Hope no-one minds me putting this here, it's a paper on d-lactate and b12 deficiency, and thought it might be of interest.
Abnormal Fecal Lactobacillus Flora and Vitamin B12 Deficiency in a Patient With Short Bowel Syndrome
Hojo, Kenichi*; Bando, Yuki†; Itoh, Yoko†; Taketomo, Naoki*; Ishii, Masahiro†
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Author Information*Food Science Institute, Meiji Dairies Corporation, Odawara, Japan
†Department of Pediatrics, Kitasato University School of Medicine, Sagamihara, Japan
Received October 10, 2006; accepted May 23, 2007.
Address correspondence and reprint requests to Kenichi Hojo, Food Science Institute, Meiji Dairies Corp, 540 Naruda, Odawara, Kanagawa 250-0862, Japan (e-mail: email@example.com
The authors report no conflicts of interest.
Lactobacillus overgrowth has been reported in patients with short bowel syndrome (SBS) (1-4). We previously reported D-lactic acidosis that Lactobacillus overgrowth caused in a male SBS patient (5). He had severe anemia related to vitamin B12 deficiency 9 years later. It has been generally accepted that B12 deficiency in SBS patients results from the inadequate absorption of B12 and/or the bacteria-host competition for uptake of B12 (6). In the present study, we suspected the recurrence of Lactobacillus overgrowth as a possible cause, because some lactobacilli require B12 for growth. Therefore, we analyzed the fecal flora and the B12 auxotrophy of Lactobacillus isolates.
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A 14-year-old boy presented in June 2005 with severe anemia and complaints of general fatigue. His medical history included an extensive small-bowel resection to treat obstructive ileus in September 1995. Subsequent malabsorption was treated with intravenous hyperalimentation and elemental diet for 2 months. The first attack of D-lactic acidosis was observed in this patient in early 1996. Similar attacks were observed 6 times in 5 months. Bacterial analyses revealed an increase of D-lactate producers, namely Lactobacillus delbrueckii subsp. lactis. We concluded that this increase was responsible for the D-lactic acidosis attack (5). Before the June 2005 presentation, he had been free from D-lactic acidosis for 9 years.
The laboratory findings on admission suggested megaloblastic anemia with pancytopemia. The serum B12 level was <50 pg/mL (reference range 180-914 pg/mL), unsaturated B12-binding capacity 2351 pg/mL (650-1340 pg/mL), serum folate 6.5 ng/mL (>3.1 ng/mL), serum iron 83 μg/dL (48-154 μg/dL), hemoglobin 4.2 g/dL (14-18 g/dL), and red blood cells 149 104 cells/mm3 (410 104-530 104 cells/mm3). The levels of other vitamins such as B1, B6, A, and E were normal. Bone marrow aspirates showed megaloblastic erythropoiesis; therefore, megaloblastic anemia due to B12 deficiency was diagnosed. He was treated with physiological doses (1000 μg/day) of B12 (Methycobal, Eisai, Tokyo, Japan) by intramuscular injection. Given 5 days of treatment, serum B12 level recovered within 2 weeks and the other hematological parameters normalized within 1 month. Subsequently, he was treated with B12 by mouth for 6 months without any other complication.
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Fecal samples were obtained in September 2001 at a periodical medical checkup (during normal conditions) and in July 2005, when the patient was hospitalized (during B12 deficiency). Bacteriological analyses were carried out using methods described in a previous study (5,7). As shown in Table 1, the number of lactobacilli and the percentage of lactobacilli in the total bacteria count during B12 deficiency (9.45 log10 CFU/g and 45.2%) were higher than during the normal conditions (9.16 log10 CFU/g and 8.6%). During B12 deficiency, the number of bacteroidaceae, eubacteria, clostridia, and anaerobic cocci was <107/g in the fecal sample (Table 1).
Image ToolsWe obtained 35 Lactobacillus isolates from the feces sampled during normal conditions, and 45 isolates from the B12-deficient sample. Lactobacillus isolates were identified by 16S rDNA sequence similarity (>98%) and a species-specific polymerase chain reaction method described by Torriani et al (8). The 16S rDNA sequence analysis could not discriminate clearly between L pentosus and L plantarum, or between L gasseri and L johnsonii. Table 2 shows that L fermentum was the predominant species, followed by L pentosus/plantarum and L gasseri/johnsonii, during the normal conditions. During B12 deficiency, L gasseri/johnsonii was predominant, followed by L mucosae and L delbrueckii subsp. lactis. We explored whether L delbrueckii subsp. lactis isolates in this study were the same strains that caused D-lactic acidosis in our previous study (5). Randomly amplified polymorphic DNA analysis (9) revealed that the isolates obtained in the present study were different from the previous isolates.
Image ToolsTo determine the B12 auxotrophy of Lactobacillus isolates, each isolate was anaerobically inoculated in a chemically defined medium (10) with or without B12 (40 μg/L) for 24 hours at 37C. As shown in Table 2, all of the isolates of L gasseri/johnsonii and L delbrueckii subsp. lactis required B12. During the normal conditions, the ratio of these B12 auxotrophs to the total bacteria was only 1% in the feces (Table 2). Surprisingly, the population of B12 auxotrophs was one fourth (25.8%) of the total fecal flora during B12 deficiency.
Short-chain fatty acids in feces, urine, and serum samples were identified using a high-pressure liquid chromatography system (Shimadzu, Kyoto, Japan) (11). D- and L-lactate concentrations in the samples were identified by an enzymatic method (Boehringer, Mannheim, Germany). The serum lactate levels during B12 deficiency (D-lactate, 0.93 mmol/L; L-lactate, 2.82 mmol/L) were excessively higher than those during the normal condition (D-lactate, 0.04 mmol/L; L-lactate, 1.51 mmol/L) (Table 3). Moreover, the fecal D- and L-lactate levels during B12 deficiency were 23.82 mmol/kg and 37.13 mmol/kg, respectively; these levels were higher than those during the normal conditions (1.13 and 2.18 mmol/kg, respectively). The pH of fecal samples during the normal conditions and B12 deficiency was 5.6 and 4.5, respectively.
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Valman and Roberts (12) measured B12 absorption in SBS patients who underwent ileal resections in childhood. They found normal B12 absorption in infants in whom the residual terminal ileum was ≥15 cm in length. In our patient, the residual terminal ileum with an intact ileocecal valve was about 52 cm in length when he was operated on for intestinal volvulus. In light of the previous article (12), the length and segment of the residual terminal ileum are sufficient for normal B12 absorption. In the present study, therefore, we examined whether intestinal bacteria were associated with B12 deficiency in our patient. Bacteriological analyses in feces revealed that the ratio of fecal lactobacilli to total bacteria during B12 deficiency (45.2%) was higher than that during normal conditions (8.6%) (Table 1). The number of bacteroidaceae, eubacteria, and clostridia, which are usually predominant in human feces (7), was <107/g. This may be related to the lactobacilli overgrowth because the overproduced lactate would suppress the other bacteria. In fact, the fecal pH during B12 deficiency was extremely low, and total lactate concentration in the feces during B12 deficiency was extremely high. From these results, we presume that Lactobacillus overgrowth recurred in this patient.
In humans, the relation between the microorganisms that constitute the intestinal microflora and B12 metabolism has been discussed. From this viewpoint, the ratio of B12 producer/consumer may be crucial for B12 uptake. In this report, L gasseri/johnsonii and L delbrueckii subsp. lactis required B12. Surprisingly, the ratio of these B12 auxotrophs during B12 deficiency (25.8%) was much higher than that during normal conditions (1.0%) (Table 2). It is known that patients with SBS have a predisposition to B12 malabsorption because of ileal resection and bacterial overgrowth (6). In the present study, we cannot rule out possible causes other than bacterial competition for uptake of B12, especially the inadequate absorption of B12, because we did not carry out a Schilling test for the determination of B12 absorption ability. Moreover, because our patient went through pubertal growth at about 11 years of age, it is not evident whether B12 deficiency was under the influence of pubertal growth. However, the possibility that lactobacilli may compete with the host for the uptake of B12 was implied by the finding that predominant Lactobacillus species required B12. The B12 auxotrophic lactobacilli apparently competed for uptake of B12 with our patient. To our knowledge, this is the first case indicating that B12 deficiency may be attributable to the overgrowth of the Lactobacillus strains.
Here, we would like to address a question: Why did our patient evade D-lactic acidosis despite the recurrence of Lactobacillus overgrowth? The serum D-lactate level during B12 deficiency (0.93 mmol/L) was higher than that during normal conditions (0.04 mmol/L) (Table 3); however, it was lower than that observed at 1 day after the attack of D-lactic acidosis in our previous study (2.32 mmol/L) (5). Before and during the episode of anemia, our patient had no typical D-lactic acidosis symptoms such as confusion, speech disturbance, and unusual behavior. We can suggest at least 2 possible explanations. One is dietary management, including carbohydrates, with related changes in the bacterial cell counts of D-lactate producers. Mayne et al (13) suggested that manipulation of carbohydrate intake may be used to treat severe, recurrent D-lactic acidosis in a patient with SBS. We had instructed our patient to avoid large amounts of simple carbohydrates after the onset of D-lactic acidosis (5). Therefore, restricted sugar administration probably contributed to the prevention of D-lactic acidosis. The second possible explanation is the difference in the bacterial cell counts of D-lactate producers. The number of lactobacilli that produced D-lactate was 9.0 log10 CFU/g in our previous study (5), whereas the number of D-lactate producers, namely, the isolates of L delbrueckii subsp. lactis, was 8.3 log10 CFU/g in the present study (Table 2).
In conclusion, it is evident that lactobacilli overgrowth recurred in our patient. Moreover, B12 auxotrophic Lactobacillus species increased during B12 deficiency. We believe that the overgrowth of these B12 auxotrophs may be implicated in B12 deficiency, although other causes were not excluded, such as the inadequate absorption of B12. From the results presented in this study, our patient seems to be predisposed to lactobacilli overgrowth; therefore, the lifelong control of intestinal flora would be required for the patient with SBS. Restricted intake of simple carbohydrates seems promising to improve and/or prevent abnormal intestinal Lactobacillus flora. In addition, routine hematological analyses, including minerals and vitamins, are needed for the detection of nutritional deficiencies before clinical symptoms occur.