Vitamin C Status in Iranian Children With Acute Lymphoblastic Leukemia: Evidence for Increased Utilization

Abstract

Vitamin C status and total antioxidant capacity (TAC) in children with acute lymphoblastic leukemia (ALL) was assessed in 28 hospitalized patients and 30 apparently healthy control subjects. It was determined that whereas vitamin C intake in patients with ALL was more than twice as much as in controls, plasma and urinary concentrations of vitamin C were more than 10 times and 2.5 times higher in controls than in the patients with ALL, respectively (P < 0.001). Accordingly, serum TAC levels in the patients with ALL were lower than in healthy subjects (P < 0.001). Vitamin C utilization is increased in children with ALL.

INTRODUCTION

Leukemia is a general term denoting a group of malignant myeloproliferative disorders that are traditionally divided into myeloid and lymphoid categories based on the cell type involved, each of which may be acute or chronic. Acute leukemia is the most common form of childhood cancer and is the primary cause of cancer-related mortality in children ⁽¹⁾. Approximately 80% of children with leukemia have acute lymphoblastic leukemia (ALL), 17% have acute myeloid leukemia, and the remainder has rare forms of chronic leukemia ⁽²⁾. Although there is not a consistent record of blood cancers in Iran, there are some reports of increasing frequency of leukemias among Iranian children ^(3,4) with an autumn/winter peak in the diagnosis of childhood ALL ⁽⁴⁾.

The etiology of leukemia is poorly understood, and both genetic and environmental factors have been implicated ^(5–8). There is increasing evidence to support the theory that even gene rearrangements leading to leukemia may originate in utero ⁽⁸⁾. It has been shown that maternal dietary factors, especially carotenoids and glutathione, may play a protective role against acute lymphoid leukemia ⁽⁹⁾. Data regarding the possible supportive role of dietary factors in treatment of leukemia are even fewer. A huge body of evidence from human and animal studies indicates altered metabolism in cancer ^(10,11). Considering the high concentration of vitamin C in leukocytes ⁽¹²⁾ and the oxidative stress that may be induced by disease and chemotherapy ^(13,14), we looked at the possibility that vitamin C utilization may be enhanced in ALL, indicating a state of increased need.

PATIENTS AND METHODS

Briefly, 28 hospitalized patients and 30 healthy control subjects were introduced into the cross-sectional study. Blood and urine samples were taken to evaluate vitamin C status and total antioxidant capacity (TAC). Dietary assessment was performed to estimate vitamin C intake. Written informed consent was obtained from all participants. The research design was scientifically and ethically approved by the Deputy of Research of Hamedan (Iran) University of Medical Sciences and the Higher Education Council of the School of Public Health and Institute of Public Health Research at Tehran University of Medical Sciences.

Subjects

A total of 28 patients with ALL (16 female and 13 male) hospitalized at Sina Hospital in Hamedan in western Iran, ages 2 to 18 years, and 30 age- and sex-matched apparently healthy controls were introduced into the study. Patients, all diagnosed within the previous 6 months and receiving routine antileukemic chemotherapy, had no clinical evidence of active infection at the time of sampling and were following a regular diet. The control group had normal complete blood cell count, including peripheral smear, no specific signs or symptoms on clinical examination, and no history of vitamin supplement intake for at least 1 month before sampling.

Laboratory Tests

Five milliliters of fasting venous blood was collected and divided into 2 test tubes, with or without heparin. The sample containing anticoagulant was covered and kept on ice for a maximum of 1 hour until plasma separation for vitamin C analysis. One volume of cold 10% trichloroacetic acid was added to an equal volume of fresh plasma to precipitate the proteins. After shaking, the tubes were centrifuged at 4°C and 2500g for 20 minutes. The supernatant was then transferred to fresh tubes and kept at −80°C awaiting vitamin C analysis using 2,4-dinitrophenol hydrazine as described elsewhere ⁽¹⁵⁾. The blood sample without heparin was kept at room temperature for 30 minutes. The serum was then recovered and transferred to fresh tubes, which were kept at −80°C for a maximum of 2 weeks to determine their TAC.

Total antioxidant capacity was determined according to Miller et al ⁽¹⁶⁾ with minor modifications. The assay is based on the ability of serum antioxidants to prevent oxidation of 2,2′-azino di-(3-ethylbenzthiazoline sulfonate, ABTS). The results are expressed as inhibition percent.

An early-morning urine sample was also collected for vitamin C analysis. The samples were transferred to the laboratory on ice, wherein samples were mixed with equal volume of 10% trichloroacetic acid and then centrifuged, just like the plasma samples. Other interfering chromogenic substances in the supernatant were then removed by adding supernatant to 0.2 g charcoal, shaking, and centrifugation. The resulting supernatant was then used to determine vitamin C concentration using 2,4-dinitrophenol hydrazine.

To estimate vitamin C intake, 24-hour dietary recall, including vitamin supplements, was undertaken on 2 different days (including 1 weekend) by way of interviews with the patients' mothers and/or other caregivers.

Statistical Analyses

Normality of data distribution was tested using the Kolmogorov-Smirnov test. Comparison of means was done with Student t test or, when the distribution was not normal, Mann-Whitney U-Wilcoxon test. Correlations were determined using the Pearson equation. The predetermined upper limit of significance throughout this investigation was P < 0.05. All statistical analyses were done with SPSS software (version 10; SPSS, Chicago, IL).

RESULTS

Data distribution for TAC and serum and urinary concentrations of vitamin C were normal, but vitamin C intake was not normally distributed. In the control group, fresh fruits and vegetables made up the predominant sources of vitamin C intake, whereas patients with ALL received vitamin C mostly through fruit juices, fresh fruits, and multivitamin supplements. Although vitamin C intake in patients with ALL was more than twice as much as in healthy control subjects (Mann-Whitney U-Wilcoxon test, P = 0.001), serum and urinary concentrations of vitamin C were more than 10-fold and 2.5-fold higher in healthy control subjects than in patients with ALL, respectively (P < 0.001). Accordingly, serum TAC of patients with ALL was lower than in healthy control subjects (P < 0.001). Although vitamin C intake was directly correlated with urinary vitamin C levels in healthy control subjects (r = 0.503; P = 0.005), there was no such correlation in the patients with ALL. Interestingly, plasma and urinary concentrations of vitamin C showed significant correlation in only healthy controls (r = 0.482; P = 0.007). TAC was also correlated with plasma (r = 0.530; P < 0.001) and urinary (r = 0.360; P < 0.015) vitamin C levels (Table 1).

TABLE 1: Distribution of variables in patients with ALL and healthy control subjects

DISCUSSION

Considering the high prevalence of malnutrition in children with malignancies, including leukemias, at the time of diagnosis (50% in developing countries and <10% in patients with ALL in developed countries) ⁽¹⁷⁾, the frequency of single and multiple nutrient deficiencies in these patients is expected to be much higher. The importance of nutritional status in children with cancer lies in its possible effect on the course of the disease, response to therapy, and survival ⁽¹⁷⁾. Many factors may be involved in malignancy-induced malnutrition, among them decreased intake, increased utilization, increased excretion, increased body need, and decreased absorption ^(10,11,18).

In the present study, lower serum TAC and serum and urinary vitamin C despite higher intake of the vitamin in the patient group suggest increased utilization of vitamin C in ALL. Although decreased vitamin C absorption in the patients receiving chemotherapy may be another possibility, several studies failed to show decreased nutrient absorption due to chemotherapy in patients with ALL ^(19–22). Higher intake of vitamin C by the patients with ALL is likely due to special dietary care such as fresh fruits, fruit juices, and fresh vegetables given by the hospital dietetic service and their other caregivers, mostly their mothers. Multivitamin supplementation was another important source of vitamin C in patients with ALL.

It has been suggested that cancer-induced antioxidant depletion may be associated with increased utilization by the tumor itself or the resulting oxidative stress ⁽²³⁾. Chemotherapy per se may also induce oxidative stress and hence increase the utilization of antioxidants ⁽²⁴⁾.

One of the other possible mechanisms of increased utilization of vitamin C may be related to its apoptotic properties against malignant blood cells ^(25,26). Increased vitamin C utilization in blood cell malignancies may therefore reflect a physiological adaptive response. Under these conditions, antioxidant intake according to Recommended Daily Allowances may not meet patients' increased requirements.

In a study recently conducted in 103 children with ALL for 6 months, it was found that greater vitamin C intake at the sixth month was associated with fewer therapy delays, less toxicity, and fewer days spent in the hospital. The intake of antioxidants and vitamin A in this group of children was mostly less than the Recommended Daily Allowance ⁽²⁷⁾.

L-ascorbic acid (LAA) has been reported to inhibit the growth of HL-60 cells by induction of its differentiation into polymorphonuclear neutrophils ⁽²⁸⁾. It is unknown whether LAA has a similar effect on malignant lymphoid cells. If this is the case, then it can explain to some extent the increased utilization of LAA in the context of malignancy. Although increased ascorbic acid utilization in malignancies was investigated in a murine model many years ago ⁽²⁹⁾, the increased urinary ascorbate excretion in this model made it difficult to interpret decreased vitamin C status as enhanced utilization of the vitamin.

CONCLUSIONS

Our findings suggest increased vitamin C utilization in ALL. This may be due to oxidative stress induced by the disease itself and/or chemotherapy or apoptotic and inhibitory functions of vitamin C against leukemic cells. The increased body need for the vitamin may not be met solely by regular diet. Further studies are needed to elucidate the safe and adequate level of intake of vitamin C in ALL.

Acknowledgments

The authors thank all the patients and especially their mothers and caregivers who cooperated with us; we also offer our condolences to those who have lost their beloved children.