Anitra C. Carr1,
Principles of Nutritional
Assessment: Vitamin C
AbstractVitamin C (ascorbic acid) is involved in a variety of biological processes, acting primarily as an electron donating enzyme cofactor and reducing agent (antioxidant). Clinical deficiency of vitamin C results in scurvy, a relatively rare disease in high-income countries. Nevertheless, moderate vitamin C deficiency (hypovitaminosis C) has been described in selected vulnerable groups such as the institutionalized elderly, with gingival inflammation and fatigue appearing to be the most sensitive clinical markers for moderate vitamin C deficiency. Excess intakes of vitamin C may produce osmotic diarrhea in some people, and although oxaluria has been reported, this measurement is prone to errors in the preparative or investigative procedure. There are currently no reliable functional indices of vitamin C nutriture. Static biochemical indices such as plasma or leukocyte ascorbic acid concentrations are most frequently used. The former reflect recent dietary intake within a limited range (30–80mg/d). Plasma ascorbic acid concentrations increase linearly with dietary intake but only up to a plateau of about 70µmol/L, and thus are unsuitable for detecting excessive intakes of vitamin C. A number of non-nutritional factors, including sex, body weight, smoking, and severe infection, affect plasma ascorbic acid concentrations. Plasma concentrations are a more reliable marker of vitamin C status than dietary intake assessments. Urinary excretion of vitamin C occurs when the renal reabsorption threshold has been reached and plasma “saturation” occurs (between about 60 and 70µmol/L). Urinary excretion is influenced by recent dietary intake. Moreover, the sensitivity and specificity of this test is low, and 24h urine samples are generally required. Leukocyte ascorbic acid concentrations are considered a relatively reliable index of tissue ascorbic acid stores in healthy people; they are less responsive than plasma to short-term fluctuations in vitamin C intakes, but their assay is difficult and requires a larger blood sample. Furthermore, leukocyte ascorbic acid concentrations may not be a reliable marker of vitamin C status in some conditions, such as severe infection. The body pool size of vitamin C has been estimated using isotope dilution techniques. More research is needed to identify useful functional tests of vitamin C status. CITE AS: Anitra C. Carr, Principles of Nutritional Assessment: Vitamin C https://nutritionalassessment.org/vitaminc/
Licensed under CC-BY-SA-4.0
19.1 Vitamin CFor centuries, the consumption of citrus fruit has been associated with the prevention of the vitamin C deficiency disease scurvy, but it was not until 1932 that the active compound was isolated (King & Waugh, 1932; Svirbely & Szent-Györgyi, 1932). A year later, ascorbic acid was characterized and became the first vitamin to be chemically synthesized. Humans are unable to synthesize vitamin C in vivo from glucose due to random genetic mutations, which occurred at an early stage in our evolution, and have resulted in loss of the terminal enzyme in the biosynthetic pathway, L‑gulonolactone oxidase (Nishikimi & Yagi, 1991; Drouin et al., 2011).
The term “vitamin C” is used here to embrace both L‑ascorbic acid and its oxidised form, L‑dehydroascorbic acid; both have approximately equal vitamin C activity as dehydroascorbic acid can be readily reduced back to the biologically active ascorbic acid in the body. Ascorbic acid is the enolic form of an α‑ketolactone (2,3-didehydro-threo-hexano-1,4-lactone), which in solution can be oxidized to the diketo form— dehydro-L‑ascorbic acid; their structures are shown in Figure 19.1. Under physiological conditions, vitamin C is present predominantly as the mono-anion, ascorbate.
19.2 Functions of Vitamin CAscorbic acid acts as an electron donating cofactor for a family of biosynthetic and regulatory metalloenzymes that carry out important hydroxylation reactions (Englard & Seifter, 1986; Kuiper & Vissers, 2014; Young et al., 2015). These include copper-containing mono-oxygenases and iron-containing dioxygenases. Of the latter, the most well-known is hydroxylation of proline and lysine residues during synthesis of collagen, the major connective tissue protein in the body. Ascorbic acid acts in these hydroxylation reactions by reducing the metal catalytic sites of procollagen proline 4‑dioxygenase (EC 188.8.131.52) and procollagen lysine 5‑dioxygenase (EC 184.108.40.206). Decreased activity of these two enzymes can lead to the defects in connective tissue that in turn cause the characteristic clinical features of scurvy. These include joint pain, tooth loss, bone and connective tissue disorders, and poor wound healing. Ascorbic acid is also involved in the hydroxylation of dopamine to noradrenaline, by acting as a cofactor for the enzyme dopamine-β-monooxygenase (EC 220.127.116.11). Noradrenaline is a neurotransmitter, and deficient dopamine hydroxylation may be associated with the mood changes, depression, and hypochondria that can occur with scurvy. The early symptoms of scurvy — fatigue, lethargy, and muscle weakness — are related to the role of ascorbic acid as a cofactor for maximal activity of two dioxygenases involved in carnitine biosynthesis (N‑trimethyllysine dioxygenase; EC 18.104.22.168, and γ‑butyrobetaine dioxygenase; EC 22.214.171.124). Carnitine is essential for the transport of activated long-chain fatty acids into the mitochondria, where they are converted to energy by way of β‑oxidation (Englard & Seifter, 1986). Several other enzymes depend on ascorbic acid as a cofactor, although their specific role in the development of scurvy is still unclear. These include peptidylglycine α‑amidating monooxygenase (EC 126.96.36.199), an enzyme required for the synthesis of numerous amidated peptide hormones which have multiple functions throughout the body (Englard & Seifter, 1986). More recent discoveries have included the hydroxylases that regulate the concentration and activity of the transcription factor hypoxia-inducible factor‑1α (i.e. hypoxia-inducible factor — proline dioxygenase; EC 188.8.131.52, and hypoxia-inducible factor — asparagine dioxygenase; EC 184.108.40.206) (Kuiper & Vissers, 2014), and the ten-eleven translocation (TET) hydroxylases and histone demethylases that modify epigenetic marks on DNA and histones (Young et al., 2015). Together, these epigenetic and transcription regulating enzymes have the potential to up- and down-regulate thousands of genes in the body, likely contributing to vitamin C's pleiotropic functions in human health and disease. Ascorbic acid also has several nonenzymatic functions based on its role as a reducing agent and antioxidant (i.e. electron donation). For example, ascorbic acid enhances the gastrointestinal absorption of nonheme iron, through its ability to reduce intraluminal iron to its more absorbable ferrous form (Hallberg et al., 1987). Ascorbic acid is a potent antioxidant, with the ability to quench a wide range of reactive oxygen species, including neutralising phagocyte-derived oxidants, thus attenuating tissue damage (Carr & Frei, 1999a). Ascorbic acid provides important antioxidant protection to plasma lipids and lipid membranes through direct scavenging of reactive oxygen species and through the regeneration of vitamin E from its oxidised form (α‑tocopheroxyl radical). Ascorbic acid can also regenerate the enzyme cofactor tetrahydrobiopterin from its oxidised form (dihydrobiopterin), thus potentially aiding synthesis of dopamine, serotonin and nitric oxide. At high intakes, vitamin C has been associated with protection against cardiovascular disease, cataracts, and cancer (Carr & Frei, 1999b), and a variety of immune-related functions (Carr & Maggini, 2017). Additional research, however, is needed to establish causal relationships between vitamin C and these degenerative diseases and immunocompetence.
19.3 Absorption and metabolism of vitamin CVitamin C is absorbed in the small intestine by an active sodium-dependent vitamin C transporter (SVCT‑1) that is dose dependent and can become saturated (Savini et al., 2008). About 70%–90% of vitamin C is absorbed when daily intakes range from 30 to 180mg, but absorption falls to ≤ 50% at intakes above 1g/d (Kallner et al., 1979; Levine et al., 1996). Indeed, the osmotic diarrhea and intestinal discomfort sometimes experienced by persons ingesting large gram doses of vitamin C is due to the presence of a large amount of unabsorbed vitamin C. Regulation of the whole body vitamin C content is achieved in part by this dose-dependent intestinal absorption of vitamin C. Renal action to conserve vitamin C (via SVCT‑1) or excrete unmetabolized vitamin C also plays an important part. Tissue uptake occurs primarily by the SVCT‑2 isoform (Savini et al., 2008). Different tissues uptake and retain vitamin C to variable extents, with neuronal and glandular tissues containing the highest concentrations (Hornig, 1975), indicating vital roles for vitamin C in these tissues. Catabolism of ascorbic acid in humans occurs through oxidation to dehydroascorbic acid and, in the absence of sufficient reducing equivalents, subsequent hydrolysis to diketogulonic acid. This can then be oxidised to smaller molecules, such as oxalate, that are excreted in urine. However, it should be noted that dehydroascorbic acid is readily taken up via cell membrane glucose transporters (GLUTs) and rapidly reduced back to ascorbic acid via various chemical and enzymatic pathways (Rumsey et al., 1997; Washko et al., 1993), thus limiting dehydroascorbic acid concentrations in vivo. With large intakes, most of the vitamin C is excreted in its unmetabolized form (Levine et al., 1996).
19.4 Deficiency of vitamin C in humansClinical manifestations of scurvy, such as weakness, petechial hemorrhages, gingival bleeding, ecchymoses and subperiosteal hemorrhage, anemia, and defects in bone development in children, are relatively rare in high-income countries. Nevertheless, vitamin C deficiency has been observed in institutionalized elderly subjects, likely arising from inadequate intake of vitamin C (Carr & Rowe, 2020). Alcoholic patients, those on restrictive diets and people in refugee camps are also vulnerable to vitamin C deficiency. Infantile scurvy is rare because breast milk provides adequate amounts of ascorbic acid and infant formulas are fortified with sufficient ascorbic acid to prevent the disease. Subclinical vitamin C deficiency may develop secondarily to some disease states such as severe respiratory infections, cancer, gastrointestinal disorders, cardiometabolic disorders and inflammatory diseases. This is likely partly due to the enhanced oxidative stress and inflammation associated with many of these diseases, as well as the use of certain drugs (e.g. aspirin) or chemotherapeutic agents that appear to affect the bioavailability or metabolism of ascorbic acid (Basu, 1982; Carr & Cook, 2018). The functional health consequences of both mild and moderate vitamin C deficiency are not well characterized. Gingival inflammation, fatigue, and irritability have been reported in moderate vitamin C deficiency induced experimentally (Leggot et al., 1986; Levine et al., 1996).
19.5 Food sources and dietary intakes of vitamin CVitamin C is synthesised in plants. However, the vitamin C content of different foods is highly variable (Carr & Rowe, 2020). Values can also depend on the growing conditions, season of the year, stage of maturity, location, and storage time prior to consumption. Exposure to high temperatures, oxidation, or cooking in large amounts of water all reduce the vitamin C content of foods. Major food sources of vitamin C in the diets of most high-income countries are fresh fruits and fruit juices and some fresh vegetables; they can contribute up to 90% of the vitamin C intake. Citrus and kiwifruit are particularly rich sources of vitamin C. Of the vegetables, potatoes can be one of the most important sources of vitamin C in the diets of adults, for example in the U.K. (Gregory et al.,1990). Most staple foods, e.g. rice, wheat, maize and other grains, contain negligible amounts of vitamin C and meat, fish, eggs, and dairy products are also poor sources (Carr & Rowe, 2020). In low-income countries, the supply of food sources of vitamin C is often seasonal, so intakes may vary widely. In The Gambia, for example, intakes ranged from zero to 115mg/d in one community, depending on the seasonal availability of mangos, oranges, and grapefruit (Bates et al., 1994). In humans, the bioavailability of vitamin C from food sources is comparable to that from supplements and is not affected by the type of food consumed (Carr & Vissers, 2013).
19.6 Nutrient reference values for vitamin CSeveral expert groups including the Institute of Medicine (IOM) in the U.S. and the European Food Safety Authority (EFSA) have derived values for Average Requirements (ARs) and Recommended Intakes (RIs) for vitamin C. The AR (U.S. equivalent, Estimated Average Requirement) is set for a particular group of individuals and is used to assess the prevalence of adequate intakes of vitamin C within that group. The RIs (U.S. equivalent, Recommended Daily Allowance (RDA); EFSA equivalent, Population Reference Intake) are derived from the ARs and its distribution and are used to assess intakes and plan diets for individuals. Recommendations for vitamin C intakes vary greatly around the world, with recommended intakes (RIs) for individuals ranging from as low as 40mg/d in the U.K. to 110mg/d by EFSA (2017). In the U.S., the RDAs established by the IOM in 2000 are 75mg/d and 90mg/d for women and men, respectively (Levine et al., 1996; IOM, 2000). The large discrepancies in the RIs have resulted in calls for harmonization (Carr & Lykkesfeldt, 2020). Higher vitamin C intakes are recommended by all expert groups for pregnant and lactating women due to the needs of the developing fetus and the transfer of vitamin C to breast milk. Furthermore, some countries (e.g., the U.S.) recommend higher intakes for smokers due an enhanced oxidative burden and resultant increase in turnover of vitamin C. Consequently, RIs for smokers may be increased by +20mg/d to +80mg/d, depending on the country. Upper levels of intake (ULs) have also been derived for vitamin C by some expert groups. The ULs represent daily intakes that if consumed chronically over time will have a very low risk of causing adverse effects. Intakes from all sources of vitamin C are considered: food and nutrient supplements. Because vitamin C is water soluble, however, any excess not required by the body is readily excreted, therefore, there is no known upper toxic concentration. Nevertheless, some countries have set ULs ranging from 1–2g/d for adults based on potential gastrointestinal disturbances at very large (> 4g/d) doses. For more details on the application of these nutrient reference values, see Chapter 8b. In addition to diarrhea and other gastrointestinal disturbances, possible adverse effects of excessive intakes of vitamin C include an increase in the absorption of dietary iron potentially leading to iron overload in people with hemochromatosis, and hemolytic anaemia in people with glucose‑6-phosphate dehydrogenase deficiency due to an inability to attenuate hydrogen peroxide generation. Current evidence is insufficient to implicate high intakes of vitamin C as a risk factor in oxalate stone formation. Nevertheless, individuals with hemochromatosis, glucose‑6-phosphate dehydrogenase deficiency, and renal disorders, may be susceptible to adverse effects from excessive intakes of vitamin C (IOM, 2000).
19.7 Indices of vitamin C statusThere are no reliable functional tests of vitamin C status. Instead, static biochemical tests, particularly the measurement of plasma or leukocyte ascorbic acid concentrations, are most frequently used to assess vitamin C status. These static biochemical tests are discussed in detail below.
19.8 Plasma ascorbic acidAscorbic acid is transported in the plasma but is not bound to any protein; almost all is present as the ascorbate monoanion. Plasma (or serum) ascorbic acid concentrations are the most frequently used and practical index of vitamin C status in studies of individuals and populations. Concentrations are influenced by recent intake of the vitamin, making fasting blood samples essential.
19.8.1 Ascorbic acid pharmacokineticsPlasma ascorbic acid concentrations exhibit a characteristic sigmoidal relationship with intake, as shown in Figure 19.2. In adults, the steepest change is evident at intakes between about 30 and 90mg/d of vitamin C. When intakes are below about 20mg/d, most of the vitamin C enters the tissues, so there is very little available for circulation in the blood. For intakes greater than 100mg/d, plasma concentrations tend to plateau between 60 and 70µmol/L, concentrations representing the renal threshold and plasma saturation (Figure 19.2). When intakes exceed about 200mg/d, intestinal absorption of vitamin C becomes the limiting factor (Levine et al., 1996). As a result of this threshold effect with only limited intestinal absorption at intakes > 200mg/d, and the excess circulating ascorbic acid being excreted in the urine, plasma ascorbic acid concentrations cannot identify people who are regularly consuming excessive amounts of vitamin C. Hence, it is not surprising that in the U.S. National Health and Nutrition Examination Survey (NHANES II), a correlation between plasma ascorbic acid and vitamin C intakes was only reported for people not taking vitamin C supplements (Loria et al., 1998). Vitamin C is one of the best markers for fruit and vegetable intake, particularly fruit intake (Block et al., 2001; Drewnowski et al., 1997). However, strong correlations between dietary intakes of vitamin C and plasma ascorbic acid concentrations have only been reported when habitual dietary intakes of ascorbic acid are relatively modest (30–80mg/d) (Bates et al., 1979). People consuming chronically low intakes of vitamin C likely have plasma ascorbic acid concentrations that reflect the ascorbic acid content of the body (Jacob et al., 1987). Indeed, in such circumstances, plasma concentrations are probably as accurate an indicator as concentrations in leukocytes. However, the relationship may be obscured in epidemiological studies by other factors that affect the absorption or metabolic turnover of vitamin C. In NHANES III, for example, only 50% of adult men had serum ascorbic acid concentrations > 38µmol/L, although more than 75% had dietary intakes higher than the estimated average requirement (i.e., 75mg/d) (IOM, 2000). Such a discrepancy may be due, in part, to the high proportion of U.S. adult males who smoke or who are exposed to environmental tobacco smoke.
19.8.2 Factors affecting plasma ascorbic acidCigarette smoking has a marked effect on plasma ascorbic acid concentrations. Many studies report lower plasma ascorbic acid concentrations in smokers (Carr & Rowe, 2020), as well as in those regularly exposed to environmental cigarette smoke (Figure 19.3). The mechanism controlling the influence of cigarette smoking on plasma ascorbic acid concentrations appears to be related to the higher metabolic turnover of vitamin C in smokers than in nonsmokers (Kallner et al., 1981), which is probably induced by increased oxidative stress from substances in smoke. Certainly, studies using isotopically labeled ascorbic acid show an increased metabolic turnover of ascorbic acid in smokers (70mg/d versus 36mg/d for nonsmokers), resulting in a 40% greater requirement for vitamin C (IOM, 2000). Smokers also tend to have lower dietary intake of vitamin C, further contributing to a poor vitamin C status (Schectman et al., 1989). Sex influences plasma ascorbic acid concentrations; women have higher plasma ascorbic acid concentrations than men who consume similar intakes of vitamin C. However, this is likely partly explained by volumetric dilution due to differences in fat-free mass (Jungert & Neuhauser-Berthold, 2015), a premise supported by a lack of sex differences in plasma ascorbic acid concentrations before adolescence. For example, in NHANES III, higher serum ascorbate concentrations in women than in men were only apparent in those aged > 19y; no sex-related differences were reported in younger age groups (IOM, 2000). Thus, sex differences in lean body mass are likely to be a contributing factor (Blanchard, 1991). Age-differences in plasma ascorbic acid concentrations have been reported. The elderly, especially those who are institutionalized or housebound, may have lower concentrations than those of younger persons (Carr & Rowe, 2020). This trend has been attributed in part to lower dietary intakes because of problems of poor dentition or chronic diseases that may influence absorption or metabolism of the vitamin. Nevertheless, at present, there is no evidence that the elderly have higher vitamin C requirements than those for young adults. Indeed, NHANES IV data has indicated a U-shaped curve for circulating vitamin C concentrations by age (Schleicher et al., 2009). Pregnancy lowers plasma ascorbic acid concentrations due to increasing body weight and hemodilution, as well as active transfer to the fetus, especially during the last trimester. Lactation results in even higher requirements due to active transfer of an average of 40mg/d vitamin C through breast milk to the growing infant (IOM, 2000). Higher body weight is typically associated with lower vitamin C status in epidemiological studies. This is likely due to volumetric dilution, whereby an identical vitamin C intake will result in lower vitamin C concentrations in a person of higher bodyweight relative to a person of lower bodyweight (Block et al., 1999). People with central obesity are likely to have even higher requirements due to enhanced oxidative stress and inflammation (Carr et al., 2022). Despite the obesity pandemic in many regions of the world, and the use of bodyweight to estimate vitamin C recommendations for women and children, there are as yet no vitamin C recommendation categories for large or overweight individuals. Acute or chronic infection lowers circulating ascorbic acid concentrations. The more severe the infection, the lower the vitamin C status, as evidenced by the high prevalence of hypovitaminosis C and deficiency in hospitalised patients with pneumonia and sepsis (Carr, 2020).
19.8.3 Interpretive criteria for plasma ascorbic acidProtracted intakes of vitamin C < 20mg/d cause plasma ascorbic acid concentrations to decline rapidly to deficient concentrations (i.e. 11µmol/L or less) (Hodges et al., 1971; Jacob et al., 1987). At the lower plasma ascorbic acid concentrations, clinical signs of scurvy such as follicular hyperkeratosis, swollen or bleeding gums, petechial hemorrhages, and joint pain occur (Hodges et al., 1969). Therefore, plasma ascorbic acid concentrations ≤ 11µmol/L are taken as indicative of vitamin C deficiency. The cut-off values for plasma ascorbic acid concentrations used to distinguish different categories of deficiency are poorly defined. NHANES II defined “clinically based categories” of plasma ascorbate concentrations in terms of mg/dL, i.e. deficient < 0.2, low 0.2–0.39, normal 0.4–0.99, and saturated ≥ 1.0mg/dL (equating to < 11, 11–22, 23–56, and ≥ 57µmol/L, respectively) (Loria et al., 1998). Of note, the low (hypovitaminosis C) cut-off is when the “sub-clinical” signs and symptoms of vitamin C deficiency start to be observed, e.g. changes in mood and energy levels. More recently, the European Food Safety Authority (EFSA) has defined ≥ 50µmol/L as “adequate” (Table 19.1; EFSA, 2013). Well-conducted pharmacokinetic studies in men and women suggest 70µmol/L as a cut-off for “saturation”, although there is variation between individuals (Levine et al., 1996; Levine et al., 2001). The criteria in Table 19.1 have been used in more recent NHANES analyses (Crook et al., 2021), that indicated that smoking, male sex and higher body weight/BMI were common indicators for lower vitamin C status.
|Status|| Plasma Ascorbic
|Hypovitaminosis C||< 23|
19.8.4 Measurement of plasma ascorbic acidFasting blood samples are required for plasma ascorbic acid analysis. The samples need to be preserved immediately after collection to avoid degradation of the ascorbic acid. Metaphosphoric acid (or trichloroacetic acid) is typically used to precipitate protein and to stabilize the ascorbic acid in the samples prior to analysis. Additionally, a reducing agent, such as dithiothreitol (DTT), can be added to preserve the ascorbic acid in a reduced state. The reducing agent TCEP (tris(2-carboxyethyl)phosphine) is versatile in that it can also work in acidified solutions with longer incubation times. A metal chelator, such as DTPA (diethylenetriaminepentaacetic acid), can be added to attenuate metal ion-dependent oxidation of ascorbic acid (particularly important if samples are hemolysed). It should be noted that EDTA (ethylenediaminetetraacetic acid), although a commonly used metal-chelator, is redox-active and can catalyse the oxidation of ascorbic acid if the samples are not handled appropriately (Pullar et al., 2018). Plasma samples for ascorbic acid analysis that have been prepared appropriately and frozen at −20°C are stable for several weeks and at −70°C for at least 1y (Margolis & Duewer, 1996). If the samples cannot be processed immediately, whole blood can be stored for up to 8h at 4°C before processing (Galan et al., 1988). Care needs to be taken with choice of anti-coagulants for collection of blood samples as the anti-coagulant EDTA, although a metal-chelator, remains redox active and can result in loss of ascorbic acid if the blood or plasma samples are not kept at 4°C (Pullar et al., 2018). Several methods are available for measuring vitamin C in both the reduced form or as total ascorbic acid. The older colourimetric or fluorometric assays use predominantly 2,4‑dinitrophenylhydrazine or o‑phenylenediamine. These methods, however, have a number of limitations (Washko et al., 1992). Their sensitivity and specificity is low, and they are often subject to interference by other biological substances and yield falsely high readings at low ascorbic acid concentrations. In some circumstances, inadvertent oxidation of ascorbic acid or hydrolysis of dehydroascorbic acid may occur. The method of choice for measuring ascorbic acid in plasma samples is high-performance liquid chromatography (HPLC); only 50µL of plasma is required for the measurement. Of the HPLC methods available, electrochemical detection is preferred, with its high selectivity and sensitivity. However, this method does require a dedicated instrument and an experienced operator (Washko et al., 1989). Other simpler methods use HPLC coupled with an ultraviolet detector, but these methods can have a lower sensitivity and specificity (Washko et al., 1992). Analysis of dehydroascorbic acid by HPLC is more difficult because direct electrochemical or UV detection of dehydroascorbic acid is not possible. Instead, samples must first be analysed for ascorbic acid and then reduced for measurement of ascorbate plus dehydroascorbic acid. Dehydroascorbic acid is then determined by subtraction. In vivo concentrations of dehydroascorbic acid are generally very low (e.g. < 2µmol/L) due to rapid uptake (via GLUTs) and intracellular reduction to ascorbic acid by cells of the vasculature. Therefore, reports of higher plasma (or serum) concentrations of dehydroascorbic acid in different disease states are likely due to artefactual ex vivo oxidation often associated with use of the older spectrophotometric methods and/or inappropriate handling and processing of the samples prior to analysis (Pullar et al., 2018).
19.9 Ascorbic acid in leukocytes and specific cell subsetsLeucocytes include lymphocytes, monocytes, and three classes of granulocytes: polymorphonuclear leukocytes or neutrophils, eosinophils, and basophils. These cell types differ in their concentration of ascorbic acid and possibly in their response to supplemental vitamin C (Levine et al., 1996; Levine et al., 2001), thus complicating the interpretation of ascorbic acid concentrations in leukocytes.
19.9.1 Leukocyte ascorbic acidConcentrations of ascorbic acid in plasma and leukocytes correlate relatively well (Figure 19.4; Sauberlich et al., 1989), even though ascorbic acid concentrations in leukocytes are at least 14 times greater than those in plasma (Levine et al., 1996; Levine et al., 2001). The ascorbic acid concentrations of mixed leukocytes range from 90–300nmol/108 cells in adults (Omaye et al., 1987), and depend in part on the heterogeneous nature of the leukocytes, and the methods used to isolate and analyse them. Leukocyte ascorbic acid concentrations are commonly believed to be a more reliable index of tissue stores of ascorbic acid than are the corresponding concentrations in the plasma, erythrocytes, or whole blood (Turnbull et al., 1981). Leukocyte ascorbic acid concentrations are less responsive than plasma to short-term fluctuations in recent vitamin C intakes. In the study shown in Figure 19.5, leukocyte ascorbic acid concentrations decreased about 33%, compared with the much larger and more rapid decline in plasma. Similar trends have been reported by others (Jacob et al., 1987). Thus, they provide a less sensitive measure of lower intakes. Concentrations of ascorbic acid in leukocytes reflect changes in tissue ascorbic acid concentrations and the ascorbic acid body pool, but not dietary intakes. Animal studies (i.e., monkeys and guinea pigs) have confirmed that leukocyte ascorbic acid concentrations provide an accurate reflection of ascorbic acid concentrations in the liver and body pool (Omaye et al., 1979). Several factors can influence the concentrations of ascorbic acid in leukocytes. It has been proposed that the platelet/leukocyte ratio can influence leukocyte ascorbic acid values (Vallance, 1986). Platelets have comparable ascorbic acid concentrations to mononuclear cells (Levine et al., 2001), and should ideally be separated from leukocytes prior to leukocyte ascorbic acid assays (Vallance, 1986). Choice of the anticoagulant used may influence binding of platelets to leukocytes (Healy & Egan, 1984). Some additional non-dietary factors that influence concentrations of ascorbic acid in leukocytes are smoking, sex, infection, and certain drugs, as discussed for plasma ascorbic acid concentrations. In some cases, these effects are due to alterations in cell populations (e.g., acute infection) or to differences in ascorbic acid uptake (Lee et al., 1988). Finally, concentrations of vitamin C in phagocytic cells, such as neutrophils, can be influenced by activation of their oxidative burst. This results in the generation of reactive oxygen species which oxidise extracellular ascorbic acid to dehydroascorbic acid that is readily transported into the cells via membrane GLUTs, where it is then rapidly reduced back to ascorbic acid (Washko et al., 1993). Higher than anticipated concentrations of vitamin C have been observed in leukocytes isolated from patients with severe sepsis, which is a condition characterised by systemic oxidative stress and activation of neutrophils (Carr et al., 2021).
19.9.2 Ascorbic acid in specific cell subsetsThe specific cell type that is most useful for assessing vitamin C status is presently uncertain. As noted earlier, both ascorbic acid concentrations in individual leukocytes and their response to vitamin C supplementation vary. Mononuclear cells (lymphocytes and monocytes) have concentrations that are two- to threefold higher than granulocytes (e.g., neutrophils). Furthermore, the ascorbic acid pool in mononuclear cells is depleted more slowly than that from other blood compartments (Jacob, 1990). In addition, it can be difficult to obtain homogeneous and reproducible fractions of specific cell types, so that their reported ascorbic acid concentrations can vary widely. Lymphocyte ascorbic acid concentrations of 120–250nmol/108 cells have been reported for healthy adults not consuming supplemental vitamin C (Evans et al., 1982; Yew, 1984). Jacob et al. (1991) measured lymphocyte ascorbic acid concentrations in a depletion-repletion study of healthy men that was designed to induce moderate vitamin C deficiency. The mean baseline lymphocyte ascorbic acid concentration in the men was 209nmol/108 cells. After 60d of depletion, lymphocyte ascorbic acid concentrations fell significantly and consistently to a mean concentration of 87nmol/108 cells. Moreover, the concentrations distinguished between the group of participants receiving 5, 10, or 20mg ascorbic acid (74–145 nmol/108 cells) versus those participants receiving 60 or 250mg/d (182–261nmol/108 cells). Strong correlations of plasma and lymphocyte ascorbic acid concentrations were noted within the individual subjects when ascorbic acid intakes ranged from 5–250mg/d (Table 19.2) There was no evidence of scorbutic symptoms in the depletion phase of this study, but there was evidence of greater oxidative damage, based on alterations in indices of oxidant status (Jacob et al., 1991).
|n|| Plasma |
|1||4||250†||8||59.6 ± 3.3a||209 ± 9a|
|2||32||5||8||6.6 ± 0.3b||117 ± 7‡b|
|3||28||10||4||5.3 ± 0.3b||87 ± 9b|
|4||28||20||4||5.9 ± 0.5b||95 ± 5c|
|5||28||60||3||26.7 ± 8.8c||201 ± 2a|
|6||28||250||5||64.2 ± 4.5a||217 ± 11a|
19.9.3 Interpretive criteria for leukocytesConfusion exists over the interpretive criteria used for leukocyte ascorbic acid concentrations, in part because they can be expressed in different ways: per 108 cells, per mL, per DNA concentration, or per unit of protein. This makes comparisons among laboratories difficult. The most common method is in terms of cell numbers (nmol/108 cells). The heterogeneous nature of the leukocytes and various technical difficulties with their analyses, are further complicating factors. Table 19.3 presents the interpretive criteria used by Jacob (1994) and given in Sauberlich (1999), expressed in terms of cell numbers. When expressed in this way, clinical signs of scurvy, such as swollen or bleeding gums or petechial hemorrhages, have been associated with leukocyte concentrations of about 11nmol/108 cells. Even at concentrations as high as 50nmol/108 cells, scorbutic changes including inflammation, tenderness, bleeding of the gums, and petechiae have sometimes been described (Sauberlich et al., 1989). For this reason, the cut-off shown in Table 19.3, indicative of deficiency, is < 57nmol/108 cells.
|Status|| Mixed leukocytes |
|Deficient||< 57||< 114|
|Adequate||> 114||> 142|