Comparison of Quick Lactose Intolerance Test in duodenal biopsies of dyspeptic patients with single nucleotide polymorphism LCT-13910 C > T associated with primary hypolactasia / lactase-persistence

pURpOSE: To analyze the usefulness of Quick Lactose Intolerance Test in relation to the genetic test based on LCT-13910C>T genotypes, previously validated for clinical practice, for primary hypolactasia/lactase-persistence diagnosis. METhOdS: Thirty-two dyspeptic patients that underwent upper gastrointestinal endoscopy entered the study. Two postbulbar duodenal biopsies were taken for the Quick test, and gastric antral biopsy for DNA extraction and LCT-13910C>T polymorphism analysis. DNA was also extracted from biopsies after being used in the Quick Test that was kept frozen until extraction. RESULTS: Nine patients with lactase-persistence genotype (LCT-13910CT or LCT-13910TT) had normolactasia, eleven patients with hypolactasia genotype (LCT-13910CC) had severe hypolactasia, and among twelve with mild hypolactasia, except for one that had LCT13910CT genotype, all the others had hypolactasia genotype. The agreement between genetic test and quick test was high (p<0.0001; Kappa Index 0.92). Most of the patients that reported symptoms with lactose-containing food ingestion had severe hypolactasia (p<0.05). Amplification with good quality PCR product was also obtained with DNA extracted from biopsies previously used in the Quick Test; thus, for the future studies antral gastric biopsies for genetic test would be unnecessary. CONCLUSION: Quick test is highly sensitive and specific for hypolactasia diagnosis and indicated those patients with symptoms of lactose intolerance.


Introduction
Lactase or lactase-phlorizin hydrolase, located in the brush border of intestinal mucosa, is responsible for the hydrolysis of lactose, the major carbohydrate present in milk, into galactose and glucose.The maximal activity of lactase is during perinatal period; however, in most humans of different ethnic groups, it declines at some point during life, emerging two groups: lactasepersistence (normolactasia, lactose digestion) and lactase nonpersistence (hypolactasia, lactose maldigestion).Undigested lactose is fermented by the intestinal flora, producing hydrogen, methane, carbon dioxide and short-chain fatty acids, which cause symptoms of lactose intolerance, flatulence, bloating, abdominal pain and diarrhea, depending on the amount of ingested lactose 1 .
Symptoms suggestive of lactose intolerance are unspecific and may be related to other causes such as irritable bowel syndrome, cow's milk pro tein allergy, bacterial overgrowth, celiac disease, and inflamma tory bowel disease, or other dietary sources of intestinal gas, such as beans, which contain two indigestible sugars, stachyose and raffinose 1 .Self-perceived lactose intolerance may lead to unnecessary avoidance of milk and dairy products, the main source of calcium in the diet, with future consequences on bone health 1 , hypertension, and diabetes 2 .Thus, diagnosis of lactose maldigestion imposes for adequate clinical management and correct lactose-restriction diet.
The diagnosis of lactose maldigestion was initially performed by lactase activity in jejunal biopsies 3 , being replaced by duodenal biopsies; nonetheless, the mean lactase activity in the duodenum is lower than in the jejunum, being less reliable 4 .Quick Lactose Intolerance Test in postbulbar duodenum samples showed sensitivity and specificity of 95% and 100%, respectively 5 , being more sensitive than lactose breath test 6,7 , and highly sensitive in a pediatric population 8 .A questionnaire of dietary lactose containing food ingestion and related symptoms was applied (Chart 1).
ChART 1 -Dietary lactose intolerance survey applied before the endoscopic procedure.

Results
There was no significant association of ethnicity and

discussion
The most important finding of this study is that comparing the genetic test with Quick lactose intolerance test, the latter indicates individuals with severe hypolactasia that are more prone to present symptoms of lactose intolerance, agreeing with previous report 5 , rather than those with mild hypolactasia, even both presenting primary hypolactasia genotype.Another finding was that mild hypolactasia was associated with hypolactasia genotype rather than with lactase-persistence genotype, as was previously described by Kuokkanen et al. 5 , although they showed that these individuals had intermediate levels of duodenal lactase activity, between those levels detected in the normolactasia group and in the severe hypolactasia group.This finding may explain the fact that some individuals with hypolactasia genotype are asymptomatic even ingesting a great amount of milk, suggesting that the levels of the physiological decline of lactase vary among different populations 12 .
The association of primary hypolactasia genotype with symptoms after lactose-containing food ingestion was not significant, in contrast to previously reported in 1900 Finnish adults that showed a significant association of hypolactasia genotype, less milk intake, and the presence of gastrointestinal symptoms 13 , the explanation for that is 50% of patients with hypolactasia genotype had mild hypolactasia.One Caucasian woman 46 years old with lactase-persistence and normolactasia, unexpectedly reported symptoms with milk ingestion that may Comparison of Quick Lactose Intolerance Test in duodenal biopsies of dyspeptic patients with single nucleotide polymorphism LCT-13910C>T associated with primary hypolactasia/lactase-persistence1 Acta Cirúrgica Brasileira -Vol.28 (supl.1) 2013 -81 be caused by irritable bowel syndrome (IBS), as was suggested there may be other components of milk besides lactose that cause symptoms among subjects with IBS 14 .
Even though the number of the patients analyzed in this study is lower than previous reported by other authors that compared the Quick Test with the genetic test, the results of sensitivity (100%) was similar 5 .Although presenting a high sensitivity the Quick Test has limitations, one is the size of the biopsies that if larger or shorter than 2 mm may give false negative or false positive results of hypolactasia, respectively.Another is the dependence on an invasive exam for collection of samples that not always is accepted by the patients that prefer a blood collection test.A biopsy-based gastrointestinal endoscopy exam is limited by the coagulation status, bleeding risks, and clinical conditions of the patient.Another point to consider is the Quick Test has to be performed immediately after collection of duodenum samples, requiring a laboratory technician in the endoscopy suite, as the incubation times have to be rigorously followed, and even being a very simple device, skillful in laboratory handling is necessary to perform the Quick Test.
The Quick Test is expensive (USD 30/individual test) for the Public Health Service compared to the genetic test, being more suitable for dyspeptic patients already undergoing endoscopy examination in private clinics for some clinical investigation.
Additionally, the genetic test indicates primary hypolactasia and lactase-persistence 8 .However, those with lactase-persistence genotype may also present transitory hypolactasia along with celiac disease, Crohn's disease, or infectious enteritis diagnosis 1 .
Outside the setting of tertiary referral centers genetic test may be considered more cumbersome than a gastrointestinal endoscopy with duodenal biopsy for the Quick Test analysis.
Nonetheless, blood samples may be shipped to this referral centers for DNA-based genetic tests that have advantages: absence of lactose intolerance symptoms, single testing, non-invasive, and low cost 8 .
In these case series an association of hypolactasia genotype with ethnic groups was not observed in contrast with our report in 567 brazilians 8 , maybe the effect of the low number of patients.

Conclusions
Quick test is highly sensitive and specific to indicate subjects with hypolactasia and lactase persistence, and those more prone to present lactose intolerance symptoms.The genetic test is simple, non invasive, highly sensitive, and has a low cost.
and other flavored milk) Others (candies, caramel, potato puree, some salad sauces, soufflés) Thirty-two dyspeptic patients (mean age 50.2±17.5years, 59.4% females), submitted to routine upper digestive endoscopy in the Digestive Endoscopy Center of Clinics Hospital of Ribeirão Preto Medical School of the University of São Paulo, entered the study.Twenty-three (72%) subjects were Caucasian and 9 were African-Brazilians.Comparison of Quick Lactose Intolerance Test in duodenal biopsies of dyspeptic patients with single nucleotide polymorphism LCT-13910C>T associated with primary hypolactasia/lactase-persistence1 Acta Cirúrgica Brasileira -Vol.28 (supl.1) 2013 -79 Quick lactose intolerance test Two specimens of biopsy of the postbulbar region (2 mm each) were taken to perform the Quick Test (Biohit, Helsinki, Finland) for lactase activity assay, according to the manufacturer instruction.The specimens were placed into the well of the test plate immediately after its collection; two drops (80 µl) of a substrate solution were added and incubated for 15 minutes.After this step 2 drops of chromogen solution (10 µl) in acetic acid and 1 drop (80 µl) of signaling enzymatic solution were added with incubation time of 5 minutes.The total reaction time was 20 minutes.The test result was colorimetric: a dark blue coloring, indicating normolactasia (lactase activity), light blue color mild hypolactasia and colorless severe hypolactasia.LCT-13910C>T single nucleotide polymorphism analysis DNA was extracted by salting out from specimens of the antrum 11 , and used in the polymerase chain reaction and restriction fragment length polymorphism analysis, as was previously described 10 .Two biopsies after being used in the Quick Test were frozen for DNA extraction in order to see viability to avoid collection of biopsies from the gastric antrum for the genetic test.Digested PCR products with HinfI were visualized on a 3% low melting point agarose gel stained by ethidium bromide.Samples showing a single band of 201 bp were classified as the LCT-13910CC genotype (hypolactasia or lactase non-persistence), a single band of 177 bp as the LCT-13910TT genotype (normolactasia, lactase-persistence), and two bands of 201 bp and 177 bp the LCT-13910CT genotype (normolactasia, lactase-persistence). Statistical analysis Statistical analysis was performed by Kappa index measure of agreement of diagnostic tests, and Chi-square or Likelihood ratio using SPSS version 15.0 for Windows (Chicago, Illinois, USA).A p value of <0.05 was considered statistically significant.

TABLE 1 -
Presence of symptoms with milk and/or other lactose-containing food consumption in patients with hypolactasia and lactase-persistence genotypes, and in groups according to the Quick Test results.

TABLE 2 -
Association of the LCT-13910C>T genotypes with the Quick Test results.