A 49-year-old man presented to hospital with severe orthostatic hypotension, gingival dysplasia and a purpuric rash involving his extremities. The orthostatic hypotension failed to respond to fluids and, on the basis of physical examination and dietary history, the patient was given a preliminary diagnosis of scurvy (ascorbic acid deficiency). Serum ascorbic acid levels were undetectable and the orthostasis was resolved within 24 h of ascorbic acid replacement. The pathogenesis of orthostatic hypotension in the setting of scurvy appears to involve impaired catecholamine synthesis and attenuated vasomotor response to α-adrenergic stimulation. We believe that this case describes a rare presentation of scurvy and highlightsa previously under-reported connection between scurvy and vasomotor instability.

The objective of this study was to determine the impact of limited ascorbate (Asc) availability on type II cell sensitivity to oxidant stress. Guinea pigs were fed diets with or without Asc for 18 days, and type II cells were isolated. Although lung Asc was decreased by 90% in deficient animals (scorbutic), type II cell Asc was decreased by 50%. Upon treatment with 250μM HO, the necrotic injury was twofold greater in scorbutic cells compared with control cells. With 100μM HOtreatment, apoptotic injury was twofold greater in scorbutic cells compared with control cells. Although there was less necrotic injury in cells exposed to 95% O, the scorbutic cells were more sensitive than control cells. Asc pretreatment protected against necrosis and apoptosis. The Asc analog isoascorbate provided partial protection and suggested that part of the protection was not chemical detoxification but was Asc specific. We conclude that limited Asc availability resulted in a functional type II cell but a cell more sensitive to oxidant-induced injury.

AIM:Our main objective was to determine the effect of ascorbate supplementation in mice unable to synthesize ascorbic acid (gulo KO) when challenged with murine B16FO cancer cells.

METHODS:Gulo KO female mice 36-40 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to subcutaneous injection of 2.5×10(6) B16FO murine melanoma cells in the right flank of mice. A control group of wild type mice were also injected with the melanoma cells and maintained on a regular murine diet. Mice were continued on their respective diets for another 2 weeks after injection. The mice were then sacrificed, blood was drawn and their tumors were measured, excised and processed for histology.

RESULTS:Mean weight of animals decreased significantly (30%, p<0.0001) in the ascorbate-restricted group but increased slightly, but insignificantly, in the ascorbate-supplemented group. The mean tumor weight in ascorbate supplemented mice was significantly reduced (by 64%, p = 0.004) compared to tumor weight in ascorbate-deprived gulo mice. The mean tumor weight of wild type mice did not differ significantly from the ascorbate-supplemented mice. Gulo KO mice supplemented with ascorbate developed smaller tumors with more collagen encapsulation and fibrous capsule interdigitation, while gulo KO mice deprived of ascorbate hosted large tumors with poorly defined borders, showing more necrosis and mitosis. Ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine IL-6 (90% decrease, p = 0.04) and IL-1β (62% decrease) compared to the levels in gulo KO mice deprived of ascorbate.

CONCLUSION:Ascorbate supplementation modulated tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors in scorbutic mice.

Severe systemic inflammatory response to infection results in severe sepsis and septic shock, which are the leading causes of death in critically ill patients. Septic shock is characterised by refractory hypotension and is typically managed by fluid resuscitation and administration of catecholamine vasopressors such as norepinephrine. Vasopressin can also be administered to raise mean arterial pressure or decrease the norepinephrine dose. Endogenous norepinephrine and vasopressin are synthesised by the copper-containing enzymes dopamineβ-hydroxylase and peptidylglycine α-amidating monooxygenase, respectively. Both of these enzymes require ascorbate as a cofactor for optimal activity. Patients with severe sepsis present with hypovitaminosis C, and pre-clinical and clinical studies have indicated that administration of high-dose ascorbate decreases the levels of pro-inflammatory biomarkers, attenuates organ dysfunction and improves haemodynamic parameters. It is conceivable that administration of ascorbate to septic patients with hypovitaminosis C could improve endogenous vasopressor synthesis and thus ameliorate the requirement for exogenously administered vasopressors. Ascorbate-dependent vasopressor synthesis represents a currently underexplored biochemical mechanism by which ascorbate could act as an adjuvant therapy for severe sepsis and septic shock.

OBJECTIVES:Recent studies have highlighted the role of oxidative stress in the pathogenesis of lichen planus (LP). In the present study the interest of the authors is focused on the investigation of ascorbic acid status in patients with LP and identification of parameters that might influence the level of this vitamin.

MATERIAL AND METHOD:We analyzed the level of urinary ascorbic acid (reflectometric method) in 77 patients with LP (cutaneous LP (CLP) - 49 cases, oral LP (OLP) - 28 cases) and 50 control subjects. The evaluation of all participants included clinical examination and laboratory and imaging tests.

RESULTS:Compared to the control group (19.82 mg/dl) the level of ascorbic acid was significantly lower both in patients with CLP (8.47mg/dl, p = 0.001) and in those with OLP (8.04 mg/dl, p = 0.001). In patients with LP it was found that the deficiency of ascorbic acid increases with age (r=-0.318, p=0.032). The urinary concentrations of ascorbic acid were significantly lower in patients with LP associated with infections compared to patients with LP without infections.

CONCLUSIONS:The urinary ascorbic acid level may be a useful parameter in identifying patients with LP who are at risk of developing viral or bacterial infections.

In this paper we demonstrate that patients treated with chemotherapy and/or hematopoietic stem cell transplantation (HSCT) have highly significant reduced serum ascorbic acid (AA) levels compared to healthy controls. We recently observed in in vitro experiments that growth of both T and NK cells from hematopoietic stem cells is positively influenced by AA. It might be of clinical relevance to study the function and recovery of immune cells after intensive treatment, its correlation to AA serum levels and the possible effect of AA supplementation.

Epidemiology of bronchial asthma (BA) indicates a marked paradox: rapid rise in the prevalence.Simultaneous decline in mortality is mostly related to improvement in the diagnosis and therapy. In many economically developed countries the BA affects more than 10 per cent of the population, while mortality related to this respiratory disorder is below 1/100,000. Factors favorably influencing mortality of BA include new more effective medications, decline in smoking and also improved nutrition, based on awareness of protective role of vitamins. Vitamin D deficiency has a number of biological effects that are potentially instrumental in the pathogenesis and severity of BA. Increased number of randomized, controlled, interventional studies is showing positive effects of vitamin D supplementation in pediatric and in adult BA. Oxidative stress is potentially an important pathogenic factor in the progression of BA. Vitamin C (ascorbic acid) belongs to the most effective nutritional antioxidants. By counteracting oxidants, reducing generation of reactive oxygen species, vitamin C may inhibit external attacks in the respiratory tract, thus modulating the development of BA (Fig. 2, Ref. 15).

BACKGROUND:Epilepsy is one of the chronic and heterogeneous epidemic neurological disorders leading to substantial mortality. The aim of the present study was to investigate the serum levels of malondialdehyde (MDA), vitamin C, and trace elements namely zinc (Zn), copper (Cu), and manganese (Mn) in epileptic patients of Bangladesh and to establish if there are any pathophysiological correlations.

METHODS:This was a case-control study with 40 generalized epileptic patients and 40 healthy subjects as controls. Epilepsy was determined by the presence of seizure events with an abnormal electroencephalography and magnetic resonance imaging report of brain.

RESULTS:Anthropometric parameters highlighted that age is a major risk factor of epilepsy and men are more prone to epilepsy than women. Blood serum analysis demonstrated significantly ( P<.001) higher values of MDA and lower level of vitamin C in the patient group (4.41± 0.76 μmol/mL and 18.31 ± 0.84 μmol/L, respectively) compared with control (1.81 ± 0.70 μmol/mL and 29.72 ± 1.06 μmol/L, respectively). Pearson's correlation analysis revealed a negative correlation between the serum level of MDA and vitamin C for both patient ( r = -0.023, P = .887) and control group ( r = -0.142, P = .383). This study also revealed that the trace elements (Zn, Cu) were significantly ( P<.05) lower in epileptics (68.32± 4.59 and 50.81 ± 2.54 μg/dL, respectively) where the level of Mn in patients (187.71 ± 9.04 μg/dL) was almost similar to that of the control group ( P>.05). The univariate analysis demonstrated that zinc<70μg/dL (odds ratio = 3.56, P<.05) and copper<50μg/dL were associated (odds ratio = 14.73, P<.001) with an increased risk of epilepsy. Establishment of interelement relationship strongly supported that there was a disturbance in the element homeostasis of epileptic patients.

CONCLUSIONS:The study results strengthen the role of lipid peroxidation, antioxidants and trace elements in the pathogenesis and warrant larger studies to investigate the association of these biochemical parameters with epilepsy.

AIM:To examine whether low circulating vitamin C concentrations and low fruit and vegetable intakes were associated with insulin resistance and other Type 2 diabetes risk markers in childhood.

METHODS:We conducted a cross-sectional, school-based study in 2025 UK children aged 9-10 years, predominantly of white European, South-Asian and black African origin. A 24-h dietary recall was used to assess fruit, vegetable and vitamin C intakes. Height, weight and fat mass were measured and a fasting blood sample collected to measure plasma vitamin C concentrations and Type 2 diabetes risk markers.

RESULTS:In analyses adjusting for confounding variables (including socio-economic status), a one interquartile range higher plasma vitamin C concentration (30.9μmol/l) was associated with a 9.6% (95% CI 6.5, 12.6%) lower homeostatic model assessment of insulin resistance value, 0.8% (95% CI 0.4, 1.2%) lower fasting glucose, 4.5% (95% CI 3.2, 5.9%) lower urate and 2.2% (95% CI 0.9, 3.4%) higher HDL cholesterol. HbA1c concentration was 0.6% (95% CI 0.2, 1.0%) higher. Dietary fruit, vegetable and total vitamin C intakes were not associated with any Type 2 diabetes risk markers. Lower plasma vitamin C concentrations in South-Asian and black African-Caribbean children could partly explain their higher insulin resistance.

CONCLUSIONS:Lower plasma vitamin C concentrations are associated with insulin resistance and could partly explain ethnic differences in insulin resistance. Experimental studies are needed to establish whether increasing plasma vitamin C can help prevent Type 2 diabetes at an early stage. This article is protected by copyright. All rights reserved.

It has been suggested that vitamin C deficiency/scurvy is associated with gingival inflammatory changes; however, the disorder is very infrequently encountered in the modern era. Here, we report a case of extensive gingival overgrowth caused by vitamin C deficiency associated with metabolic syndrome and severe periodontal infection.

BACKGROUND:Vitamin C (ascorbate) is likely to be essential for skeletal muscle structure and function via its role as an enzyme cofactor for collagen and carnitine biosynthesis. Vitamin C may also protect these metabolically active cells from oxidative stress.

OBJECTIVE:We investigated the bioavailability of vitamin C to human skeletal muscle in relation to dietary intake and plasma concentrations and compared this relation with ascorbate uptake by leukocytes.

DESIGN:Thirty-six nonsmoking men were randomly assigned to receive 6 wk of 0.5 or 2 kiwifruit/d, an outstanding dietary source of vitamin C. Fasting blood samples were drawn weekly, and 24-h urine and leukocyte samples were collected before intervention, after intervention, and after washout. Needle biopsies of skeletal muscle (vastus lateralis) were carried out before and after intervention.

RESULTS:Baseline vastus lateralis ascorbate concentrations were ~16 nmol/g tissue. After intervention with 0.5 or 2 kiwifruit/d, these concentrations increased ~3.5-fold to 53 and 61 nmol/g, respectively. There was no significant difference between the responses of the 2 groups. Mononuclear cell and neutrophil ascorbate concentrations increased only ~1.5- and ~2-fold, respectively. Muscle ascorbate concentrations were highly correlated (P<0.001) with dietary intake (R = 0.61) and plasma concentrations (R = 0.75) in the range from 5 to 80μmol/L.

CONCLUSIONS:Human skeletal muscle is highly responsive to vitamin C intake and plasma concentrations and exhibits a greater relative uptake of ascorbate than leukocytes. Thus, muscle appears to comprise a relatively labile pool of ascorbate and is likely to be prone to ascorbate depletion with inadequate dietary intake. This trial was registered at the Australian New Zealand Clinical Trials Registry (www.anzctr.org.au) as ACTRN12611000162910.

AIM:l-ascorbic acid (vitamin C) insufficiency is considered one of the major risk factors for the development of liver disease. However, its specific effects and related mechanisms in vivo are largely unknown. The objective of this study was to investigate the in vivo protective role of vitamin C and its related mechanisms in liver injury with Gulo(-/-) mice that cannot synthesize vitamin C like humans due to the lack of l-gulonolactone-γ-oxidase (Gulo), an essential enzyme for vitamin C synthesis.

RESULTS:When liver injury was induced in Gulo(-/-) mice by injection of concanavalin A (Con A), there was greater extensive liver damage accompanied by an increased number of apoptotic hepatocytes in vitamin C-insufficient Gulo(-/-) mice. Additionally, the plasma and hepatic levels of the proinflammatory cytokines, such as TNF-α and IFN-γ, were much higher in the vitamin C-insufficient Gulo(-/-) mice than in the control mice. Moreover, increased numbers of liver-infiltrating T-cells in the vitamin C-insufficient Gulo(-/-) mice were related to the increased hepatic levels of IFN-inducible factor (IP-10). Although the vitamin C-insufficient Gulo(-/-) mice had higher amounts of interleukin-22 (IL-22), a hepatoprotective cytokine, a defect in IL-22Rα expression and its downstream STAT3 activation in hepatocytes were found.

INNOVATION:We first demonstrate the novel in vivo action mechanisms of vitamin C on the prevention of disease development in the liver, through the regulation of excessive immune activation and maintenance of the IL-22Rα signaling pathways.

CONCLUSION:These results suggest that severe liver damage induced by inflammation could be prevented by sufficient supplementation with vitamin C.

Cancer progression is characterized by loss of extracellular matrix (ECM) integrity, which is a precondition for tumor growth and metastasis. In order to elucidate the precise mechanisms of ECM degradation in cancer we used a genetically modified mouse mimicking two distinct human metabolic features associated with carcinogenesis, the lack of endogenous vitamin C synthesis and the production of human Lp(a). Female Lp(a)+; Gulo(-/-) and control wild-type Balb/c mice without these two metabolic features were orthotopically inoculated with 4T1 breast cancer cells (5x105). The transgenic and control mice were divided into 4 different dietary groups in respect to dietary vitamin C intake: i) low ascorbate intake for 6 weeks; ii) high ascorbate intake for 6 weeks; iii) low ascorbate intake for 3 weeks followed by high ascorbate for 3 weeks; iv) high ascorbate intake for 3 weeks followed by low ascorbate for 3 weeks. After 6 weeks, all wild-type mice developed tumors. In contrast, Lp(a)+; Gulo(-/-) mice developed one third less primary tumors (low ascorbate diet) or no primary tumors at all (high ascorbate diet). Significantly, tumors from Lp(a)+; Gulo(-/-) mice immunostained positively for Lp(a) and their size was inversely proportional to Lp(a) serum levels. The results implicate that Lp(a) may play a role in controlling tumor growth and expansion. The most likely mechanism is the competitive inhibition of plasmin-induced ECM degradation due to the homology of Lp(a) components to plasminogen. The confirmation of this pathomechanism could lead to a universal therapeutic target for the prevention and treatment of cancer.

Activation of the transcription factor hypoxia-inducible factor (HIF)-1 allows solid tumors to thrive under conditions of metabolic stress. Because HIF-1 is switched off by hydroxylation reactions that require ascorbate, inadequate intracellular ascorbate levels could contribute to HIF-1 overactivation. In this study, we investigated whether the ascorbate content of human endometrial tumors [known to be driven by HIF-1 and vascular endothelial growth factor (VEGF)] influenced HIF-1 activity and tumor pathology. We measured protein levels of HIF-1alpha and three downstream gene products [glucose transporter 1 (GLUT-1), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), and VEGF], as well as the ascorbate content of tumor and patient-matched normal endometrial tissue samples. HIF-1alpha and its downstream gene products were upregulated in tumor tissue, with the highest levels being present in high-grade tumors. High-grade tumors also had reduced capacity to accumulate ascorbate compared with normal tissue; however, all grades contained tumors with low ascorbate content. Tumors with the highest HIF-1alpha protein content were ascorbate deficient. Low ascorbate levels were also associated with elevated VEGF, GLUT-1, and BNIP3 protein levels and with increased tumor size, and there was a significant association between low tissue ascorbate levels and increased activation of the HIF-1 pathway (P = 0.007). In contrast, tumors with high ascorbate levels had lesser levels of HIF-1 activation. This study shows for the first time a likely in vivo relationship between ascorbate and HIF-1, with low tumor tissue ascorbate levels being associated with high HIF-1 activation and tumor growth.

Curcuma longa has long been used in China and India as anti-inflammatory agent to treat a wide variety of conditions and also as a spice for varied curry preparations. The chemoprofile of the Curcuma species exhibits the presence of varied phytochemicals with curcumin being present in all three species but AA only being shown in C. longa. This study explored the effect of a curcumin/AA combination on human cancer cell lines. The curcumin/AA combination was assessed by isobologram analysis using the Loewe additivity drug interaction model. The drug combination showed additive cytotoxicity toward CCRF-CEM and CEM/ADR5000 leukemia cell lines and HCT116p53(+/+) and HCT116p53(-/-) colon cancer cell line, while the glioblastoma cell lines U87MG and U87MG.ΔEGFR showed additive to supra-additive cytotoxicity. Gene expression profiles predicting sensitivity and resistance of tumor cells to induction by curcumin and AA were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Numerous genes involved in transcription (TFAM, TCERG1, RGS13, C11orf31), apoptosis-regulation (CRADD, CDK7, CDK19, CD81, TOM1) signal transduction (NR1D2, HMGN1, ABCA1, DE4ND4B, TRIM27) DNA repair (TOPBP1, RPA2), mRNA metabolism (RBBP4, HNRNPR, SRSF4, NR2F2, PDK1, TGM2), and transporter genes (ABCA1) correlated with cellular responsiveness to curcumin and ascorbic acid. In conclusion, this study shows the effect of the curcumin/AA combination and identifies several candidate genes that may regulate the response of varied cancer cells to curcumin and AA.

Back pain brings about one of the heaviest burden of disease. Despite much research, this condition remains poorly understood and effective treatments are frustratingly elusive. Thus, researchers in the field need to consider new hypotheses. Vitamin C (ascorbic acid) is an essential co-factor for collagen crosslinks, a key determinant of ligament, tendon and bone quality. Recent studies have reported high frequency of hypovitaminosis C in the general population. We hypothesized that lack of vitamin C contributes to poor collagen properties and back pain. We conducted this study to examine the associations between serum concentration of vitamin C and the prevalence of spinal pain and related functional limitations in the adult general population.This study used nationwide cross-sectional data from the U.S. National Health and Nutrition Examination Survey (NHANES) 2003-2004. Data were available for 4,742 individuals aged≥20 years.Suboptimal serum vitamin C concentrations were associated with the prevalence of neck pain (adjusted odds ratio (aOR): 1.5; 95% confidence interval (CI): 1.2-2.0), low back pain (aOR: 1.3; 95% CI: 1.0-1.6), and low back pain with pain below knee (aOR: 1.3; 95% CI: 1.0-1.9) in the past three months, self-reported diagnosis of arthritis/rheumatism (aOR: 1.4; 95% CI: 1.2-1.7), and related functional limitations' score (adjusted difference of means (aB): 0.03; 95% CI: 0.00-0.05).The prevalence of hypovitaminosis C in the general population is high. Our study shows associations betweenvitamin C and spinal pain that warrant further investigation to determine the possible importance of vitamin C in the treatment of back pain patients.

Vitamin C (ascorbic acid) plays important roles as an anti-oxidant and in collagen synthesis. These important roles, and the relatively large amounts of vitamin C required daily, likely explain why most vertebrate species are able to synthesize this compound. Surprisingly, many species, such as teleost fishes, anthropoid primates, guinea pigs, as well as some bat and Passeriformes bird species, have lost the capacity to synthesize it. Here, we review the genetic bases behind the repeated losses in the ability to synthesize vitamin C as well as their implications. In all cases so far studied, the inability to synthesize vitamin C is due to mutations in the L-gulono-γ-lactone oxidase (GLO) gene which codes for the enzyme responsible for catalyzing the last step of vitamin C biosynthesis. The bias for mutations in this particular gene is likely due to the fact that losing it only affects vitamin C production. Whereas the GLO gene mutations in fish, anthropoid primates and guinea pigs are irreversible, some of the GLO pseudogenes found in bat species have been shown to be reactivated during evolution. The same phenomenon is thought to have occurred in some Passeriformes bird species. Interestingly, these GLO gene losses and reactivations are unrelated to the diet of the species involved. This suggests that losing the ability to make vitamin C is a neutral trait.

BACKGROUND:The hyperglycemia and hyperoxidation that characterize diabetes lead to reduced vitamin C (VC) in diabetic humans and experimentally diabetic animals. Herein, we access the effects of VC deficiency on the diabetic kidney injury and explore the underlying mechanism.

METHODS:l-gulonolactone oxidase conventional knockout (Gulo-/-) mice genetically unable to synthesize VC were subjected to streptozotocin-induced diabetic kidney injury and the role of VC deficiency was evaluated by biochemical and histological approaches. Rat mesangial cells were cultured to investigate the underlying mechanism.

RESULTS:Functionally, VC deficiency aggravates the streptozotocin-induced renal insufficiency, exhibiting the increased urine albumin, water intake, and urine volume in Gulo-/- mice. Morphologically, VC deficiency exacerbates the streptozotocin-induced kidney injury, exhibiting the increased glomerular expansion, deposition of Periodic Acid-Schiff- and Masson-positive materials, and expression ofα-smooth muscle actin, fibronectin and type 4 collagen in glomeruli of Gulo-/- mice. Mechanistically, VC activates protein kinase B (Akt) to destabilize Ski and thereby induce the expression of Smad7, resulting in suppression of TGF-β/Smad signaling and extracellular matrix deposition in mesangialcells.

CONCLUSIONS:VC is essential for the renal function maintenance in diabetes.

GENERAL SIGNIFICANCE:Compensation for the loss of VC could be an effective remedy for diabetic kidney injury.

This study was designed to determine the effects of vitamin C deficiency on the immune response to infection with influenza virus. l-Gulono-gamma-lactone oxidase gene-inactivated mice (gulo-/- mice) require vitamin C supplementation for survival. Five-wk-old male and female gulo-/- mice were provided water or water containing 1.67 mmol/L vitamin C for 3 wk before inoculation with influenza A/Bangkok/1/79. There were no differences in lung influenza virus titers between vitamin C-adequate and -deficient mice; however, lung pathology in the vitamin C-deficient mice was greater at 1 and 3 d after infection but less at d 7 compared with vitamin C-adequate mice. Male vitamin C-deficient mice had higher expression of mRNA for regulated upon activation normal T expressed and secreted (RANTES), IL-1beta, and TNF-alpha in the lungs at d 1 after infection compared with male controls. However, at d 3 after infection, male vitamin C-deficient mice had less expression of mRNA for RANTES, monocyte chemotactic protein-1 (MCP-1), and IL-12 compared with male controls. None of these differences were observed in female mice. Vitamin C-deficient male mice also had greater nuclear factor-kappaB activation as early as 1 d after infection compared with male controls. These data suggest that vitamin C is required for an adequate immune response in limiting lung pathology after influenza virus infection.

The human aging process is often accompanied by significant increases in degenerative spine disease. The pathophysiology of intervertebral disc degeneration has been extensively studied, but the etiology of this aging-related problem remains poorly understood. The elderly often have lower daily vitamin C intakes and circulating ascorbic acid values than younger people because of problems with poor dentition or mobility, and also are more likely to have underlying sub-clinical diseases that can reduce plasma ascorbate concentrations. Ascorbate is essential for collagen production, and vitamin C deficiency will result in defective connective tissue, including reductions in collagen synthesis and structural stability. It is hypothesised that vitamin C deficiencies may be a key contributing factor in the development of degenerative disk disease (DDD) in the elderly. Once degenerative disc disease has begun, the tissue inflammation that accompanies DDD may further increase vitamin C requirements in the affected patient, thereby creating a cascade of positive feedbacks that potentially accelerates and contributes to further disc degeneration and low-back pain. Aggressive monitoring of patient ascorbate status, as well as more finely-calibrated RDAs for vitamin C that explicitly take into account the patient's age, may be required if aging-related degenerative disk disease is to be minimised.