronucleus test in mouse bone marrow cells, respectively) were also performed with Mirasol PRT-treated products. Human platelets treated with the Mirasol PRT System gave negative results in all genotoxicity experiments.

### **3.3. Neoantigenicity and cytotoxicity**

evaluation program in support of the Mirasol PRT System, designed to investigate all potential sources of concern, was conducted as part of the overall development program. In vivo animal and in vitro toxicity studies were performed using RB, lumichrome and photolyzed

To obtain a consistent test article in as humane a fashion as possible for those studies, speciesspecific plasma was used rather than platelets. The photochemistry of RB yields equivalent photoproduct profiles in plasma products and in platelet products (which consist mainly of plasma). The absence of platelets eliminates the possibility of detecting toxic alterations to the platelet surface; however, that issue was addressed in the neoantigenicity and <sup>14</sup>C-RB binding studies.

No toxicologically significant findings were observed in any of the studies of acute toxicity. In the repeated-dose toxicity study, the levels of RB and lumichrome in blood samples from animals receiving Mirasol PRT-treated products were below the limits of quantification, as were the levels in blood samples from animals receiving untreated plasma. These results were consistent with the observed rapid clearance of RB after IV administration, both in the literature [9] and in the pharmacokinetic study with 14C-RB in Mirasol PRT-treated products. RB and its photoproducts naturally occur in human blood, see **Figure 2**. All photoproducts were found to be present in apheresis platelet products that had not undergone any photochemical treatment, although at a much lower concentration. The presence of these agents in human blood, the ubiquitous nature of RB exposure, its presence in human diets and the ability of humans to metabolize it

and manage its inherent photochemistry suggests a low risk profile for this product.

• Acute Toxicity\* Negative • Neoantigenicity\* Negative • Ames Mutagenicity# Negative • CHO Clastogenicity# Negative • Cytotoxicity# Negative • Reproductive Toxicity\* Negative • Subchronic Toxicity\* Negative • MMN Genotoxicity\* Negative • Blood Compatibility# Passed • Leachables and Extractables# Passed

No developmental toxicity was observed in the embryo-fetal development study. All fetuses were examined for malformations and developmental variations. No mutagenicity was

**3.2. Developmental toxicity and genotoxicity**

RB (see **Table 1**).

132 B Group Vitamins - Current Uses and Perspectives

**3.1. Systemic toxicity**

**Table 1.** In vivo\* and in vitro#

toxicology.

Results of studies using <sup>14</sup>C-labeled RB and exposure of platelets and plasma to UV light did not demonstrate any detectable binding of RB or its photoproducts to platelets or to plasma proteins. No evidence of neoantigenicity was observed with the Ouchterlony assay, indicating that no new antigens were formed during treatment with the Mirasol PRT System. Treatment with the Mirasol PRT System did not result in greater immunoglobulin G binding than what was observed in comparison with untreated controls, when assessed with the Capture-P assay. In the tests of lumichrome cytotoxicity, and of the cytotoxicity of Mirasol PRT-treated products, no cytotoxicity was observed.

### **3.4. Hemocompatibility**

In tests of hemocompatibility, no hemolysis was observed. In functional assessments, when mixed with thrombocytopenic whole blood, the function of Mirasol PRT-treated platelets was well preserved, in comparison with controls [37]. Treated platelets displayed no evidence of hyperactivation or hypercoagulability.

### **3.5. Pharmacokinetics of photolyzed 14C-RB in rats**

After a single IV administration of Mirasol treated plasma containing photolyzed <sup>14</sup>C-RB, the radioactivity was well distributed from the whole blood to tissues selected for assay within the first hour postdose. Most of the excreted urinary radioactivity was recovered by 12 h postdose, and more than half of all radioactivity was excreted in urine. Blood levels of radioactivity declined rapidly post-dose, as expected from studies of RB metabolism and excretion in humans [9]. Measurements of the radioactivity associated with the 14C-RB-treated plasma indicated rapid initial apparent distribution (and/or clearance) from the systemic circulation that appeared to be complete within the first 8 to 48 h postdose.

Though quite effective for the treatment of plasma, neither of these methods could be used for cellular blood products. Two newer technologies have been developed, both using UV light and two distinct chemical compounds to enable irreversible breakage of nucleic acids and blocking further replication of cells and pathogens. One technology uses amotosalen hydrochloric acid or S-59 as the photoactive-compound, which together with its photoproducts need to be removed from the blood component post-illumination due to its high toxic profile [47]. The second system uses riboflavin, a natural vitamin (vitamin B2) of which both photoproducts and catabolites are found endogenously in the normal blood and therefore do

Vitamin B2 and Innovations in Improving Blood Safety http://dx.doi.org/10.5772/intechopen.78260 135

Pathogen reduction is a proactive strategy to mitigate the risk of transfusion-transmitted infections. The Mirasol PRT System consists of an illumination/storage bag, RB solution, and an Illuminator that delivers UV light to cause permanent damage to nucleic acids of pathogens and leukocytes (see **Figure 3**). The system has been shown to be effective against clinically relevant pathogens [50, 51] and inactivates leukocytes [52] without significantly compromising the efficacy of the product [53–55] or resulting in product loss. The process involves transferring the blood component to the Illumination/Storage bag and adding 35 ± 5 mL of RB solution (500 μM). The product is then placed into the Mirasol Illuminator and exposed to UV light. After illumination, the final PRT treated product can be transfused immediately or stored without the need for additional filtration or processing. Treated plasma products are transferred to a storage bag appropriate for freezing. The Mirasol PRT System has been developed with the flexibility of treating plasma and platelet components, as well as whole blood.

not need to be eliminated from the blood component before transfusion [6, 48, 49].

**5. The Mirasol PRT process**

**Figure 3.** Mirasol PRT System.

### **3.6. Leachables and extractables**

The leachables and extractables analyses detected no polymeric material in either test or control platelet products. The Mirasol illumination/storage bag does not contain the plasticizer di (2-ethylhexyl)phthalate (DEHP), and testing verified that this plasticizer was not present in treated and stored products. No toxicologically relevant concentrations of metals were found. These results correlate with those from the biocompatibility testing of the Mirasol illumination/storage bag elements—all elements were biocompatible.

### **4. Safety of blood**

Blood transfusion safety is considered by the World Health Organization an integral part of each country's national health care policy and infrastructure [38]. In the last four decades safety of blood has been positively impacted by technological, economic and social improvements [2]. Improvements in blood processing and storage as per good manufacturing practices (GMP), introduction of policies discouraging paid blood donation and successive addition of screening tests for known transmissible pathogens, as Hepatitis B virus (HBV), Human Immunodeficiency viruses 1 and 2 (HIV-1/2) and Hepatitis C virus (HCV) are among the most successful measures to increase quality and safety of blood transfusion worldwide [39, 40]. From the late nineties onwards, introduction of nucleic acid testing (NAT) was able to minimize the window period of detection of these three viruses in asymptomatic blood donors to single days [41, 42].

Yet, in the last 20 years attention has been drawn to blood safety threats by recently known and/or re-emergent pathogens such as, Severe Acute Respiratory Syndrome virus (SARS), West-Nile Fever virus (WNV), Chikungunya virus (CHIKV), Dengue virus (DENV) or most recently ZIKA virus (ZIKV). Epidemics of these diseases are geographical or seasonal in nature and may not necessarily require universal reactive measures [2, 43, 44].

These unpredictable threats, as well as the long-recognized risk of bacterial transmission through platelet transfusion, may be effectively countered through the novel proactive approach with broad applicability and effectiveness, the pathogen inactivation/reduction technology (PRT) [4]. PRT has first been used to treat plasma and focused on destroying the structural elements of potential pathogens by the solvent-detergent method [45]. By the midnineties, the nucleic acid binding properties of Methylene Blue (MB) became exploited in a pathogen inactivation system for fresh frozen plasma using visible light [46].

Though quite effective for the treatment of plasma, neither of these methods could be used for cellular blood products. Two newer technologies have been developed, both using UV light and two distinct chemical compounds to enable irreversible breakage of nucleic acids and blocking further replication of cells and pathogens. One technology uses amotosalen hydrochloric acid or S-59 as the photoactive-compound, which together with its photoproducts need to be removed from the blood component post-illumination due to its high toxic profile [47]. The second system uses riboflavin, a natural vitamin (vitamin B2) of which both photoproducts and catabolites are found endogenously in the normal blood and therefore do not need to be eliminated from the blood component before transfusion [6, 48, 49].

### **5. The Mirasol PRT process**

the first hour postdose. Most of the excreted urinary radioactivity was recovered by 12 h postdose, and more than half of all radioactivity was excreted in urine. Blood levels of radioactivity declined rapidly post-dose, as expected from studies of RB metabolism and excretion in humans [9]. Measurements of the radioactivity associated with the 14C-RB-treated plasma indicated rapid initial apparent distribution (and/or clearance) from the systemic circulation

The leachables and extractables analyses detected no polymeric material in either test or control platelet products. The Mirasol illumination/storage bag does not contain the plasticizer di (2-ethylhexyl)phthalate (DEHP), and testing verified that this plasticizer was not present in treated and stored products. No toxicologically relevant concentrations of metals were found. These results correlate with those from the biocompatibility testing of the Mirasol illumina-

Blood transfusion safety is considered by the World Health Organization an integral part of each country's national health care policy and infrastructure [38]. In the last four decades safety of blood has been positively impacted by technological, economic and social improvements [2]. Improvements in blood processing and storage as per good manufacturing practices (GMP), introduction of policies discouraging paid blood donation and successive addition of screening tests for known transmissible pathogens, as Hepatitis B virus (HBV), Human Immunodeficiency viruses 1 and 2 (HIV-1/2) and Hepatitis C virus (HCV) are among the most successful measures to increase quality and safety of blood transfusion worldwide [39, 40]. From the late nineties onwards, introduction of nucleic acid testing (NAT) was able to minimize the window period of detection of these three viruses in asymptomatic blood

Yet, in the last 20 years attention has been drawn to blood safety threats by recently known and/or re-emergent pathogens such as, Severe Acute Respiratory Syndrome virus (SARS), West-Nile Fever virus (WNV), Chikungunya virus (CHIKV), Dengue virus (DENV) or most recently ZIKA virus (ZIKV). Epidemics of these diseases are geographical or seasonal in

These unpredictable threats, as well as the long-recognized risk of bacterial transmission through platelet transfusion, may be effectively countered through the novel proactive approach with broad applicability and effectiveness, the pathogen inactivation/reduction technology (PRT) [4]. PRT has first been used to treat plasma and focused on destroying the structural elements of potential pathogens by the solvent-detergent method [45]. By the midnineties, the nucleic acid binding properties of Methylene Blue (MB) became exploited in a

nature and may not necessarily require universal reactive measures [2, 43, 44].

pathogen inactivation system for fresh frozen plasma using visible light [46].

that appeared to be complete within the first 8 to 48 h postdose.

tion/storage bag elements—all elements were biocompatible.

**3.6. Leachables and extractables**

134 B Group Vitamins - Current Uses and Perspectives

**4. Safety of blood**

donors to single days [41, 42].

Pathogen reduction is a proactive strategy to mitigate the risk of transfusion-transmitted infections. The Mirasol PRT System consists of an illumination/storage bag, RB solution, and an Illuminator that delivers UV light to cause permanent damage to nucleic acids of pathogens and leukocytes (see **Figure 3**). The system has been shown to be effective against clinically relevant pathogens [50, 51] and inactivates leukocytes [52] without significantly compromising the efficacy of the product [53–55] or resulting in product loss. The process involves transferring the blood component to the Illumination/Storage bag and adding 35 ± 5 mL of RB solution (500 μM). The product is then placed into the Mirasol Illuminator and exposed to UV light. After illumination, the final PRT treated product can be transfused immediately or stored without the need for additional filtration or processing. Treated plasma products are transferred to a storage bag appropriate for freezing. The Mirasol PRT System has been developed with the flexibility of treating plasma and platelet components, as well as whole blood.

**Figure 3.** Mirasol PRT System.

### **5.1. Platelets and plasma**

PRT-treated plasma products have been on the market in Europe for more than a decade and were issued a CE Mark in 2007 and 2008 for platelets and plasma respectively. FFP intended for transfusion to patients with multiple coagulation factor deficiencies (e.g. massive transfusion), emergency reversal of warfarin as well as for therapeutic plasma exchange must contain adequate functional levels of coagulation factors and other therapeutically valuable proteins. Protein levels should be as close as possible to those found in fresh plasma. Blood component processing can affect the quality of plasma products, particularly labile coagulation factors such as factors V and VIII.

2015 and is a significant step forward ensuring blood safety where whole blood transfusions

The Mirasol PRT System pathogen reduction process has been evaluated for performance against several pathogens. **Table 2** summarizes the pathogen reduction results. The data show reduction factors ranging from 2 to 6 log (99.0–99.9999% reduction) for each pathogen tested with the Mirasol PRT System. Log reduction values reported in the table were calculated by determining the number of virus particles present in infectious form prior to treatment and the number of virus particles present after treatment. The level of log reduction is reported as the starting titer expressed in units of 10× per mL minus the level after treatment expressed as the titer in 10× per mL. Because volume was constant in the samples before and after treat-

For example, a sample containing 1,000,000 infectious virus particles per mL would of course

level of 99.99%. Because values are reported in log units, 100% reduction is never achievable.

Despite the fact that bacterial contamination of blood products poses one of the greatest risks for transfusion, there are currently no standards in place that establish a panel of species to test or a method to evaluate technology for pathogen reduction. A panel based on published hemovigilance studies incorporating those species responsible for the majority of morbidity and mortality in transfusion-associated reactions was utilized to guide study targets. Two complementary test methods were developed, as described below, to measure bacterial

To assess bacterial reduction efficiency of the system, two complementary test methods, known as "High Spike Bacterial Titer" and "Low Spike Bacterial Titer" tests, for bacterial reduction have been developed to measure the Mirasol PRT System performance. Both methods involve inoculation of known titers of bacteria (a "spiking" study) into platelet products followed by PRT treatment and subsequent measurement of the presence of bacteria. The objective of the High Spike Bacterial Titer experiments is to determine the overall bacterial reduction ability of the system against a severely contaminated platelet product. These studies may not, however,

**Pathogen type Typical performance** Viruses (enveloped, non-enveloped; intracellular, extracellular) ~2 to 6 log (99.0–99.9999%)

Bacteria (Gram +, Gram −) ~2 to 5 log (99.0–99.999%)

Parasites (Malaria, Chagas, Babesiosis, Leishmaniasis, Scub typhus) ≥ 3.0 to ≥ 5.0 (≥ 99.9% to ≥ 99.999%)

virus particles per mL. If after treatment, only 100 particles per mL were measured in

virus particles per mL. The log

or 4 logs. This corresponds to a reduction in virus

Vitamin B2 and Innovations in Improving Blood Safety http://dx.doi.org/10.5772/intechopen.78260 137

are routine, such as sub-Saharan Africa and in far-forward combat situations.

ment, the unit of volume cancels, resulting in a reported value of log reduction.

tissue culture infectivity assays, this would correspond to 102

reduction reported for this system would be 104

**6. Pathogen reduction performance**

have 106

reduction performance.

**Table 2.** Pathogen reduction performance.

Mirasol-treated FFP shows high overall protein retention under a broad range of blood banking conditions. Mirasol-treated FFP meets the European guidelines [56], showing on average factor VIIIc levels of 0.8 ± 0.2 IU/ml post treatment. Protein content meets guidelines even when whole blood is held overnight at room temperature and plasma is separated up to 18 h or frozen up to 24 h after collection. Additionally, anticoagulant factors such as protein C and protein S are well preserved after treatment with a 96% retention reported post treatment for both proteins. Extended storage of treated plasma at −30°C for up to 2 years does not significantly decrease protein quality [57].

Platelet products derived from apheresis or whole-blood can be treated with the system and products can be stored either in plasma or platelet additive solutions (PAS) for up to 7 days under standard blood banking conditions. It is critical that Mirasol-treated platelets remain viable and hemostatically effective. A series of in vitro studies have been performed to assess platelet quality after Mirasol treatment, and a correlation between in vitro parameters and in vivo performance was established [58]. In these studies pH and lactate production rate were found to be most strongly correlated with the in vivo recovery and survival of Mirasol-treated platelets. Glucose consumption rate and swirl also showed some correlation with these in vivo parameters, though to a lesser extent. P-selectin, pO<sup>2</sup> and pCO<sup>2</sup> expression in Mirasol-treated platelets, however, were poorly correlated with in vivo platelet recovery and survival. Changes in cell quality parameters do occur, cellular metabolism is up-regulated in treated platelets, and treatment induces some degree of platelet activation. However, shear-induced adhesion is maintained in Mirasol-treated platelets, and mitochondrial function is preserved [37, 59].

### **5.2. Whole blood: military and developing world**

The Mirasol system was further developed for the treatment of whole blood, providing a single pathogen reduction and leukocyte inactivation step, followed by the use of the product as whole blood or pRBCs. The treatment of RBCs or whole blood has been more challenging due to the absorption of light by hemoglobin. Although the peak absorption of hemoglobin (400–450 nm) is outside the spectral region of the Mirasol lamp output, the UV light energy dose delivered to units of whole blood is normalized for RBC volume (J/mlRBC). In vitro cell quality studies have verified that adequate quality and functionality of the RBCs and plasma components post treatment and throughout 21 days of storage is preserved [49]. In addition, crossmatch compatibility of the products is preserved. PRT treatment of whole blood has received CE marking in 2015 and is a significant step forward ensuring blood safety where whole blood transfusions are routine, such as sub-Saharan Africa and in far-forward combat situations.

### **6. Pathogen reduction performance**

**5.1. Platelets and plasma**

136 B Group Vitamins - Current Uses and Perspectives

such as factors V and VIII.

cantly decrease protein quality [57].

parameters, though to a lesser extent. P-selectin, pO<sup>2</sup>

**5.2. Whole blood: military and developing world**

PRT-treated plasma products have been on the market in Europe for more than a decade and were issued a CE Mark in 2007 and 2008 for platelets and plasma respectively. FFP intended for transfusion to patients with multiple coagulation factor deficiencies (e.g. massive transfusion), emergency reversal of warfarin as well as for therapeutic plasma exchange must contain adequate functional levels of coagulation factors and other therapeutically valuable proteins. Protein levels should be as close as possible to those found in fresh plasma. Blood component processing can affect the quality of plasma products, particularly labile coagulation factors

Mirasol-treated FFP shows high overall protein retention under a broad range of blood banking conditions. Mirasol-treated FFP meets the European guidelines [56], showing on average factor VIIIc levels of 0.8 ± 0.2 IU/ml post treatment. Protein content meets guidelines even when whole blood is held overnight at room temperature and plasma is separated up to 18 h or frozen up to 24 h after collection. Additionally, anticoagulant factors such as protein C and protein S are well preserved after treatment with a 96% retention reported post treatment for both proteins. Extended storage of treated plasma at −30°C for up to 2 years does not signifi-

Platelet products derived from apheresis or whole-blood can be treated with the system and products can be stored either in plasma or platelet additive solutions (PAS) for up to 7 days under standard blood banking conditions. It is critical that Mirasol-treated platelets remain viable and hemostatically effective. A series of in vitro studies have been performed to assess platelet quality after Mirasol treatment, and a correlation between in vitro parameters and in vivo performance was established [58]. In these studies pH and lactate production rate were found to be most strongly correlated with the in vivo recovery and survival of Mirasol-treated platelets. Glucose consumption rate and swirl also showed some correlation with these in vivo

platelets, however, were poorly correlated with in vivo platelet recovery and survival. Changes in cell quality parameters do occur, cellular metabolism is up-regulated in treated platelets, and treatment induces some degree of platelet activation. However, shear-induced adhesion is maintained in Mirasol-treated platelets, and mitochondrial function is preserved [37, 59].

The Mirasol system was further developed for the treatment of whole blood, providing a single pathogen reduction and leukocyte inactivation step, followed by the use of the product as whole blood or pRBCs. The treatment of RBCs or whole blood has been more challenging due to the absorption of light by hemoglobin. Although the peak absorption of hemoglobin (400–450 nm) is outside the spectral region of the Mirasol lamp output, the UV light energy dose delivered to units of whole blood is normalized for RBC volume (J/mlRBC). In vitro cell quality studies have verified that adequate quality and functionality of the RBCs and plasma components post treatment and throughout 21 days of storage is preserved [49]. In addition, crossmatch compatibility of the products is preserved. PRT treatment of whole blood has received CE marking in

and pCO<sup>2</sup>

expression in Mirasol-treated

The Mirasol PRT System pathogen reduction process has been evaluated for performance against several pathogens. **Table 2** summarizes the pathogen reduction results. The data show reduction factors ranging from 2 to 6 log (99.0–99.9999% reduction) for each pathogen tested with the Mirasol PRT System. Log reduction values reported in the table were calculated by determining the number of virus particles present in infectious form prior to treatment and the number of virus particles present after treatment. The level of log reduction is reported as the starting titer expressed in units of 10× per mL minus the level after treatment expressed as the titer in 10× per mL. Because volume was constant in the samples before and after treatment, the unit of volume cancels, resulting in a reported value of log reduction.

For example, a sample containing 1,000,000 infectious virus particles per mL would of course have 106 virus particles per mL. If after treatment, only 100 particles per mL were measured in tissue culture infectivity assays, this would correspond to 102 virus particles per mL. The log reduction reported for this system would be 104 or 4 logs. This corresponds to a reduction in virus level of 99.99%. Because values are reported in log units, 100% reduction is never achievable.

Despite the fact that bacterial contamination of blood products poses one of the greatest risks for transfusion, there are currently no standards in place that establish a panel of species to test or a method to evaluate technology for pathogen reduction. A panel based on published hemovigilance studies incorporating those species responsible for the majority of morbidity and mortality in transfusion-associated reactions was utilized to guide study targets. Two complementary test methods were developed, as described below, to measure bacterial reduction performance.

To assess bacterial reduction efficiency of the system, two complementary test methods, known as "High Spike Bacterial Titer" and "Low Spike Bacterial Titer" tests, for bacterial reduction have been developed to measure the Mirasol PRT System performance. Both methods involve inoculation of known titers of bacteria (a "spiking" study) into platelet products followed by PRT treatment and subsequent measurement of the presence of bacteria. The objective of the High Spike Bacterial Titer experiments is to determine the overall bacterial reduction ability of the system against a severely contaminated platelet product. These studies may not, however,


**Table 2.** Pathogen reduction performance.

represent a clinically relevant finding in that viable bacteria remaining after treatment may grow to high titers through the storage period. The objective of the "Low Spike Bacterial Titer Experiments" is to spike a platelet product with a more clinically relevant bacterial titer, treat the product using the Mirasol PRT System, and evaluate the platelet product using a standard culture system through a 7-day storage period to determine if it has remained culture negative, indicating that the platelet product meets release criteria for transfusion. The system demonstrated 98% effectiveness in these studies against a broad range of bacteria [60]. The combined data from these studies demonstrates the bacterial reduction capability of the system under conditions that are still substantially higher challenges than may be anticipated in an actual clinical setting.

PRT treatment of blood components is regarded as the next step to increase blood safety and support the credibility of blood institutions and health policy makers. However, there is a lack of consistency in the decision-making criteria used by regulatory bodies and blood operators regarding PRT implementation. The European Directorate for the Quality of Medicines & Healthcare of the Council of Europe in its Guide to the Preparation, Use and Quality Assurance of Blood Components, 19th Edition defines properties and requirements for therapeutic plasma, platelet concentrates and cryoprecipitate treated with PRT [56], yet PRT treatment of blood components is mandated in very few countries in Europe. Belgium, Switzerland and France have made the use of pathogen-inactivation mandatory for the treatment of platelet concentrates. Plasma treated by PRT is mandatory in Belgium, whereas the use of solvent/detergent treated plasma is more widespread in Europe but not mandated by

Vitamin B2 and Innovations in Improving Blood Safety http://dx.doi.org/10.5772/intechopen.78260 139

The Mirasol PRT system has been gradually adopted in Europe, Asia and Latin-America. A hemovigilance program, based on the collection of passive hemovigilance data of Mirasoltreated components in multiple blood transfusion centers in Europe started in 2010. By 2015 data about 94,509 transfused platelet concentrates and 96,115 plasma transfusions were recorded in the program [64]. By 2017 over 750,000 disposable treatment sets have been distributed world-

It is reasonable to envisage a future when all labile blood components will be PR treated to ensure a safe and sustainable blood supply in accordance with regional transfusion best practices. PR treatment of WB represents the most efficient implementation path to achieve this goal. It has been recently demonstrated through a clinical trial in a malaria-endemic country that a WB PR technology based upon riboflavin and UV light does reduce the risk of transfusion-transmitted malaria [61]. RBCs derived from PR-treated WB have shown good quality and recovery in health subjects and are currently being evaluated in a pivotal

Parts of the chapter were taken from previously published papers by Goodrich RP and co-

Marcia Cardoso and Susanne Marschner are employees of Terumo BCT, the manufacturer of

wide and 225,000 transfusion data have been recorded in the hemovigilance program.

national agencies.

**9. Future**

clinical trial [65].

**Acknowledgements**

**Conflict of interest**

the Mirasol PRT System.

workers et al., and we have the permission to re-use it.

### **7. Clinical performance**

There have been 11 completed clinical studies with the Mirasol PRT System for Platelets stored in 100% plasma or platelet additive solution (PAS). There are two ongoing clinical studies in the United States, one study with platelets and one with RBCs derived from Mirasol-Treated Whole Blood. Primary outcome measure in most clinical studies has been levels of circulating platelets in thrombocytopenic patient's blood after prophylactic transfusion. Both CI (count increment) and CCI (corrected count increment) are accepted as surrogate markers of platelet transfusion efficacy, but they do not necessarily account for platelet function or bleeding outcomes in patients and they rely upon the assumption that a sufficient number of circulating, intact platelets will provide protection against bleeding. Patient factors and platelet product variability have been shown in published studies to affect increments, limiting the sensitivity of the CCI as a clinical efficacy measure. The CCI at one and 24 h after transfusion is decreased in patients receiving PRT treated products, compared to patients receiving control products. In two recent clinical trials Grade 2 or higher bleeding was the primary endpoint [55, 66]. Although lower CCIs were observed in these 2 studies, no difference in clinically meaningful bleeding in thrombocytopenic patients was observed.

The clinical evaluation of the Mirasol Whole Blood system includes a clinical trial in patients assessing the incidence of transfusion transmitted *Plasmodium* spp. infection that was conducted in Kumasi, Ghana [61]. Treatment of whole blood reduced significantly the incidence of transfusion-transmitted malaria. The safety profile and clinical outcomes were similar between test and control groups.

### **8. Current adoption for routine use**

Since 2007, when the Canadian Consensus Conference on Pathogen Inactivation (PI) concluded that a proactive approach in accordance to the precautionary principle would reduce the theoretical risk and help sustain public confidence in the blood supply, many national and international committees, such as the Advisory Committee on Blood Safety and Availability (ACBSA), USA and the European Committee on Blood Transfusion of the Council of Europe discussed the accumulating evidence about the efficacy and safety of PRT [62, 63].

PRT treatment of blood components is regarded as the next step to increase blood safety and support the credibility of blood institutions and health policy makers. However, there is a lack of consistency in the decision-making criteria used by regulatory bodies and blood operators regarding PRT implementation. The European Directorate for the Quality of Medicines & Healthcare of the Council of Europe in its Guide to the Preparation, Use and Quality Assurance of Blood Components, 19th Edition defines properties and requirements for therapeutic plasma, platelet concentrates and cryoprecipitate treated with PRT [56], yet PRT treatment of blood components is mandated in very few countries in Europe. Belgium, Switzerland and France have made the use of pathogen-inactivation mandatory for the treatment of platelet concentrates. Plasma treated by PRT is mandatory in Belgium, whereas the use of solvent/detergent treated plasma is more widespread in Europe but not mandated by national agencies.

The Mirasol PRT system has been gradually adopted in Europe, Asia and Latin-America. A hemovigilance program, based on the collection of passive hemovigilance data of Mirasoltreated components in multiple blood transfusion centers in Europe started in 2010. By 2015 data about 94,509 transfused platelet concentrates and 96,115 plasma transfusions were recorded in the program [64]. By 2017 over 750,000 disposable treatment sets have been distributed worldwide and 225,000 transfusion data have been recorded in the hemovigilance program.

### **9. Future**

represent a clinically relevant finding in that viable bacteria remaining after treatment may grow to high titers through the storage period. The objective of the "Low Spike Bacterial Titer Experiments" is to spike a platelet product with a more clinically relevant bacterial titer, treat the product using the Mirasol PRT System, and evaluate the platelet product using a standard culture system through a 7-day storage period to determine if it has remained culture negative, indicating that the platelet product meets release criteria for transfusion. The system demonstrated 98% effectiveness in these studies against a broad range of bacteria [60]. The combined data from these studies demonstrates the bacterial reduction capability of the system under conditions that are still substantially higher challenges than may be anticipated in an actual clinical setting.

There have been 11 completed clinical studies with the Mirasol PRT System for Platelets stored in 100% plasma or platelet additive solution (PAS). There are two ongoing clinical studies in the United States, one study with platelets and one with RBCs derived from Mirasol-Treated Whole Blood. Primary outcome measure in most clinical studies has been levels of circulating platelets in thrombocytopenic patient's blood after prophylactic transfusion. Both CI (count increment) and CCI (corrected count increment) are accepted as surrogate markers of platelet transfusion efficacy, but they do not necessarily account for platelet function or bleeding outcomes in patients and they rely upon the assumption that a sufficient number of circulating, intact platelets will provide protection against bleeding. Patient factors and platelet product variability have been shown in published studies to affect increments, limiting the sensitivity of the CCI as a clinical efficacy measure. The CCI at one and 24 h after transfusion is decreased in patients receiving PRT treated products, compared to patients receiving control products. In two recent clinical trials Grade 2 or higher bleeding was the primary endpoint [55, 66]. Although lower CCIs were observed in these 2 studies, no difference in clinically meaningful

The clinical evaluation of the Mirasol Whole Blood system includes a clinical trial in patients assessing the incidence of transfusion transmitted *Plasmodium* spp. infection that was conducted in Kumasi, Ghana [61]. Treatment of whole blood reduced significantly the incidence of transfusion-transmitted malaria. The safety profile and clinical outcomes were similar

Since 2007, when the Canadian Consensus Conference on Pathogen Inactivation (PI) concluded that a proactive approach in accordance to the precautionary principle would reduce the theoretical risk and help sustain public confidence in the blood supply, many national and international committees, such as the Advisory Committee on Blood Safety and Availability (ACBSA), USA and the European Committee on Blood Transfusion of the Council of Europe

discussed the accumulating evidence about the efficacy and safety of PRT [62, 63].

**7. Clinical performance**

138 B Group Vitamins - Current Uses and Perspectives

bleeding in thrombocytopenic patients was observed.

between test and control groups.

**8. Current adoption for routine use**

It is reasonable to envisage a future when all labile blood components will be PR treated to ensure a safe and sustainable blood supply in accordance with regional transfusion best practices. PR treatment of WB represents the most efficient implementation path to achieve this goal. It has been recently demonstrated through a clinical trial in a malaria-endemic country that a WB PR technology based upon riboflavin and UV light does reduce the risk of transfusion-transmitted malaria [61]. RBCs derived from PR-treated WB have shown good quality and recovery in health subjects and are currently being evaluated in a pivotal clinical trial [65].

### **Acknowledgements**

Parts of the chapter were taken from previously published papers by Goodrich RP and coworkers et al., and we have the permission to re-use it.

### **Conflict of interest**

Marcia Cardoso and Susanne Marschner are employees of Terumo BCT, the manufacturer of the Mirasol PRT System.

### **Author details**

Raymond P. Goodrich<sup>1</sup> , Marcia Cardoso<sup>2</sup> and Susanne Marschner2 \*

\*Address all correspondence to: susanne.marschner@terumobct.com


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recommendations/resjan08.pdf

## *Edited by Jean Guy LeBlanc and Graciela Savoy de Giori*

B-group vitamins are involved in numerous metabolic reactions and their widespread deficiency can cause a large series of health problems. The aim of this book is to provide an update on the current use and perspectives of B-group vitamins. Novel methods to detect folates in pregnant women, the use and role of folate dentistry, the use of genotype notification to modify food intake behavior, thiamin metabolism in Archaea and its role in plants and in crop improvement, the use of riboflavin in blood safety and niacin in metabolic stress and resistance in dairy cows are some of the subjects that are described in this multitopic book written by authors from seven different countries.

Published in London, UK © 2018 IntechOpen © Viktoriya Kuzmenkova / iStock

B Group Vitamins - Current Uses and Perspectives

B Group Vitamins

Current Uses and Perspectives

*Edited by Jean Guy LeBlanc* 

*and Graciela Savoy de Giori*