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Mechanism of hypercalcemia in HL has long been suggested to involve extra-renal activation of 1α-hydroxylase leading to production of 1, 25(OD)2 Vitamin D3 or Calcitriol, an active metabolite of Vitamin D, which leads to increased re-absorption of calcium and phosphate from intestine, increased osteoclast activation and bone resorption as well as increased phosphate re-absorption in renal tubules

Hypercalcemia of malignancy involves three mechanisms: 1. Humoral hypercalcemia mediated by PTHrP—seen in solid tumors like breast cancer and adult T cell leukemia/lymphoma (ATLL), 2. Direct osteoclast mediated bone resorption due to bony metastasis—seen in solid tumors and multiple myeloma, 3. Calcitriol mediated hypercalcemia—seen in Hodgkin’s and non-Hodgkin’s lymphoma as well as granulomatous disorders like tuberculosis, sarcoidosis, leprosy and disseminated Candidiasis

Hypercalcemia in HL is rare and its incidence has been reported as 0.9, 1.6 and 5.4 % in different series

The source of 1α-hydroxylase in HL has been postulated as monocytes and macrophages infiltrating the tumor akin to tuberculosis or sarcoidosis and is stimulated by IFN-γ secreted by T-lymphocytes

Like sarcoidosis, patients with HL exhibit increased sensitivity to Vitamin D supplements and sunlight, which have been found to precipitate hypercalcemia in these patients

Classical biochemical profile in Calcitriol mediated hypercalcemia include: an elevated calcium, normal/slightly elevated phosphate, normal 25(OH) Vitamin D, suppressed PTHrP and PTH, elevated Calcitriol and a normal/increased tubular reabsorption of phosphate

not been associated with a poorer prognosis and tends to subside after treatment of the underlying disease

Our data show that ascorbic acid selectively killed cancer but not normal cells, using concentrations that could only be achieved by i.v. administration

Ascorbate-mediated cell death was due to protein-dependent extracellular H2O2 generation, via ascorbate radical formation from ascorbate as the electron donor. Like glucose, when ascorbate is infused i.v., the resulting pharmacologic concentrations should distribute rapidly in the extracellular water space (42). We showed that such pharmacologic ascorbate concentrations in media, as a surrogate for extracellular fluid, generated ascorbate radical and H2O2. In contrast, the same pharmacologic ascorbate concentrations in whole blood generated little detectable ascorbate radical and no detectable H2O2. These findings can be accounted for by efficient and redundant H2O2 catabolic pathways in whole blood (e.g., catalase and glutathione peroxidase) relative to those in media or extracellular fluid

ascorbic acid administered i.v. in pharmacologic concentrations may serve as a pro-drug for H2O2 delivery to the extracellular milieu

H2O2 generated in blood is normally removed by catalase and glutathione peroxidase within red blood cells, with internal glutathione providing reducing equivalents

The electron source for glutathione is NADPH from the pentose shunt, via glucose-6-phosphate dehydrogenase. If activity of this enzyme is diminished, the predicted outcome is impaired H2O2 removal causing intravascular hemolysis, the observed clinical finding.

Only recently has it been understood that the discordant clinical findings can be explained by previously unrecognized fundamental pharmacokinetics properties of ascorbate

Intracellular transport of ascorbate is tightly controlled in relation to extracellular concentration

Intravenous ascorbate infusion is expected to drastically change extracellular but not intracellular concentrations

For i.v. ascorbate to be clinically useful in killing cancer cells, pharmacologic but not physiologic extracellular concentrations should be effective, independent of intracellular ascorbate concentrations.

It is unknown why ascorbate, via H2O2, killed some cancer cells but not normal cells.

There was no correlation with ascorbate-induced cell death and glutathione, catalase activity, or glutathione peroxidase activity.

H2O2, as the product of pharmacologic ascorbate concentrations, has potential therapeutic uses in addition to cancer treatment, especially in infections

Neutrophils generate H2O2 from superoxide,

i.v. ascorbate is effective in some viral infections

H2O2 is toxic to hepatitis C

Use of ascorbate as an H2O2-delivery system against sensitive pathogens, viral or bacterial, has substantial clinical implications that deserve rapid exploration.

Recent pharmacokinetics studies in men and women show that 10 g of ascorbate given i.v. is expected to produce plasma concentrations of nearly 6 mM, which are >25-fold higher than those concentrations from the same oral dose

As much as a 70-fold difference in plasma concentrations is expected between oral and i.v. administration,

Complementary and alternative medicine practitioners worldwide currently use ascorbate i.v. in some patients, in part because there is no apparent harm

Human Burkitt's lymphoma cells

We first investigated whether ascorbate in pharmacologic concentrations selectively affected the survival of cancer cells by studying nine cancer cell lines

Clinical pharmacokinetics analyses show that pharmacologic concentrations of plasma ascorbate, from 0.3 to 15 mM, are achievable only from i.v. administration

plasma ascorbate concentrations from maximum possible oral doses cannot exceed 0.22 mM because of limited intestinal absorption

For five of the nine cancer cell lines, ascorbate concentrations causing a 50% decrease in cell survival (EC50 values) were less than 5 mM, a concentration easily achievable from i.v. infusion

All tested normal cells were insensitive to 20 mM ascorbate.

Lymphoma cells were selected because of their sensitivity to ascorbate

As ascorbate concentration increased, the pattern of death changed from apoptosis to pyknosis/necrosis, a pattern suggestive of H2O2-mediated cell death

Apoptosis occurred by 6 h after exposure, and cell death by pyknosis was ≈90% at 14 h after exposure

In contrast to lymphoma cells, there was little or no killing of normal lymphocytes and monocytes by ascorbate

Ascorbate is transported into cells as such by sodium-dependent transporters, whereas dehydroascorbic acid is transported into cells by glucose transporters and then immediately reduced internally to ascorbate

Whether or not intracellular ascorbate was preloaded, extracellular ascorbate induced the same amount and type of death.

extracellular ascorbate in pharmacologic concentrations mediates death of lymphoma cells by apoptosis and pyknosis/necrosis, independently of intracellular ascorbate.

H2O2 as the effector species mediating pharmacologic ascorbate-induced cell death

Superoxide dismutase was not protective

Because these data implicated H2O2 in cell killing, we added H2O2 to lymphoma cells and studied death patterns using nuclear staining (19, 28). The death patterns found with exogenous H2O2 exposure were similar to those found with ascorbate

For both ascorbate and H2O2, death changed from apoptosis to pyknosis/necrosis as concentrations increased

Sensitivity to direct exposure to H2O2 was greater in lymphoma cells compared with normal lymphocytes and normal monocytes

There was no association between the EC50 for ascorbate-mediated cell death and intracellular glutathione concentrations, catalase activity, or glutathione peroxidase activity

H2O2 generation was dependent on time, ascorbate concentration, and the presence of trace amounts of serum in media

ascorbate radical is a surrogate marker for H2O2 formation.

whatever H2O2 is generated should be removed by glutathione peroxidase and catalase within red blood cells, because H2O2 is membrane permeable

The data are consistent with the hypothesis that ascorbate in pharmacologic concentrations is a pro-drug for H2O2 generation in the extracellular milieu but not in blood.

The occurrence of one predicted complication, oxalate kidney stones, is controversial

In patients with glucose-6-phosphate dehydrogenase deficiency, i.v. ascorbate is contraindicated because it causes intravascular hemolysis

ascorbate at pharmacologic concentrations in blood is a pro-drug for H2O2 delivery to tissues.

ascorbate, an electron-donor in such reactions, ironically initiates pro-oxidant chemistry and H2O2 formation

data here showed that ascorbate initiated H2O2 formation extracellularly, but H2O2 targets could be either intracellular or extracellular, because H2O2 is membrane permeant

More than 100 patients have been described, presumably without glucose-6-phosphate dehydrogenase deficiency, who received 10 g or more of i.v. ascorbate with no reported adverse effects other than tumor lysis

IR also indirectly induces DNA damage by stimulating reactive oxygen species (ROS) production

IR is known to induce EMT in vitro

p53 is activated in response to IR-induced DNA damage

IR paradoxically also promotes tumour recurrence and metastasis

DNA double-strand breaks (DSBs)

cancer cells undergoing EMT acquire invasive and metastatic properties

changes in the tumour microenvironment (TME)

IR seems to induce EMT and CSC phenotypes by regulating cellular metabolism

EMT, stemness, and oncogenic metabolism are known to be associated with resistance to radiotherapy and chemotherapy

Hanahan and Weinberg proposed ten hallmarks of cancer that alter cell physiology to enhance malignant growth: 1) sustained proliferation, 2) evasion of growth suppression, 3) cell death resistance, 4) replicative immortality, 5) evasion of immune destruction, 6) tumour-promoting inflammation, 7) activation of invasion and metastasis, 8) induction of angiogenesis, 9) genome instability, and 10) alteration of metabolism

EMT is a developmental process that plays critical roles in embryogenesis, wound healing, and organ fibrosis

IR is known to induce stemness and metabolic alterations in cancer cells

transforming growth factor-β [TGF-β], epidermal growth factor [EGF]) and their associated signalling proteins (Wnt, Notch, Hedgehog, nuclear-factor kappa B [NF-κB], extracellular signal-regulated kinase [ERK], and phosphatidylinositol 3-kinase [PI3K]/Akt

activate EMT-inducing transcription factors, including Snail/Slug, ZEB1/δEF1, ZEB2/SIP1, Twist1/2, and E12/E47

Loss of E-cadherin is considered a hallmark of EMT

IR has been shown to induce EMT to enhance the motility and invasiveness of several cancer cells, including those of breast, lung, and liver cancer, and glioma cells

IR may increase metastasis in both the primary tumour site and in normal tissues under some circumstance

sublethal doses of IR have been shown to enhance the migratory and invasive behaviours of glioma cells

ROS are known to play an important role in IR-induced EMT

High levels of ROS trigger cell death by causing irreversible damage to cellular components such as proteins, nucleic acids, and lipids, whereas low levels of ROS have been shown to promote tumour progression—including tumour growth, invasion, and metastasis

hypoxia-inducible factor-1 (HIF-1) is involved in IR-induced EMT

Treatment with the N-acetylcysteine (NAC), a general ROS scavenger, prevents IR-induced EMT, adhesive affinity, and invasion of breast cancer cells

Snail has been shown to play a crucial role in IR-induced EMT, migration, and invasion

IR activates the p38 MAPK pathway, which contributes to the induction of Snail expression to promote EMT and invasion

NF-κB signalling that promotes cell migration

ROS promote EMT to allow cancer cells to avoid hostile environments

HIF-1 is a heterodimer composed of an oxygen-sensitive α subunit and a constitutively expressed β subunit.

Under normoxia, HIF-1α is rapidly degraded, whereas hypoxia induces stabilisation and accumulation of HIF-1α

levels of HIF-1α mRNA are enhanced by activation of the PI3K/Akt/mammalian target of rapamycin (mTOR)

IR is known to increase stabilisation and nuclear accumulation of HIF-1α, since hypoxia is a major condition for HIF-1 activation

IR induces vascular damage that causes hypoxia

ROS is implicated in IR-induced HIF-1 activation

IR causes the reoxygenation of hypoxic cancer cells to increase ROS production, which leads to the stabilisation and nuclear accumulation of HIF-1

IR increases glucose availability under reoxygenated conditions that promote HIF-1α translation by activating the Akt/mTOR pathway

The stabilised HIF-1α then translocates to the nucleus, dimerizes with HIF-1β, and increases gene expression— including the expression of essential EMT regulators such as Snail—to induce EMT, migration, and invasion

TGF-β signalling has been shown to play a crucial role in IR-induced EMT

AP-1 transcription factor is involved in IR-induced TGF-β1 expression

Wnt/β-catenin signalling is also implicated in IR-induced EMT

Notch signalling is known to be involved in IR-induced EMT

IR also increases Notch-1 expression [99]. Notch-1 is known to induce EMT by upregulating Snail

PAI-1 signalling is also implicated in IR-induced Akt activation that increases Snail levels to induce EMT

EGFR activation is known to be associated with IR-induced EMT, cell migration, and invasion by activating two downstream pathways: PI3K/Akt and Raf/MEK/ERK

ROS and RNS are also implicated in IR-induced EGFR activation

IR has also been shown to activate Hedgehog (Hh) signalling to induce EMT

IR has been shown to induce Akt activation through several signalling pathways (EGFR, C-X-C chemokine receptor type 4 [CXCR4]/C-X-C motif chemokine 12 [CXCL12], plasminogen activator inhibitor 1 [PAI-1]) and upstream regulators (Bmi1, PTEN) that promote EMT and invasion

CSCs possess a capacity for self-renewal, and they can persistently proliferate to initiate tumours upon serial transplantation, thus enabling them to maintain the whole tumour

Conventional cancer treatments kill most cancer cells, but CSCs survive due to their resistance to therapy, eventually leading to tumour relapse and metastasis

identification of CSCs, three types of markers are utilised: cell surface molecules, transcription factors, and signalling pathway molecules

CSCs express distinct and specific surface markers; commonly used ones are CD24, CD34, CD38, CD44, CD90, CD133, and ALDH

Transcription factors, including Oct4, Sox2, Nanog, c-Myc, and Klf4,

signalling pathways, including those of TGF-β, Wnt, Hedgehog, Notch, platelet-derived growth factor receptor (PDGFR), and JAK/STAT

microRNAs (miRNAs), including let-7, miR-22, miR-34a, miR-128, the miR-200 family, and miR-451

Non-CSCs can be reprogrammed to become CSCs by epigenetic and genetic changes

EMT-inducing transcription factors, such as Snail, ZEB1, and Twist1, are known to confer CSC properties

Signalling pathways involved in EMT, including those of TGF-β, Wnt, and Notch, have been shown to play important roles in inducing the CSC phenotype

TGF-β1 not only increases EMT markers (Slug, Twist1, β-catenin, N-cadherin), but also upregulates CSC markers (Oct4, Sox2, Nanog, Klf4) in breast and lung cancer cells

some CSC subpopulations arise independently of EMT

IR has been shown to induce the CSC phenotype in many cancers, including breast, lung, and prostate cancers, as well as melanoma

Genotoxic stress due to IR or chemotherapy promotes a CSC-like phenotype by increasing ROS production

IR has been shown to induce reprogramming of differentiated cancer cells into CSCs

In prostate cancer patients, radiotherapy increases the CD44+ cell population that exhibit CSC properties

IR also induces the re-expression of stem cell regulators, such as Sox2, Oct4, Nanog, and Klf4, to promote stemness in cancer cells

EMT-inducing transcription factors and signalling pathways, including Snail, STAT3, Notch signalling, the PI3K/Akt pathway, and the MAPK cascade, have been shown to play important roles in IR-induced CSC properties

STAT3 directly binds to the Snail promoter and increases Snail transcription, which induces the EMT and CSC phenotypes, in cisplatin-selected resistant cells

Other oncogenic metabolic pathways, including glutamine metabolism, the pentose phosphate pathway (PPP), and synthesis of fatty acids and cholesterol, are also enhanced in many cancers

metabolic reprogramming

HIF-1α, p53, and c-Myc, are known to contribute to oncogenic metabolism

metabolic reprogramming

tumour cells exhibit high mitochondrial metabolism as well as aerobic glycolysis

occurring within the same tumour

CSCs can be highly glycolytic-dependent or oxidative phosphorylation (OXPHOS)-dependen

mitochondrial function is crucial for maintaining CSC functionality

cancer cells depend on mitochondrial metabolism and increase mitochondrial production of ROS that cause pseudo-hypoxia

HIF-1 then enhances glycolysis

CAFs have defective mitochondria that lead to the cells exhibiting the Warburg effect; the cells take up glucose, and then secrete lactate to 'feed' adjacent cancer cells

lactate transporter, monocarboxylate transporter (MCT)

nutrient microenvironment

Epithelial cancer cells express MCT1, while CAFs express MCT4. MCT4-positive, hypoxic CAFs secrete lactate by aerobic glycolysis, and MCT1-expressing epithelial cancer cells then uptake and use that lactate as a substrate for the tricarboxylic acid (TCA) cycle

MCT4-positive cancer cells depend on glycolysis and then efflux lactate, while MCT1-positive cells uptake lactate and rely on OXPHOS

metabolic heterogeneity induces a lactate shuttle between hypoxic/glycolytic cells and oxidative/aerobic tumour cells

bulk tumour cells exhibit a glycolytic phenotype, with increased conversion of glucose to lactate (and enhanced lactate efflux through MCT4), CSC subsets depend on oxidative phosphorylation; most of the glucose entering the cells is converted to pyruvate to fuel the TCA cycle and the electron transport chain (ETC), thereby increasing mitochondrial ROS production

the major fraction of glucose is directed into the pentose phosphate pathway, to produce redox power through the generation of NADPH and ROS scavengers

HIF-1α, p53, and c-Myc, are known to contribute to oncogenic metabolism

regulatory molecules involved in EMT and CSCs, including Snail, Dlx-2, HIF-1, STAT3, TGF-β, Wnt, and Akt, are implicated in the metabolic reprogramming of cancer cells

HIF-1 induces the expression of glycolytic enzymes, including the glucose transporter GLUT, hexokinase, lactate dehydrogenase (LDH), and MCT, resulting in the glycolytic switch

HIF-1 represses the expression of pyruvate dehydrogenase kinase (PDK), which inhibits pyruvate dehydrogenase (PDH), thereby inhibiting mitochondrial activity

STAT3 has been implicated in EMT-induced metabolic changes as well

TGF-β and Wnt play important roles in the metabolic alteration of cancer cells

Akt is also implicated in the glycolytic switch and in promoting cancer cell invasiveness

EMT, invasion, metastasis, and stemness

pyruvate kinase M2 (PKM2), LDH, and pyruvate carboxylase (PC), are implicated in the induction of the EMT and CSC phenotypes

decreased activity of PKM2 is known to promote an overall shift in metabolism to aerobic glycolysis

LDH catalyses the bidirectional conversion of lactate to pyruvate

High levels of LDHA are positively correlated with the expression of EMT and CSC markers

IR has been shown to induce metabolic changes in cancer cells

IR enhances glycolysis by upregulating GAPDH (a glycolysis enzyme), and it increases lactate production by activating LDHA, which converts pyruvate to lactate

IR enhances glycolysis by upregulating GAPDH (a glycolysis enzyme), and it increases lactate production by activating LDHA, which converts pyruvate to lactate

IR also elevates MCT1 expression that exports lactate into the extracellular environment, leading to acidification of the tumour microenvironment

IR increases intracellular glucose, glucose 6-phosphate, fructose, and products of pyruvate (lactate and alanine), suggesting a role for IR in the upregulation of cytosolic aerobic glycolysis

Lactate can activate latent TGF-

lactate stimulates cell migration and enhances secretion of hyaluronan from CAF that promote tumour metastasis

promote tumour survival, growth, invasion, and metastasis; enhance the stiffness of the ECM; contribute to angiogenesis; and induce inflammation by releasing several growth factors and cytokines (TGF-β, VEGF, hepatocyte growth factor [HGF], PDGF, and stromal cell-derived factor 1 [SDF1]), as well as MMP

tumours recruit the host tissue’s blood vessel network to perform four mechanisms: angiogenesis (formation of new vessels), vasculogenesis (de novo formation of blood vessels from endothelial precursor cells), co-option, and modification of existing vessels within tissues.

immunosuppressive cells such as tumour-associated macrophages (TAM), MDSCs, and regulatory T cells, and the immunosuppressive cytokines, TGF-β and interleukin-10 (IL-10)

immunosuppressive cells such as tumour-associated macrophages (TAM), MDSCs, and regulatory T cells, and the immunosuppressive cytokines, TGF-β and interleukin-10 (IL-10)

intrinsic immunogenicity or induce tolerance

cancer immunoediting’

three phases: 1) elimination, 2) equilibrium, and 3) escape.

The third phase, tumour escape, is mediated by antigen loss, immunosuppressive cells (TAM, MDSCs, and regulatory T cells), and immunosuppressive cytokines (TGF-β and IL-10).

IR can elicit various changes in the TME, such as CAF activity-mediated ECM remodelling and fibrosis, cycling hypoxia, and an inflammatory response

IR activates CAFs to promote the release of growth factors and ECM modulators, including TGF-β and MMP

TGF-β directly influences tumour cells and CAFs, promotes tumour immune escape, and activates HIF-1 signalling

MMPs degrade ECM that facilitates angiogenesis, tumour cell invasion, and metastasis

IR also promotes MMP-2/9 activation in cancer cells to promote EMT, invasion, and metastasis

IR-induced Snail increases MMP-2 expression to promote EMT

Radiotherapy has the paradoxical side-effect of increasing tumour aggressiveness

IR promotes ROS production in cancer cells, which may induce the activation of oncogenes and the inactivation of tumour suppressors, which further promote oncogenic metabolism

Metabolic alterations

oncogenic metabolism

elicit various changes in the TME

Although IR activates an antitumour immune response, this signalling is frequently suppressed by tumour escape mechanisms

Thus, while glucose metabolism is negatively regulated by phosphofructokinase, fructose can continuously enter the glycolytic pathway. Therefore, fructose can uncontrollably produce glucose, glycogen, lactate, and pyruvate, providing both the glycerol and acyl portions of acyl-glycerol molecules. These particular substrates, and the resultant excess energy flux due to unregulated fructose metabolism, will promote the over-production of TG (reviewed in [53]).

Glycemic excursions and insulin responses were reduced by 66% and 65%, respectively, in the fructose-consuming subjects

reduction in circulating leptin both in the short and long-term as well as a 30% reduction in ghrelin (an orexigenic gastroenteric hormone) in the fructose group compared to the glucose group.

A prolonged elevation of TG was also seen in the high fructose subjects

Both fat and fructose consumption usually results in low leptin concentrations which, in turn, leads to overeating in populations consuming energy from these particular macronutrients

Chronic fructose consumption reduces adiponectin responses, contributing to insulin resistance

A definite relationship has also been found between metabolic syndrome and hyperhomocysteinemia

the liver takes up dietary fructose rapidly where it can be converted to glycerol-3-phosphate. This substrate favours esterification of unbound FFA to form the TG

Fructose stimulates TG production, but impairs removal, creating the known dyslipidemic profile

the effects of fructose in promoting TG synthesis are independent of insulinemia

Although fructose does not appear to acutely increase insulin levels, chronic exposure seems to indirectly cause hyperinsulinemia and obesity through other mechanisms. One proposed mechanism involves GLUT5

If FFA are not removed from tissues, as occurs in fructose fed insulin resistant models, there is an increased energy and FFA flux that leads to the increased secretion of TG

In these scenarios, where there is excess hepatic fatty acid uptake, synthesis and secretion, 'input' of fats in the liver exceed 'outputs', and hepatic steatosis occurs

Carbohydrate induced hypertriglycerolemia results from a combination of both TG overproduction, and inadequate TG clearance

fructose-induced metabolic dyslipidemia is usually accompanied by whole body insulin resistance [100] and reduced hepatic insulin sensitivity

Excess VLDL secretion has been shown to deliver increased fatty acids and TG to muscle and other tissues, further inducing insulin resistance

the metabolic effects of fructose occur through rapid utilization in the liver due to the bypassing of the regulatory phosphofructokinase step in glycolysis. This in turn causes activation of pyruvate dehydrogenase, and subsequent modifications favoring esterification of fatty acids, again leading to increased VLDL secretion

High fructose diets can have a hypertriglyceridemic and pro-oxidant effect

Oxidative stress has often been implicated in the pathology of insulin resistance induced by fructose feeding

Administration of alpha-lipoic acid (LA) has been shown to prevent these changes, and improve insulin sensitivity

LA treatment also prevents several deleterious effects of fructose feeding: the increases in cholesterol, TG, activity of lipogenic enzymes, and VLDL secretion

Fructose has also been implicated in reducing PPARα levels

PPARα is a ligand activated nuclear hormone receptor that is responsible for inducing mitochondrial and peroxisomal β-oxidation

decreased PPARα expression can result in reduced oxidation, leading to cellular lipid accumulation

fructose diets altered the structure and function of VLDL particles causing and increase in the TG: protein ratio

LDL particle size has been found to be inversely related to TG concentration

therefore the higher TG results in a smaller, denser, more atherogenic LDL particle, which contributes to the morbidity of the metabolic disorders associated with insulin resistance

High fructose, which stimulates VLDL secretion, may initiate the cycle that results in metabolic syndrome long before type 2 diabetes and obesity develop

A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, disturbs normal hepatic carbohydrate metabolism leading to two major consequences (Figure 2): perturbations in glucose metabolism and glucose uptake pathways, and a significantly enhanced rate of de novo lipogenesis and TG synthesis, driven by the high flux of glycerol and acyl portions of TG molecules coming from fructose catabolism

In the liver, fructose bypasses the two highly regulated steps of glycolysis, catalyzed by glucokinase/hexokinase and phosphofructokinase both of which are inhibited by increasing concentrations of their byproducts. Instead, fructose enters the pathway at a level that is not regulated and is metabolized to fructose-1-phosphate primarily by fructokinase or ketohexokinase

Fructokinase has no negative feedback system, and ATP is used for the phosphorylation process. As a result, continued fructose metabolism results in intracellular phosphate depletion, activation of AMP deaminase, and uric acid generation which is harmful at the cellular level

Uric acid, a byproduct of fructose degradation,

Uric acid inhibits endothelial NO both in vivo and in vitro, [15] and directly induces adipocyte dysfunction

Serum uric acid increases rapidly after ingestion of fructose, resulting in increases as high as 2 mg/dL within 1 hour

Uncontrolled fructose metabolism leads to postprandial hypertriglyceridemia, which increases visceral adipose deposition. Visceral adiposity contributes to hepatic triglyceride accumulation, protein kinase C activation, and hepatic insulin resistance by increasing the portal delivery of free fatty acids to the liver

Several reviews have concluded that intake of both fructose and HFCS by children and adults was associated with an increased risk of obesity and metabolic syndrome

Sucrose is a disaccharide that is comprised of fructose and glucose

Figure 2

Non-recombinant urate oxidase (Uricozyme®)

recombinant urate oxidase (Rasburicase®)

urine alkalisation may induce calcium phosphate deposition

renal replacement therapy should be started on an emergency basis when hydration fails to produce a prompt metabolic improvement or when ARF develops

Up to 50% of patients with newly diagnosed multiple myeloma have renal failure and up to 10% require dialysis

renal ultrasonography remains the method of choice for investigating extra-renal obstruction

The relief of the obstruction, either by percutaneous nephrostomy or through a ureteral stent, is the cornerstone of treatment

TMA may be associated with the cancer itself, with cancer chemotherapy, or with allogeneic BMT

thrombotic microangiopathy (TMA)

it may be as high as 5%

Most of the cases occur in patients with solid tumours, the most common type being adenocarcinoma (stomach, breast and lung)

The pathophysiology of the TMA-malignancy association remains controversial, although many studies suggest an insult to the vascular endothelium

mitomycin C. Subsequently, TMA has been reported with many anti-cancer agents, including gemcita-bine, bleomycin, cisplatin, CCNU, cytosine arabinoside, daunorubicin, deoxycoformycin, 5-FU, azathioprine and interferon α

Plasma exchanges have been shown to improve prognosis in the general population of patients with TMA

Causative factors should be looked for and antihypertensive treatment given. Lastly, in the absence of guidelines, we believe that plasma exchange should be proposed in patients with severe cancer treatment-associated TMA

The most widely used protective measure is saline infusion to induce solute diuresis

During methotrexate infusion and elimination, fluids should be given to maintain a high urinary output and urinary alkalisation should be performed to keep the urinary pH above 7.5. Rescue with folinic acid (50 mg four times a day) should be started 24 hours after each high-dose metho-trexate infusion and serum methotrexate concentrations should be measured every day

cyclophosphamide and ifosfamide

metabolic activity, oxygen transport, and DNA synthesis

Iron is found in the human body in the form of haemoglobin in red blood cells and growing erythroid cells.

macrophages contain considerable quantities of iron

iron is taken up by the majority of cells in the form of a transferrin (Tf)-Fe(III) complex that binds to the cell surface receptor transferrin receptor 1 (TfR1)

excess iron is retained in the liver cells

the endosomal six transmembrane epithelial antigen of the prostate 3 (STEAP3) reduces Fe(III) (ferric ion) to Fe(II) (ferrous ion), which is subsequently transferred across the endosomal membrane by divalent metal transporter 1 (DMT1)

labile iron pool (LIP)

LIP is toxic to the cells owing to the production of massive amounts of ROS.

DHA is quickly converted to Vit-C within the cell, by interacting with reduced glutathione (GSH) [45,46,47]. NADPH then recycles the oxidized glutathione (glutathione disulfide (GSSG)) and converts it back into GSH

Fe(II) catalyzes the formation of OH• and OH− during the interaction between H2O2 and O2•− (Haber–Weiss reaction)

Ascorbate can efficiently reduce free iron, thus recycling the cellular Fe(II)/Fe(III) to produce more OH• from H2O2 than can be generated during the Fenton reaction, which ultimately leads to lipid, protein, and DNA oxidation

Vit-C-stimulated iron absorption

reduce cellular iron efflux

high-dose Vit-C may elevate cellular LIP concentrations

ascorbate enhanced cancer cell LIP specifically by generating H2O2

Vit-C produces H2O2 extracellularly, which in turn inhibits tumor cells immediately

tumor cells have a need for readily available Fe(II) to survive and proliferate.

Tf has been recognized to sequester most labile Fe(II) in vivo

Asc•− and H2O2 were generated in vivo upon i.v Vit-C administration of around 0.5 g/kg of body weight and that the generation was Vit-C-dose reliant

free irons, especially Fe(II), increase Vit-C autoxidation, leading to H2O2 production

iron metabolism is altered in malignancies

increase in the expression of various iron-intake pathways or the downregulation of iron exporter proteins and storage pathways

Fe(II) ion in breast cancer cells is almost double that in normal breast tissues

macrophages in the cancer microenvironment have been revealed to increase iron shedding

Advanced breast tumor patients had substantially greater Fe(II) levels in their blood than the control groups without the disease

increased the amount of LIP inside the cells through transferrin receptor (TfR)

Warburg effect, or metabolic reprogramming,

Warburg effect is aided by KRAS or BRAF mutations

Vit-C is supplied, it oxidizes to DHA, and then is readily transported by GLUT-1 in mutant cells of KRAS or BRAF competing with glucose [46]. DHA is quickly converted into ascorbate inside the cell by NADPH and GSH [46,107]. This decrease reduces the concentration of cytosolic antioxidants and raises the intracellular ROS amounts

increased ROS inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

ROS activates poly (ADP-ribose) polymerase (PARP), which depletes NAD+ (a critical co-factor of GAPDH); thus, further reducing the GAPDH associated with a multifaceted metabolic rewiring

Hindering GAPDH can result in an “energy crisis”, due to the decrease in ATP production

high-dose Vit-C recruited metabolites and increased the enzymatic activity in the pentose phosphate pathway (PPP), blocked the tri-carboxylic acid (TCA) cycle, and increased oxygen uptake, disrupting the intracellular metabolic balance and resulting in irreversible cell death, due to an energy crisis

mega-dose Vit-C influences energy metabolism by producing tremendous amounts of H2O2

Due to its great volatility at neutral pH [76], bolus therapy with mega-dose DHA has only transitory effects on tumor cells, both in vitro and in vivo.

Multiple inflammatory inputs contribute to metabolic dysfunction, including increases in circulating cytokines (10), decreases in protective factors (e.g., adiponectin; ref. 11), and communication between inflammatory and metabolic cells

adipose tissue macrophage (ATM)

well-known regulators of lipid metabolism and mitochondrial activity

increasing adiposity results in a shift in the inflammatory profile of ATMs as a whole from an M2 state to one in which classical M1 proinflammatory signals predominate (21–23).

The M2 activation state is intrinsically linked to the activity of PPARδ and PPARγ

Physiologic enhancement of the M2 pathways (e.g., eosinophil recruitment in parasitic infection) also appears to be capable of reducing metainflammation and improving insulin sensitivity (27).

Independent of obesity, hypothalamic inflammation can impair insulin release from β cells, impair peripheral insulin action, and potentiate hypertension (63–65).

inflammation in pancreatic islets can reduce insulin secretion and trigger β cell apoptosis leading to decreased islet mass, critical events in the progression to diabetes (33, 34)

Since an estimated excess of 20–30 million macrophages accumulate with each kilogram of excess fat in humans, one could argue that increased adipose tissue mass is de facto a state of increased inflammatory mass

JNK, TLR4, ER stress)

NAFLD is associated with an increase in M1/Th1 cytokines and quantitative increases in immune cells

Lipid infusion and a high-fat diet (HFD) activate hypothalamic inflammatory signaling pathways, resulting in increased food intake and nutrient storage (57)

DIO, metabolites such as diacylglycerols and ceramides accumulate in the hypothalamus and induce leptin and insulin resistance in the CNS (58, 59)

saturated FAs, which activate neuronal JNK and NF-κB signaling pathways with direct effects on leptin and insulin signaling (60)

Upon stimulation by LPS and IFN-γ, macrophages assume a classical proinflammatory activation state (M1) that generates bactericidal or Th1 responses typically associated with obesity

Maternal obesity is associated with endotoxemia and ATM accumulation that may affect the developing fetus (73)

Placental inflammation is a characteristic of maternal obesity

a risk factor for obesity in offspring, and involves inflammatory macrophage infiltration that can alter the maternal-fetal circulation (74

Of these PRRs, TLR4 has received the most attention, as this receptor can be activated by free FAs to generate proinflammatory signals and activate NF-κB

Nod-like receptor (NLR) family of PRRs

ceramides and sphingolipids

The adipokine adiponectin has long been recognized to have positive benefits on multiple cell types to promote insulin sensitivity and deactivate proinflammatory pathways.

adiponectin stimulates ceramidase activity and modulates the balance between ceramides and sphingosine-1-phosphate

Inhibition of ceramide production blocks the ability of saturated FAs to induce insulin resistance (101)

NF-κB, obesity also activates JNK in insulin-responsive tissues

Approximately 95% of the body's creatine is stored in skeletal muscle

About two thirds of the creatine found in skeletal muscle is stored as phosphocreatine (PCr) while the remaining amount of creatine is stored as free creatine

The body breaks down about 1 – 2% of the creatine pool per day (about 1–2 grams/day) into creatinine in the skeletal muscle

The magnitude of the increase in skeletal muscle creatine content is important because studies have reported performance changes to be correlated to this increase

"loading" protocol. This protocol is characterized by ingesting approximately 0.3 grams/kg/day of CM for 5 – 7 days (e.g., ≃5 grams taken four times per day) and 3–5 grams/day thereafter [18,22]. Research has shown a 10–40% increase in muscle creatine and PCr stores using this protocol

Additional research has reported that the loading protocol may only need to be 2–3 days in length to be beneficial, particularly if the ingestion coincides with protein and/or carbohydrate

A few studies have reported protocols with no loading period to be sufficient for increasing muscle creatine (3 g/d for 28 days)

Cycling protocols involve the consumption of "loading" doses for 3–5 days every 3 to 4 weeks

Most of these forms of creatine have been reported to be no better than traditional CM in terms of increasing strength or performance

Recent studies do suggest, however, that adding β-alanine to CM may produce greater effects than CM alone

These investigations indicate that the combination may have greater effects on strength, lean mass, and body fat percentage; in addition to delaying neuromuscular fatigue

creatine phosphate has been reported to be as effective as CM at improving LBM and strength

Green et al. [24] reported that adding 93 g of carbohydrate to 5 g of CM increased total muscle creatine by 60%

Steenge et al. [23] reported that adding 47 g of carbohydrate and 50 g of protein to CM was as effective at promoting muscle retention of creatine as adding 96 g of carbohydrate.

It appears that combining CM with carbohydrate or carbohydrate and protein produces optimal results

Studies suggest that increasing skeletal muscle creatine uptake may enhance the benefits of training

Nearly 70% of these studies have reported a significant improvement in exercise capacity,

Long-term CM supplementation appears to enhance the overall quality of training, leading to 5 to 15% greater gains in strength and performance

Nearly all studies indicate that "proper" CM supplementation increases body mass by about 1 to 2 kg in the first week of loading

short-term adaptations reported from CM supplementation include increased cycling power, total work performed on the bench press and jump squat, as well as improved sport performance in sprinting, swimming, and soccer

Long-term adaptations when combining CM supplementation with training include increased muscle creatine and PCr content, lean body mass, strength, sprint performance, power, rate of force development, and muscle diameter

subjects taking CM typically gain about twice as much body mass and/or fat free mass (i.e., an extra 2 to 4 pounds of muscle mass during 4 to 12 weeks of training) than subjects taking a placebo

The gains in muscle mass appear to be a result of an improved ability to perform high-intensity exercise via increased PCr availability and enhanced ATP synthesis, thereby enabling an athlete to train harder

there is no evidence to support the notion that normal creatine intakes (< 25 g/d) in healthy adults cause renal dysfunction

no long-term side effects have been observed in athletes (up to 5 years),

One cohort of patients taking 1.5 – 3 grams/day of CM has been monitored since 1981 with no significant side effects

Reactive oxygen species (ROS) refer to a class of radical or non-radical oxygen-containing molecules that have high oxidative reactivity with lipids, proteins, and nucleic acids

a large measure of intracellular ROS comes from the leakage of mitochondrial electron transport chain (ETC)

Another major source of intracellular ROS is the intentional generation of superoxides by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase

there are other ROS-producing enzymes such as cyclooxygenases, lipoxygenases, xanthine oxidase, and cytochrome p450 enzymes, which are involved with specific metabolic processes

To counteract the toxic effects of molecular oxidation by ROS, cells are equipped with a battery of antioxidant enzymes such as superoxide dismutases, catalase, peroxiredoxins, sulfiredoxin, and aldehyde dehydrogenases

intracellular oxidative stress has been indicated to contribute to metabolic syndrome and related diseases, including T2D [72; 73], CVDs [74-76], neurodegenerative diseases [69; 77-80], and cancers

intracellular oxidative stress is highly associated with the development of neurodegenerative diseases [69] and brain aging

dietary obesity was found to induce NADPH oxidase-associated oxidative stress in rat brain

mitochondrial dysfunction in hypothalamic proopiomelanocortin (POMC) neurons causes central glucose sensing impairment

Endoplasmic reticulum (ER) is the cellular organelle responsible for protein synthesis, maturation, and trafficking to secretory pathways

unfolded protein response (UPR) machinery

ER stress has been associated to obesity, insulin resistance, T2D, CVDs, cancers, and neurodegenerative diseases

brain ER stress underlies neurodegenerative diseases

under environmental stress such as nutrient deprivation or hypoxia, autophagy is strongly induced to breakdown macromolecules into reusable amino acids and fatty acids for survival

intact autophagy function is required for the hypothalamus to properly control metabolic and energy homeostasis, while hypothalamic autophagy defect leads to the development of metabolic syndrome such as obesity and insulin resistance

prolonged oxidative stress or ER stress has been shown to impair autophagy function in disease milieu of cancer or aging

TLRs are an important class of membrane-bound pattern recognition receptors in classical innate immune defense

Most hypothalamic cell types including neurons and glia cells express TLRs