PRACA ORYGINALNA

ORIGINAL ARTICLE

Khrystyna B. Kvit1,3, Natalya V. Kharchenko2, Vyacheslav V. Kharchenko2, Olga I. Chornenka3, Romania I. Chornovus3, Uljana S. Dorofeeva3, Oksana B. Draganchuk3, Oksana M. Slaba4

1Department of Therapy №1 and Medical Diagnostics, Faculty Postgraduate Teaching, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine

2Department of Gastroenterology, Dietology and Endoscopy, Shupyk National Medical Academy of Postgraduate Teaching, Kyiv, Ukraine

3Medical Department, “Medicover Ukraine”, Lviv, Ukraine

4Department of Therapeutic Dentistry, Faculty Postgraduate Teaching, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine

Abstract

Introduction: Small intestinal bacterial overgrowth may cause the hyperlipidemia appearance by enterohepatic circulation disturbance which evolves on the background of the early bile acids deconjugation with further endotoxin production and oxidative stress in the liver with hyperproduction of cholesterol and atherogenic lipoproteins.

The aim: the determination of prevalence and features of SIBO in a series of patients with hyperlipidemia and in control subjects.

Materials and methods: Nineteen patients with hyperlipidemia and ten control subjects were studied. Small intestinal bacterial overgrowth was assessed by a lactulose breath test. Such biochemical markers as CRP, ALT, AST, GGTP, apolipoprotein B, bilirubin, cholesterol and lipid profile were determined. Except the routine interpretation of lactulose breath test, which contains the SIBO detection, small intestinal transit time and hydrogen level evaluation with next comparison between groups of patients was realized.

Results: Small intestinal bacterial overgrowth was present in 78.9% of patients with hyperlipidemia and 40% in control subjects. The maximal dose of H2 was particularly higher in patients with hyperlipidemia in comparison with control group (94,7±13,69 vs. 36,13±5,4). There was a strong correlation between AST level and SIBO existence in both groups (r=1). Positive connection between LDL, TG, VLDL and the dose of exhaled hydrogen on 120 minute (r=0.6, r= 0.62, r=0.7 respectively) and strong negative correlation between HDL and 120 minute dose (r=-0.74) in main group was marked.

Conclusions: Patients with hyperlipidemia have a higher prevalence of small intestinal bacterial overgrowth and there is a relationship between H2 rate and LDL, TG, VLDL.

KEY WORDS: hyperlipidemia; hypercholesterolemia; syndrome of bacterial overgrowth; small intestinal bacterial overgrowth; lactulose breath test

Wiad Lek 2019, 72, 4, 645-649

Introduction

Numerous studies in recent years have proved the pathogenetic relationship of the intestinal microbiota with such diseases as hyperlipidemia, atherosclerosis, arterial hypertension, steatohepatitis, diabetes mellitus [1]. It is known that on the background of the syndrome of excessive bacterial growth in the intestine, proatherogenic changes in lipid spectrum are possible [2].

Microorganisms which colonize the intestine are capable to impact on cholesterol metabolism by affecting the key stages of its synthesis. Excessive microbial growth of anaerobes in the jejunum, which is typical for syndrome of bacterial overgrowth (SIBO), leads to its damage, with the development of system inflammation, that causes early deconjugation of bile acids with the formation of their toxic salts and impaired enterohepatic circulation [3,4]. Deconjugated bile acids can also damage the epithelium of the small intestinal mucosa by its detergent properties.

As a result, synthesis and sorption of enzymes on its surface are reduced, which leads to disruption of membrane digestion and absorption of fats and fat-soluble vitamins A, D, E, K, amino acids and carbohydrates [5,6]. Moreover, there was a data, linking SIBO with subclinical atherosclerosis, through the vitamin K-dependent activity of the matrix GIa-protein (MGP), that maintains arterial structure and function through prevention of calcification of vessel walls and the regulation of the extracellular matrix [7].

Bile acids induce impaired sodium absorption, increase the secretion of chlorides and water into the intestinal lumen, accelerate peristalsis of the small intestine, which aggravates diarrhea syndrome [8]. It should also be noted that deconjugated bile acids are rapidly absorbed, which prematurely turns them off from digestion processes [9,10]. In this case, induction of hypercholesterolemia is possible, especially in individuals with hereditary predisposition [11].

The only class of lipids with anti-atherogenic activity is high-density lipoproteins (HDL), that are synthesized in the liver and small intestine. An increasing amount of endotoxins that is produced by gram-negative microflora of intestine, leads to declining the anti-atherogenic HDL [12,13].

Permanent inflammation also causes the changes, that impact on cholesterol catabolism reducing and its excretion in the liver by decreasing the expression of matrix ribonucleic acids and the activity of bile acid synthesis key enzymes – CYP7A1, CYP27A1 and CYP7B1 [14,15].

Taking into account these facts, there is a big interest in searching the way of hyperlipidemia development, where the SIBO is one of the essential factors. The main attention is dedicated to enterohepatic circulation disturbance which evolves on the background of the early bile acids deconjugation with further endotoxin production and oxidative stress in the liver with hyperproduction of cholesterol and atherogenic lipoproteins [16,17].

The aim

The aim of this study therefore was to determine the prevalence and features of SIBO in a series of patients with hyperlipidemia and in control subjects.

Materials and methods

Nineteen patients with hyperlipidemia (9 men and 10 women) aged from 24 to 50 years (average age 33,69±1.73 years) with average BMI 24,4±1.54 were examined in “Medicover Ukraine” (Lviv, Ukraine). The diagnostic criteria, except the hyperlipidemia, which were used for patient including into the program of examination were: BMI not more that 25, waist circumference <94 cm for male, <80 cm for female, no significant alcohol consumption, defined as no greater than 20 g of alcohol per day. Ten control subjects (4 men and 6 women) aged from 24 to 34 years (an average 29,9±0.68 years) and average BMI 24,2±1.21 were matched with main group patients by age and metabolic characteristics. All control subjects had normal lipid range and no history of coronary disease. None of both groups subjects was taking drugs known to affect lipid profile or microbiota composition, including antibacterial medicines 1 month before and during the data.

Both groups of patients underwent biochemical evaluation of serum that included blood cell count and lipid profile. For the evaluation of the inflammation, as one of the pathogenetically ways for hyperlipidemia formation due the SIBO activity, C-reactive protein (CRP) was measured in serum, obtained on the day of SIBO testing. Another biochemical tests included alaninaminotransferase (ALT), aspartataminotransferase AST, gamma glutamyl transpeptidase (GGTP), bilirubin (total, direct, indirect), apolipoprotein B (apo B). Biochemical tests were carried out using commercially available test kits.

Additionally, the data plan involved the determination of calprotectin in feces, which positive result was the reason of excluding the patient from the data. Values >50 μg/g were considered as increased.

Ultrasound examination was proved to all patients of both groups. with aim to exclude the patients with fatty liver disease as one of the reasons of increased cholesterol level and aggravation factor for SIBO presence. The ultrasound criteria for fatty infiltration existence was a diffuse increase in the echogenicity of the liver parenchyma, decreased attenuation on the liver and ratio between the brightness level of the liver and the right kidney that was calculated for the hepato-renal index (HRI) determination [18].

All subjects were examined by a lactulose breath test what is one of the most diagnostically valuable methods for determining excessive bacterial growth under clinical conditions [19]. The test allows to determine the concentration of hydrogen (H2) in exhaled air, what is growing up when there are a lot of hydrogen-producing bacteria in the small intestine [20,21]. The patient was given from 10 g of lactulose. A change in the level of exhaled hydrogen gas above 20 parts per million (ppm) within 120 minutes from a basal value was the basis for SIBO diagnosis [22,23]. Furthermore, after the recording SIBO by enormous H2 growing (more than 30 points), the test could be stopped before 120 minute [24,25]. Before the test, subjects were asked to brush their teeth and rinse mouth with antiseptic mouth wash and tap water, to eliminate an early hydrogen peak due to action of oral bacteria on test sugars [26,27]. Patients were required to comply with a low residue diet the day before the test and not to smoke within two hours of the test to prevent high basal levels of H2 [28,29].

Lactulose breath test was carried out on the “Gastro+Gastrolyzer” (Bedfont® Scientific Ltd) device in the laboratory of “Medicover Ukraine”. Except the standart interpretation, we have analyzed the difference between the basal and the highest range of exhaled H2 during the 120 minutes of test in subjects of both groups. An estimate of small intestinal transit time was calculated, where possible, by observing the time taken from ingestion of lactulose to the appearance of the H2 peak, indicating colonic catabolism of lactulose.

Statistical analysis was carried out using Statistica 5.0 for Windows software (Statsoft Inc, Tulsa, USA). Comparisons between groups for parametric data were performed using the Student’s t test. Differences were considered statistically significant for p£0,05. The correlation between the values was measured by Person correlation coefficient.

Results

Patient characteristics are shown in Table I. Due to the results, the statistically significant difference was matched between the cholesterol level, low density lipoproteins, very low density lipoproteins of main and control groups. The level of CRP was more than in 1,4 significantly higher in group with hyperlipidemia in contrast to the controls.

The measurement of SIBO by lactulose test showed the equal result of the basal dose of hydrogen in both groups. In contrast, the maximal dose was particularly higher in patients with hyperlipidemia in comparison with control group (94,7±13,69 vs. 36,13±5,4) (Table II).

According to the fact, that SIBO existence is based on the hydrogen level increasing more that 20 ppt, not depending the amount of H2, we have analyzed the prevalence of intestinal bacterial overgrow and the small intestinal transit time in both groups. On the other hand, the fact of difference in result of H2 between group of patients with hyperlipidemia and without was essential.

The prevalence of SIBO in hyperlipidemia group was 78.9%. Small intestinal transit time amounted 100 minutes. Meanwhile, the SIBO occurrence in controls was 40% with average time of small intestine transit 140 minutes.

We have analyzed the data, where different methods of small intestine transit time were compared and found the substantial remark, that lactulose is non-physiologic for small intestine time transit measurement since it accelerates small bowel transit, presumably due its osmotic activity. Based on this evidence, we did not accent on the difference of transit time between both groups, because in both groups it was shorter than normal range. On the other hand, we could not ignore the fact of meaningful difference in SIBO existence between main and control groups.

In accordance to this result, we have calculated the correlation coefficient between different biochemical markers and lactulose test results with the purpose to find the influencing factors in each group that could be the reason of SIBO occurrence.

The results showed that there is a strong correlation between AST level and SIBO existence in both groups (r=1). Moreover, the correlative connection was marked between AST/ALT ratio and bacterial overgrowth in main group (r=0,59). An interesting detail was found during the correlation analysis – CRP, that is strongly connected with SIBO in different data, did not interrelate with bacterial overgrowth in both groups. On the other hand, the relationship between cholesterol, LDL and CRP in patients with hyperlipidemia has been found (r=0.58, r=0.59 respectively). Regarding to lipid profile – there was remarkable positive connection between LDL, TG, VLDL and the dose of exhaled hydrogen on 120 minute (r=0.6, r= 0.62, r=0.7 respectively) and strong negative correlation between HDL and 120 minutes dose (r=-0.74) in main group.

Discussion

One of the essential findings of this study was a significantly higher prevalence of SIBO in patients with hyperlipidemia compared with controls (78.9% vs. 40%). Furthermore, an interesting point was found during the analysis of main and control group results – the highest range of exhaled hydrogen during the lactulose test in patients with hyperlipidemia was in above 2.5 times higher than in controls (94,7±13,69 compared to 36,13±5,4 ppm), in while the basal level was equal in both groups (10,7±0,93 and 11,1±1,2 ppm).

Apparently, the way of hyperlipidemia development on the SIBO background could be realized by next steps. Under the influence of SIBO, the protective mechanisms of small intestine mucous membrane are injured, which causes both local and systemic pathological processes, complemented by inflammation, that are closely interrelated. The bacterial pool of colonic flora, which has, in case of SIBO, the properties of conditionally pathogenic flora, by causing the violation of the small intestine barrier function on the background of inflammation, induces the bacterial hydrolysis of proteins with the formation of ammonia and ketone acids, the oxidation of fatty acids, deconjugation of bile acids and the formation of short-chain fatty acids from carbohydrates.

However, CRP, as one of the main markers of system inflammation, that could impact on SIBO occurrence, did not exceed the upper limit of norm, in both groups, not depending the significant difference between it in patients of main and control groups. Thus, the way of SIBO development could be connected not only with injured intestine, but with another way of pathological process that is associated with liver. Due the results of this study, the singular sign that had strong correlation with SIBO presence or absence was AST, which is always associated not only with cardiac muscle, but with liver parenchyma injuring. This indicator was connected with bacterial overgrowth in both groups, but there was more specific strong correlation between de Ritis ratio and SIBO existing in patients of main group, that could be the second point of definitely including liver and cardiovascular system in SIBO manifestation in patients with hyperlipidemia.

Finally, the analysis of correlation between the lipids, CRP and SIBO demonstrated, that increasing of LDL, TG and VLDL is interlinked with higher dose of exhaled hydrogen in main group and with CRP increasing. In contrast, there was no connection in both groups between CRP and H2 growth on any minute of lactulose test. It could be the explanation of considerably higher rate of H2 in patients with hyperlipidemia – there are strong connection between the lipoproteins and SIBO manifestation. Between this, maybe CRP increasing is not before SIBO occurrence, but after its development and is the result of SIBO, not the reason.

That could be the answer for the relationship presence between LDL, VLDL, TG with H2 and CRP, with absence of correlation between CRP and SIBO. High hydrogen rate leads to LDL, VLDL and TG increasing, and HDL decreasing, that provokes the inflammation with next CRP growing. In that way, the main target organ becomes the liver. Thus, it could be the next “vicious circle”: disruption of intestinal microecology ® SIBO occurrence ® accumulation of endotoxins in the intestine ® violation of enterohepatic circulation of bile acids ® impairing of liver function ® impairing of lipid metabolism ® impairing of liver structure (fatty infiltration, fibrosis) ® impairing of lipid metabolism ® maintaining (aggravating) disturbed intestinal dysbiosis.

Conclusions

1. The prevalence of SIBO in patients with hyperlipidemia is predominantly higher than in patients without lipid metabolism disturbance.

2. The hydrogen level is significantly higher in patients with hyperlipidemia in comparison with controls.

3. There is an axis between high LDL, VLDL and TG level and hydrogen rate in patients with hyperlipidemia.

4. CRP is not interrelated with SIBO, but is strongly connected with LDL and cholesterol level in patients with hyperlipidemia.

References

1. Wigg A., Roberts-Thomson I., Dymock R., Mccarthy P., Grose R., Cummins A. The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis., Gut, 2001, 48(2) : 206-211.

2. Adkins c., Rezaie A. Small intestinal bacterial overgrowth and coronary artery disease: what is in the cards? Digestive diseases and sciences, 2018, 63(2) : 271–272

3. Olof H, Sundin A., Zeng M. The human jejunal microbiome has a distinctive bacterial flora, with streptococcus tigurinus as its signature species, and an increased fraction of gram-negative phyla in patients with small intestinal bacterial overgrowth. Gastroenterology, 2016, 150 : 689.

4. Ferolla S., Armiliato G., Couto C., Ferrari T. The role of intestinal bacteria overgrowth in obesity-related nonalcoholic fatty liver disease. Nutrients, 2014, 6(12) : 5583–5599.

5. Jung S., Joo Ns, Han Ks, Kim Kh. Obesity is inversely related to hydrogen-producing small intestinal bacterial overgrowth in non-constipation irritable bowel syndrome. J korean med sci., 2017, 32(6) : 948–953.

6. Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. Role of bile acids and bile acid receptors in metabolic regulation. Physiol rev., 2009; 89 : 147–191.

7. Ponziani F, Pompili M, Di Stasio E, Zocco M, Gasbarrini A, Flore R. Subclinical atherosclerosis is linked to small intestinal bacterial overgrowth via vitamin k2-dependent mechanisms. World j gastroenterol., 2017; 23 : 1241–1249.

8. Thomas C, Auwerx J, Schoonjans K. Bile acids and the membrane bile acid receptor tgr5 – connecting nutrition and metabolism. Thyroid 2008;18 : 167–174.

9. Kvit K., Kharchenko N. Gut microbiota changes as a risk factor for obesity. Wiadomosci lekarskie, 2017, 70(2) : 231-235.

10. Thomas C, Pellicciari R, Pruzanski M, Auwerx J, Schoonjans K. Targeting bile-acid signalling for metabolic diseases. Nat rev drug discov., 2008; 7: 678–693.

11. Trauner M., Claudel T., Fickert P., Moustafa T., Wagner M. Bile acids as regulators of hepatic lipid and glucose metabolism. Dig dis., 2010; 28(1): 220-224

12. Bures J., Cyrany J., Kohoutova D., Förstl M., Rejchrt S., Kvetina J. Small intestinal bacterial overgrowth syndrome. World j gastroenterol., 2010, 16(24) : 2978–2990.

13. Kvit K., Kharchenko V. Role of gut microbiota in lipid metabolism. Asian journal of pharmaceutical and clinical research, 2018, 11(4) : 4-8.

14. Libby P. History of discovery: inflammation in atherosclerosis. Arterioscler thromb vasc biol., 2012, 32(9) : 2045-2051.

15. Dukowicz A., Lacy B., Levine G. Small intestinal bacterial overgrowth. A comprehensive review. Gastroenterol hepatol., 2007, 3(2) : 112–122.

16. Chu H., Williams B., Schnabl B. Gut microbiota, fatty liver disease, and hepatocellular carcinoma. Liver research., 2018, 2(1) : 43-51

17. Kvit K., Kharchenko V. The influence of environmental factors on nonalcoholic fatty liver disease and obesity. In : development trends in medical science and practice: the experience of countries of eastern europe and prospects of ukraine. Latvia : izdevnieciba baltija, 2018 : 96-115.

18. Muriel Webb, Hanny Yeshua, Shira Zelber-Sagi. Diagnostic Value of a Computerized Hepatorenal Index for Sonographic Quantification of Liver Steatosis Muriel Webb1, Hanny Yeshua1 2, Shira Zelber-Sagi. American Journal of Roentgenology., 2009.- 192 (4).-909-914.

19. Rhodes Jm, Middleton P, Jewell Dp. The lactulose hydrogen breath test as a diagnostic test for small-bowel bacterial overgrowth. Scand j gastroenterol., 1979; 14 : 333–336.

20. Pimentel M, Chow Ej, Lin Hc. Normalization of lactulose breath testing correlates with symptom improvement in irritable bowel syndrome. A double-blind, randomized, placebo-controlled study. Am j gastroenterol., 2003; 98 : 412–419

21. Gasbarrini A, Corazza Gr, Gasbarrini G. Methodology and indications of h2-breath testing in gastrointestinal diseases: the rome consensus conference. Aliment pharmacol ther., 2009; 29 (1) : 1–49.

22. Khoshini R, Dai Sc, Lezcano S. A systematic review of diagnostic tests for small intestinal bacterial overgrowth. Dig dis sci., 2008; 53 : 1443–1454

23. Donald Ip, Kitchingmam G, Donald F et al. The diagnosis of small bowel bacterial overgrowth in elderly patients. J am geriatr soc., 1992; 40 : 692–696

24. Riordan Sm, Mciver Cj, Walker BM. The lactulose breath hydrogen test and small intestinal bacterial overgrowth. Am j gastroenterol., 1996; 91 : 1795–1803.

25. Ghoshal U. How to interpret hydrogen breath tests. J neurogastroenterol motil., 2011, 17(3) : 312–317

26. Rezaie A., Buresi M., Lembo A., Lin H., Mccallum R., Rao S. Hydrogen and methane-based breath testing in gastrointestinal disorders: the north american consensus. Am j gastroenterol., 2017, 112(5) : 775-784

27. Jacobs C., Coss Adame E., Attaluri A., Valestin J., Rao S. Dysmotility and ppi use are independent risk factors for small intestinal bacterial and/or fungal overgrowth. Aliment pharmacol ther., 2013, 37(11) : 1103-1111.

28. Pourmorady J, Shah E, Rezaie A. Breath testing for small intestinal bacterial overgrowth in irritable bowel syndrome: a meta-analysis. Am j gastroenterol., 2015; 110 : 762.

29. Ghoshal Uc, Ghoshal U, Das K. et al. Utility of hydrogen breath tests in diagnosis of small intestinal bacterial overgrowth in malabsorption syndrome and its relationship with oro-cecal transit time. Indian j gastroenterol., 2006; 25 : 6–10.

Authors’ contributions:

According to the order of the Authorship.

Conflict of interest:

The Authors declare no conflict of interest.

CORRESPONDING AUTHOR

Khrystyna Kvit

Pekarska Str., 69, Lviv, 79010, Ukraine

tel: +380674788881

e-mail: akskris88@gmail.com

Received: 13.01.2019

Accepted: 01.04.2019

Table I. Clinical and biochemical variables for patients with hyperlipidemia (n=19) and controls (n=10)

Variable (normal range)

Main group (19)

Control group (10)

p

Age (years)

33,69±1.73

29,9±0.68

³0,05

BMI, kg/m2

24,4±1.54

24,2±1.21

³0,05

Apo B, g/l

(normal range 0.66-1.33-men, 0,6-1,17 women)

1.19±1.03

0.81±1.6

³0,05

Bilirubin total, mmol/l (normal range <21)

15.13±1.83

12.53±0.85

³0,05

Direct bilirubin, mmol/l (normal range <5)

3.47±0.48

4±0.14

³0,05

Indirect bilirubin, mmol/l

(normal range <75% of bilirubin total)

12.03±1.7

9.2±0.07

³0,05

AST, IU/L (normal range <40)

29.6±3.5

23±1.35

³0,05

ALT, IU/L (normal range <41)

43.54±10.35

29.13±3.52

³0,05

AST/ALT ratio (normal range 0,91-1,75)

1.05±0.14

0.94±0.35

³0,05

GGTP, IU/L

(normal range <55 men, <38 women)

41,78±11.03

23.45±2.48

³0,05

CRP, mg/l (normal range £5)

2,75±0,36

1,9±0,03

£0,05

Cholesterol, mmol/l (normal range £5,2)

6,71±0,27

4,63±0,23

£0,05

Triglycerides, mmol/l (normal range n£1,7)

1.45±0,25

1,12±0,1

³0,05

LDL, mmol/l (normal range £2,59)

4,14±0,25

2,45±0,1

£0,05

VLDL, mmol/l (normal range =0.26-1,0)

0,93±0,13

0,47±0,01

£0,05

HDL, mmol/l (normal range =³1,56)

1,44±0,05

1,58±0,06

³0,05

Table II. H2 level during the lactulose breath test in patients of main and control groups

Main group (19)

Control group (10)

p

Basal dose, ppm

10,7±0,93

11,1±1,2

³0,05

Maximal dose, ppm

94,7±13,69

36,13±5,4

£0,05