Book Anitra C. Carr1  Principles of Nutritional
Assessment: Vitamin C

3rd Edition
September, 2022


Vitamin 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
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19.1 Vitamin C

For 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.

Figure 19.1
Figure 19.1. Structure of L‑ascorbic acid and dehydroascorbic acid.
Under physiological conditions, vitamin C is present pre­dom­inantly as the mono-anion, ascor­bate.

19.2 Functions of Vitamin C

Ascorbic 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 and pro­col­lagen lysine 5‑dioxy­gen­ase (EC 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 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, and γ‑butyro­betaine dioxy­genase; EC 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, 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, and hypoxia-inducible factor — asparagine dioxy­genase; EC (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 C

Vitamin 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 humans

Clinical 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 C

Vitamin 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 C

Several 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 status

There 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 acid

Ascorbic 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 pharmacokinetics

Plasma 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).
Figure 19.2
Figure 19.2. The relationship of plasma ascor­bate relative to daily vitamin C intake. Redrawn from Bates et al. (1997).
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 acid

Cigarette 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).
Figure 19.3
Figure 19.3. The effects of cigarette-smoke exposure on plasma ascorbic acid concen­trations in 141 premenopausal women who were active or passive smokers, or nonexposed nonsmokers. Three concen­trations of dietary vitamin C intake are represented in the results. Data from Tribble et al. (1993).
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 acid

Protracted 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.
Table 19.1. Current interpretive criteria for plasma ascorbic acid concen­trations.
Status Plasma Ascorbic
Acid (µmol/L)
Deficient < 11
Hypovitaminosis C< 23
Inadequate < 50
Adequate ≥ 50
Saturating ≥ 70

Of note, the mean vitamin C status of many high-income popu­la­tions globally is typically around 50µmol/L, with the noted sex differ­ences (Rowe & Carr, 2020). The prev­alence of hypo­vitamin­osis C and outright deficiency also tends to be relatively low in these countries. In contrast, many low-middle income popu­la­tions typically have lower mean vitamin C status, and a higher prev­alence of hypo­vitamin­osis C and deficiency, largely explained by lower dietary intakes. Vitamin C status and intake data from national surveys (e.g. U.S. NHANES and U.K. National Diet and Nutrition Survey) can be found in the review of Rowe and Carr (2020) and the cited references.

19.8.4 Measurement of plasma ascorbic acid

Fasting 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 subsets

Leucocytes 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 acid

Concen­trations of ascorbic acid in plasma and leukocytes correlate relatively well (Figure 19.4;
Figure 19.4
Figure 19.4. The relationship of mean plasma and leukocyte ascorbic acid concen­trations measured at various stages during a depletion and repletion study of a group of six adult females. Data from Sauberlich et al., 1989),
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).
Figure 19.5
Figure 19.5. Mean plasma and leukocyte ascorbic acid concen­trations during a depletion and repletion study of a group of six adult females. Ascorbic acid intakes were ad libitum for days 1–6; 0mg/d for days 6–30; 30mg/d for days 31–40; 60mg/d for days 41–50; and 90 mg/d for days 51–60. Data from Sauberlich et al., 1989),

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 subsets

The 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).

Table 19.2. Ascorbic acid concen­trations of healthy men after periods of various ascorbic acid intakes. Ascorbic acid concen­trations are means ± SEM at the end of each period, n = number of participants. Means within vertical columns not sharing the same subscript letter are significantly different (< 0.05) by t-test. † Participants consumed a supplement of 250mg ascorbic acid in addition to their free-living diet for 1–2wk before entering the study. ‡n = 7, one value deleted because of platelet contam­ination. Data From Jacob et al., (1991)
Ascorbic acid
intake (mg/d)
n Plasma
(nmol/108 cells)
1 4 250†8 59.6 ± 3.3a 209 ± 9a
2 32 58 6.6 ± 0.3b 117 ± 7‡b
3 28 104 5.3 ± 0.3b 87 ± 9b
4 28 204 5.9 ± 0.5b 95 ± 5c
5 28 603 26.7 ± 8.8c 201 ± 2a
6 28 2505 64.2 ± 4.5a 217 ± 11a

In later ascorbic acid depletion-repletion studies in men and women, Levine et al. (1996, 2001) measured concen­trations of ascorbic acid in purified neutro­phils, mono­cytes and lympho­cytes. Neutro­phil ascorbic acid concen­trations increased markedly in response to supple­mental vitamin C intakes of between 30 and 100mg/d but showed little further increase at higher doses. This trend is similar to that observed for plasma ascorbic acid concen­trations (Figure 19.2), although the latter reach a plateau at higher daily doses (about 200mg/d). The concen­trations of ascorbic acid in purified mono­cytes and lympho­cytes followed a similar trend, again reaching a plateau at doses of 100mg/d.

19.9.3 Interpretive criteria for leukocytes

Confusion 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.
Table19.3. Interpretive criteria for leukocyte ascorbic acid concen­trations. From Jacob (1994).
Status Mixed leukocytes
(nmol/108 cells)
(nmol/108 cells)
Deficient < 57 < 114
Low 57–114 114–142
Adequate > 114 > 142

Cut-off values for ascorbic acid concen­trations in specific cell types such as neutro­phils, mono­cytes, and lympho­cytes are less certain. Levine et al. (1996, 2001) showed that intra-cellular ascorbic acid concen­trations in neutro­phils were less than half of corresponding mono­nuclear leuk­ocyte (lympho­cyte and mono­cyte) concen­trations. The inter­pret­ation of leuko­cyte ascorbic acid concen­trations may be par­ticu­larly difficult in surgical patients or in cases of severe infection; these conditions are often associated with leuko­cytosis, activ­ation of neutro­phils, and systemic oxidative stress (Schorah et al., 1986; Vallance, 1988; Carr et al., 2021).

19.9.4 Measurement of leukocyte ascorbic acid

The isolation and assay of leukocytes for ascorbic acid is technically difficult, and presently requires relatively large blood samples (2–5mL), making the assay unsuitable for serial measure­ments on infants. Leuko­cytes can be isolated with density-gradient sediment­ation (Boyum, 1968). Mono­clonal anti­bodies to various cell types can also be used (Field, 1996). Analysis of ascorbic acid in leuko­cytes or specific cell types is best performed by HPLC with electro­chemical detection, after deprotein­ization (Washko et al., 1989).

19.10 Ascorbic acid in erythrocytes and whole blood

Erythrocyte ascorbic acid concen­trations are not widely used as an index of ascorbic acid status. Concen­trations are roughly comparable to plasma but only respond to changes in vitamin C intake over a narrow range. Hence, they are not as sensitive as plasma ascorbic acid concen­trations (Hodges et al., 1971). Because erythro­cyte concen­trations are less responsive to recent dietary intake, they could potentially be used to assess ascorbic acid status in non-fasting individuals (Pullar et al., 2020). However, both within- and between-variation and analytical variance can be greater than for plasma ascorbic acid (Jacob et al., 1987). Erythro­cyte ascorbic acid is technic­ally more challenging to measure due to potential for oxidation of ascorbic acid by hemo­globin-associated iron; the hemo­globin is ideally removed via centri­fugal filter units (Li et al., 2012). It should also be noted that conditions of vitamin C deficiency can result in enhanced erythro­cyte fragility and hemol­ysis (Tu et al., 2015).

Whole blood ascorbic acid concen­trations have also been investigated in ascorbic acid depletion-repletion studies. Again, concen­trations in whole blood are not as sensitive as plasma for assessing ascorbic acid depletion (Jacob et al., 1987). Concen­trations < 17µmol/L are considered deficient because they have been associated with scorbutic signs (Sauberlich et al., 1989); those of 17–28µmol/L are considered low. Concen­trations > 28µmol/L are inter­preted as acceptable (Sauberlich, 1999). Presently, whole blood ascorbic acid concen­trations are rarely used.

19.11 Urinary excretion of ascorbic acid and metabolites

Urine is the major excretory route for absorbed ascorbic acid. Most ascorbic acid is excreted in its un­metabol­ized form (Levine et al., 1996). When intakes are > 1g/d, there is some increase in oxalate excretion (Olson & Hodges, 1987). However, reports of high oxalate excretion are likely due to arte­factual ex vivo oxidation of excreted ascorbic acid prior to analysis (Chalmers et al., 1985), therefore careful handling of urine samples is required.

Urinary excretion of ascorbic acid reflects recent dietary intake. Experi­mental depletion-repletion studies have demon­strated that concen­trations in the urine decline progres­sively with increasing depletion of vitamin C until, in persons with scurvy, concen­trations are undetect­able (Jacob et al., 1987). Never­theless, urinary excretion is not a very sensitive index of ascorbic acid status; differ­ences between persons with adequate or deficient intakes of ascorbic acid are small. For example, in the depletion-repletion study of 11 young men shown in Figure 19.6,
Figure 19.6
Figure 19.6. Mean urinary ascorbic acid during times of varied ascorbic acid intake. Data from a study of 11 adult men by Jacob et al. (1987).
although urinary ascorbic acid excretion was significantly lower during the depletion periods (weeks 3–6) than in the initial 2wk 65mg/d baseline period, the differ­ences were small.

More recent pharma­cokinetic investigation has indicated a renal threshold of about 60µmol/L plasma ascorbic acid for a majority of healthy individ­uals (Ebenuwa et al., 2022), a concen­tration attained by intakes of ≥ 100mg/d (Levine et al., 1996, 2001). Higher concen­trations of ascorbic acid are observed in urine when plasma “saturation” is attained (i.e. intakes of ≥ 200mg/d). The vitamin C require­ments of an indi­vidual can potent­ially be deter­mined by admin­istering a test dose of vitamin C and monitoring the resultant excretion.

The specificity of urinary ascorbic acid, however, is also low. The drugs amino­pyrine, aspirin, barbit­urates, hydantoins, and para­ldehyde can all increase urinary ascorbic acid excretion (Sauberlich, 1981). An additional disadvantage of this test as a measure of ascorbic acid status in humans is the requirement for 24h urine speci­mens. The latter, although useful in clin­ical or research settings, are less practical in field studies.

19.11.1. Measurement of urinary ascorbic acid

Ascorbic acid is unstable in urine and samples should ideally be kept at 4°C and stabil­ized with a metal chelator (e.g. DTPA) and acid­ific­ation (e.g. with meta­phos­phoric acid) as soon as possible after collec­tion. HPLC with electro­chemical detection is the preferred method of analysis. Values can be presented as amount excreted per hour or day, or relative to urine creat­inine concen­trations for spot collections, to account for urine concen­tration.

19.12 Ascorbic acid in other cells and fluids

Ascorbic acid has been detected in a number of other body fluids. The concen­trations in saliva and alveolar lining fluid are generally relatively low and do not appear to correlate with concen­trations of ascorbic acid in plasma, leuko­cytes, or dietary intakes (Leggott et al., 1986; Jacob et al., 1987; Bui et al., 1992). In contrast, ascorbic acid concen­trations in cerebro­spinal fluid are typically higher than those found in plasma (Tallaksen et al., 1992). Ascorbic acid is able to bypass the blood-brain barrier via SVCTs in the choroid plexus (Angelow et al., 2003), and is believed to have important functions in the central nervous system (May, 2012). Of note, con­cen­trations of ascorbic acid in seminal fluid can be up to 10‑fold higher than those in plasma (Thiele et al., 1995). Lower seminal ascorbic acid concen­trations were observed in infer­tile men and this appeared to be related to elevated oxi­dative stress (Thiele et al., 1995; Lewis et al., 1997).

Buccal cell ascorbic acid concen­trations have been invest­igated as a potential bio­marker of vitamin C status. In a vitamin C depletion-repletion study in healthy men, buccal cell ascorbic acid concen­trations were signif­icantly lower in subjects receiving low ascorbic acid intakes (5, 10, 20mg/d) compared with repletion intakes (60 or 250mg/d) (Jacob et al., 1991). Neverthe­less, the invest­igators concluded that buccal cells are probably not suitable as a marker of ascorbic acid status over a broad range of popu­lation intakes. In contrast, muscle tissue obtained by needle biopsy has been shown to correlate relatively well with plasma concen­trations and dietary intake (Carr et al., 2013). Muscle biopsy is a highly invasive procedure and is not typically undertaken outside of research settings.

19.13 Body pool size

Isotope dilution techniques can be used to determine the size of the body pool of vitamin C. The method involves admin­istration of an oral dose of 14C‑labeled or 13C‑labeled ascorbic acid, followed by the measure­ment of the specific activity of blood or urine ascor­bate within 24–48h (Baker et al., 1971; Kallner et al., 1977). Of the two radioactive isotopes, 14C has a long half-life, so 13C, a shorter-lived isotope, is preferred.

In young healthy male adults, the pool size of ascorbic acid has been estimated to be about 1500mg (i.e., 20mg/kg body weight) (Baker et al., 1971; Kallner et al., 1977). In adult men, the total body pool of vitamin C ranges in size from < 300mg to about 3000mg, depending on the daily intake of L‑ascorbate. When pool sizes are < 600mg of vitamin C, psychological abnormalities have been reported (Kinsman & Hood, 1971), whereas at concen­trations < 300mg, scurvy symptoms have been observed (Baker et al., 1971).

19.14 Functional tests of vitamin C status

There are currently no specific functional tests for vitamin C status. Bleeding into the skin to form petechia, purpura and ecchy­moses are a sign of the vitamin C deficiency disease scurvy. Capillary fragility has been used in the past as a functional test of vitamin C deficiency (Vilter, 1967). However, this test can produce inconsistent results in individ­uals with vitamin C deficiency and is not specific to vitamin C deficiency states as other diseases may also increase capil­lary fragility (Vilter, 1967). Erythro­cyte fragility could be an alternative functional test of deficiency (Tu et al., 2015), however, once again, this is not specific for vitamin C deficiency. Consequently, altern­ative functional tests of ascorbic acid status need to be developed.

Specific markers of ascorbic acid’s enzyme cofactor functions have been proposed. An example relates to collagen cross­linking (Munday et al., 2005). In this study, urinary excretion of specific cross­link ratios were higher in British children versus Gambian children, and in Gambian children during the dry season (character­ised by high vitamin C intake and status) versus the rainy season. A supplement­ation study (100mg/d of vitamin C) in Gambian children during the rainy season did not, however, alter the crosslink ratio. This may have been due to the supplementation period (7wks) being insufficient, as collagen can have a long turnover in some tissues.

Another possibility is assess­ment of vitamin C-dependent epi­genetic marks (Young et al., 2015), for example in leuko­cytes. One prelim­inary study has demon­strated positive correl­ations between vitamin C status and hydroxy­methyl­cytosine and hydroxy­methyl­uracil marks in leuko­cyte DNA (Starczak et al., 2018). Participants with vitamin C concen­trations > 40µmol/L exhibited signif­icantly higher concen­trations of these epi­genetic marks than those with plasma concen­trations < 20µmol/L. As such, more research in this area appears warranted.


ACC wishes to thank Rosalind Gibson for her help in drafting the section on “Nutrient reference values for vitamin C”.