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Safety of D(‐)‐Lactic Acid Producing Bacteria in the Human Infant

2005; Lippincott Williams & Wilkins; Volume: 41; Issue: 4 Linguagem: Inglês

10.1097/01.mpg.0000176179.81638.45

1536-4801

Eamonn Connolly, Thomas Abrahamsson, Bengt Björkstén,

Digestive system and related health

INTRODUCTION The gut of human infants is normally rapidly colonized after birth, and this colonization is considered not only to form a protective barrier against pathogenic bacteria but also to direct maturation of the infant immune system (1,2). In recent years, exogenous preparations of probiotic lactic acid bacteria have been administered to newborn infants in attempts to strengthen various positive effects associated with colonization with lactobacilli. Indeed, insufficient colonization with lactic acid producing bacteria in Western as compared with Eastern Europe may be associated with the increased incidence of allergy in the former countries (3,4). Furthermore, recent studies indicate that administration of probiotic lactobacilli to newborn infants (5) may be beneficial in directing maturation of the immune system. Concerns have been raised that the use of D(-)-lactic acid producing bacteria as probiotic ingredients in infants formulas may be harmful through a postulated increase in blood D(-)-lactic acid levels (6). There are no reports of any healthy humans of any age with abnormal D(-)-lactic acid levels, however, and certainly none arising as a result of the ingestion of D(-)-lactic acid producing bacteria (7,8). Lactobacillus reuteri ATCC 55730 (also called SD2112) is a proven probiotic with the capacity to reduce infections and diarrhea in infants (9-11), and it has been suggested that this probiotic may also have immunomodulatory effects against allergy. It is one of many species of Lactobacillus known to produce both L(+)-lactic acid and D(-)-lactic acid through normal sugar fermentation. Safety evaluations on L. reuteri ATCC 55730 have been performed in controlled clinical trials on healthy volunteers (12) and immunocompromised (HIV-infected) adults (13), children, and infants (10,11,14) without adverse effects. The present study investigates the levels of D(-)-lactic acid in the blood at the age of 6 and 12 months in infants who have been supplemented daily with the probiotic from birth. MATERIALS AND METHODS This study forms part of a double blind, placebo controlled, multicenter clinical trial in infants with a family history of allergy, conducted at the Departments of Paediatrics at the county hospitals of Jönköping, Motala and Norrköping and the University Hospital in Linköping, Sweden. The aims of this main study are related to the effect of exposure to L. reuteri on the development of allergic symptoms during the first year of life. The main study objectives are not related to the aims of the substudy we report here and will thus be reported separately when the study is complete. After receiving written consent from both parents, pregnant women from families with a history of allergic disease were enrolled and randomized to the L. reuteri or placebo groups. The infants were supplemented from birth with either L. reuteri ATCC 55730 suspended in an oil (75% refined MCT/25% protein-free peanut oil) or a placebo preparation identical in both appearance and taste but lacking the bacteria (BioGaia AB, Stockholm, Sweden). Both study products were produced by the sponsor of the trial and were supplied by their pharmacist blinded to the clinics. A dose of 1 × 108 colony forming units (CFU) or placebo was given (in 5 drops of oil) to the infants every day for 12 months starting from the first day of life. From the entire clinical trial (232 infants), a sample group of 24 infants was randomly chosen for the analysis of D(-)-lactic acid in the blood at 6 months of age (after 6 months of supplementation). Blood was drawn and frozen at −20°C before being shipped on dry ice for analysis of plasma D(-)-lactic acid content at the Centre for Inherited Metabolic Diseases, Karolinska University Hospital, (Stockholm, Sweden), using a validated and regularly used D(-)-lactic acid analytical method based on the method of Gawehn (15). The samples were extracted with perchloric acid before the assay. D-lactate was determined in the extracts with the enzyme D-lactate dehydrogenase (D-LDH), which is specific to the D-form of lactate. D-LDH converts D-lactate to pyruvate under the reduction of NAD+ to NADH. The amount of NADH formed was determined at 340 nm in a spectrophotometer and is proportional to the sample content of D-lactate. To ensure complete degradation of D-lactate, the assay was performed in the presence of the enzyme glutamate pyruvate transaminase, which continuously removes the pyruvate formed by D-LDH. Control samples containing 2 mmol/L D-lactate were also analyzed and gave adequate responses (coefficient of variation <3%). The main clinical trial is still blinded, and none of the investigators or the pharmacies at the trial sites has access to the code. To make the analysis of D(-)-lactic acid in the present study in each group, the patient codes and their corresponding analysis results were sent to an independent statistician at the University Hospital Linköping, Sweden for analysis independent from the physicians and study nurses. Stool samples were collected in sterile plastic containers by the parents in the home and immediately placed in the freezer. Within 3 days, they were brought to the hospital (frozen on ice) and stored at −70°C. The samples were pooled and transported (frozen on dry ice) to the Biogaia laboratory (Lund, Sweden) for microbiologic analysis of L. reuteri content. Thawed samples were diluted and plated on Mann Rogosa Sharp + 2% sodium acetate (MRS-3) agar containing 50 mg/L vancomycin. Plates were incubated anaerobically at 37°C for 48 hours, after which colonies were confirmed as L. reuteri using a method based on the generation of reuterin (a metabolite specifically produced by L. reuteri) from a glycerol substrate (W. Dobrogosz and S.E. Lindgren, 4 October 1994, US Patent 5,352,586). The protocol was approved by the Scientific Ethical Committee of Linköping University Hospital before start of the trial and was performed in accordance with the Declaration of Helsinki. RESULTS AND DISCUSSION Twenty-four infants were included in the study, of which 14 were supplemented with L. reuteri and 10 with placebo. The weights (mean ± SD) of the babies at 6 months in the L. reuteri and placebo groups were 8,075 ± 876 g and 7,662 ± 991 g, and at 12 months 10,196 ± 1,214 g and 10,096 ± 1,075 g, respectively. The lengths of the babies at 6 months in the L. reuteri and placebo groups were 68 ± 2 and 67 ± 3 cm, respectively in the L. reuteri group and at 12 months were 76 ± 3 and 75 ± 3 cm, respectively. Compliance was primarily monitored using diaries filled in by the mothers because the fecal level is a poor monitor of colonization of the human strointestinal tract (16). Fecal colonization with live L. reuteri was, however, analyzed. In the L. reuteri group, average fecal levels at 6 and 12 months were 7.25 × 105 and 1.13 × 105 CFU per gram wet weight, respectively, and in the placebo group, not detected and 5.2 × 103 CFU/g, respectively. All 24 infants had very low blood levels of D(-)-lactic acid, ranging from 20 to 130 μM and there was no difference in this respect between infants who had ingested L. reuteri for 6 or 12 months and those who had received placebo (Table 1 and Fig. 1). Thus, the highest level observed was well within the normal range seen in humans (which is 20- 250 μM) (17-19) and far below levels associated with D-lactic acidosis in humans (over 3 mmol/L) (7). The physicians responsible reported no safety problems in any of the infants participating in this study and, in particular, they have observed no symptoms that would normally be associated with acidosis.TABLE 1: Plasma D(-)-lactic acid levels in infants after supplementation from birth with Lactobacillus reuteri ATCC 55730FIG. 1: Levels of D(-)-lactic acid levels (mean and SD) at 6 months in relation to daily intake of 108 CFU of Lactobacillus reuteri.Three previous studies in human infants have addressed the potential risks of giving lactic acid in infant formulas acidified in the production process by fermentation with lactic acid bacteria. They were all published between 1957 and 1965. In the first of these studies, no effect was observed on the rate of weight gain of 80 full-term infants given a feed formula containing 0.4% DL-lactic acid between 2 and 4 weeks of age (20). No negative effects were recorded. In the second study, healthy babies were fed cow's milk formula acidified with 0.4% to 0.5% mixed DL-lactic acid for periods of 10 days during the first 3 months of life, and there was some indication of acidosis and reduced weight gain in some infants (21). The third study (22) looked at administration of formula with 0.35% DL-lactic acid (20% D[-]-lactic acid and 80% L[+]-lactic acid) to healthy babies between the 10th and 20th day of life. A 3-fold increase in urinary excretion of L(+)-lactic acid and a 12-fold increase in excretion of D(-)-lactic acid were observed. It was concluded that D(-)-lactic acid appeared to be less well metabolized than L(+)-lactic acid. In none of the studies were the lactic acid levels measured in blood and in only one in excretions. On the basis of these three inconclusive studies, the Codex Standard for Infant Formula (CODEX STAN 72-1981) defines a limitation allowing only the use of "L(+)-lactic acid producing cultures" as technical aids for the adjustment of pH during manufacture of an infant formula (23,24). A pH adjusting fermenting Lactobacillus leading to high levels of lactic acid in the product, however, is different from adding a freeze-dried Lactobacillus, which is dormant in the formula and which will not produce any lactic acid until it is in the gastrointestinal tract. Thus, the formula with added freeze-dried lactobacilli will not contain any lactic acid derived from the bacteria at all. In recent years, the health benefits of adding live lactobacilli as freeze-dried probiotic ingredients to infant diets, quite distinctly and entirely different from pH adjusting agents, has been increasingly discussed. In this context, the CODEX guidelines have sometimes been interpreted as a hinder for the addition of D(-)-lactic acid producing lactobacilli to the infant diet (6). One of them, L. reuteri ATCC 55730, was isolated from human breast milk and is one of several commensal bacteria species in human milk (25). Furthermore, L. reuteri is indigenous to the human gastrointestinal tract and is present from shortly after birth and throughout life (2,3,26). There is thus reason to believe that these bacteria normally play a role in the colonization of the human infant. Clinical trials in infants and young children have confirmed that L. reuteri also confer real health benefit (e.g., in preventing gastrointestinal infection in day care centers (27) and in treating acute diarrhea (28,29)). Our study is of importance because no factual evidence on the levels of D(-)-lactic acid in infants supplemented with a D(-)-lactic acid-producing probiotic lactobacilli has previously been available to address this issue. In a recent review of D(-)-lactic acid probiotics and infants, the lack of data on blood D(-)-lactate acid levels in infants administered these probiotics was pointed out as limiting and that studies are needed (30). This was particularly unfortunate because the potential for modifying disease presentation and expression through the consumption of probiotics may be greater in infants than in any other age group. We observed no adverse events after long-term dietary supplementation with L. reuteri in the newborn infants participating in this study. This is in line with the literature because only in cases of short-bowel syndrome, where significant commensal flora overgrowth occurs spontaneously, is there possibly a risk of excessive spill over of D(-)-lactic acid into the blood and consequent D-lactic acidosis (7,30). Even in such cases, however, D(-)-lactic acid producing L. plantarum supplements have in fact been demonstrated to reverse D-lactic acidosis (31). Furthermore, infants supplemented with L. reuteri ATCC 55730 at doses up to 1011 CFU/day show no evidence of overgrowth of total lactobacilli or symptoms associated with D-lactic acidosis (10,11). In conclusion, our study provides evidence that there is no elevation of D(-)-lactic acid in the blood of infants given L. reuteri at a dose of 108 CFU/day from birth daily for 12 months. From this observation and after a careful review of the literature, we conclude that this D(-)-lactic acid producing probiotic can be safely given to infants.