Routine Disaccharidase Testing: are we there yet?

Abstract

Purpose of Review

Disaccharidase testing, as applied to the evaluation of gastrointestinal disturbances is available, but it is not routinely considered in the diagnostic work-up. The purpose of this review was to determine if disaccharidase testing is clinically useful and to consider how the results could alter patient management.

Recent Findings:

indicate that carbohydrate maldigestion could contribute functional bowel disorders and negatively impact the fecal microbiome. Diagnostic techniques include enzyme activity assays performed on random endoscopically-obtained small intestinal biopsies, immunohistochemistry, stable isotope tracer and non-enriched substrate load breath testing, and genetic testing for mutations. More than 40 sucrase-isomaltase-gene variants coding for defective or reduced enzymatic activity have been reported and deficiency conditions are more common than previously thought.

Summary

The rationale for disaccharidase activity testing relates to a need to fully assess unexplained recurrent abdominal discomfort and associated symptoms. All disaccharidases share the same basic mechanism of mucosal expression and deficiency has far reaching consequences. Testing for disaccharidase expression appears to have an important role in symptom evaluation, but there are accuracy and logistical issues that should be considered. It is likely that specific recommendations for patient management, dietary modification and enzyme supplementation would come from better testing methods.

Introduction

Carbohydrates constitute a large portion of the human diet and consist of starches, dextrins, oligosaccharides, and disaccharides [1, 2]. These substrates must be completely hydrolyzed by small intestinal, apical-surface-mucosal disaccharidases (on the ‘brush border’), and by pancreatic alpha-amylase (in the cases of starches) to ultimately be absorbed as monomeric sugars (e.g., glucose, galactose, and fructose) by transporter-dependent means. Larger starch molecules must first be partially hydrolyzed by alpha-amylase to be made sufficiently soluble to engage the mucosal-anchored disaccharidases. The disaccharidases lactase, sucrase, maltase, and trehalase are embedded on the microvilli ultrastructure of small intestinal enterocytes and function to complete the luminal glycolysis to monomers from their respective substrates [3]. This process ideally permits rapid, transporter-mediated absorption before any microorganisms can theoretically and excessively proliferate (overgrowth) in the small intestine and competitively consume the freed sugars or be passed on to the colon [4].

When small intestinal, carbohydrate-dense chyme exceeds the disaccharidase glycolytic ability and enterocyte absorptive capacity, the maldigested oligosaccharides that pass to the colon become subject to excessive fermentation. In excess, this epiphenomenon could affect colonic homeostasis and adversely alter the microbiota (dysbiosis) [5]. Symptoms thought to be caused by dysbiosis have been linked to increased by-products of colonic fermentation (including osmotically active particles) and a change in luminal pH known to have neuromotor consequences [6]. Resultant clinical features include abdominal distention to luminal retention of water and gases, discomfort, variable somatic complaints, and bothersome changes in bowel habits. There is extensive symptom-overlap with maldigestion and other potentially serious conditions that are frequently considered before the evaluation of mucosal disaccharidase activity is undertaken.

There appears to be a balance between tolerable (and potentially beneficial) and incomplete carbohydrate digestion and intolerable or noxious maldigestion due in part to disaccharidase deficiency or other conditions such as pancreatic insufficiency or motility issues. In the former and favorable case, some short-chain fatty acids (SCFA), such as acetate, butyrate, and propionate esters, are produced in moderation from carbohydrates by fecal species in the Firmicutes, Bacteroides phyla and others. The mix of SCFA appears to be dependent upon the diversity of the colonic microbiota, which in turn is dependent upon released nutrients that pass to the colon [7, 8]. Butyrate and propionate are typically absorbed and constitute a caloric salvage mechanism, provide nourishment to the colonic mucosa, and regulate inflammatory responses [9, 10]. It also appears that the SCFA mix also plays an important key role in the prevention and treatment of the metabolic syndrome, functional bowel disorders, and cancer through inhibition of nuclear factor kappa B activation and histone deacetylation [11]. For example, the inhibition of histone deacetylases (HDACs) and activation of G-protein-coupled receptors (GPCRs) have been linked to two SCFA signaling mechanisms [12]. Since the optimal production of SCFA is, in part, dependent upon upstream disaccharidase digestion, repercussions from any deficiency conditions could have harmful consequences on how HDACs regulate carcinogenic gene expression in the colon [13].

Maldigestion can indirectly lead to decreased reabsorption of bile acids, which also can deleteriously alter the colonic flora [14, 15]. Furthermore, the preferment of certain strains of Bacteroides fragilis may result in inflammatory diarrhea and may interfere with DNA-repair mechanisms that are important to the prevention of colorectal cancer development [16]. Nevertheless, more understandings are needed about the modulation of colonic floral milieu by dietary substrate diversity with attention given to disease prevention or pathogenesis. Specifically, how the various proximally maldigestion conditions, including disaccharidase deficiency, could influence the disease cascade is the subject of ongoing investigations.

It appears that diets that are low in fermentable substrates [17] are associated with decreased frequency of abdominal pain in adults and children with irritable bowel syndrome [18, 19]. The putative dietary substrates that are thought to contribute to increased ‘functional’ symptoms include maldigested fructans [20], lactose, sucrose, [21], fructose [22], oligosaccharides and starch using processes that can be dependent on effective disaccharidase activity. Excessive colonic carbohydrate fermentation can also result in the production of lactate that results in a decrease in colonic luminal pH and potentially alter 5-HT-related biological processes [23, 24]. This is accompanied by the production of gases such as hydrogen and methane [25, 26], which have been linked with bloating and altered colonic motility [27–29]. Also, an excessive release of various small-molecules in the colon may evoke inflammatory responses and elicit or suppress reflexive motor actions [30, 31]. As such, disaccharidase activity appears to be a critical factor in health and digestive pathophysiology.

Disaccharidase Insufficiency

Disaccharidase insufficiency, as a cause for maldigestion-related complaints, has been recognized for more than a century [32] and can co-exist with organic diseases or manifest with the natural aging processes. The clinical manifestations are highly variable, although none of the features are substrate-specific. Lactase expression, in part, defines the mammalian class of vertebrates and lactase insufficiency is the quintessential clinical model for this mucosal disaccharidase shortfall. It calls attention to the importance of the other types of disaccharidase insufficiency. Typically, symptoms of abdominal discomfort first suggest other organic causes for symptoms including small intestinal bacterial overgrowth (SIBO) [33], celiac disease [34], infectious enteritis [35], radiation enteritis [36], somatostatinoma [37], and ‘functional’ bowel syndrome [38, 39]; and any of these conditions could co-exist with disaccharidase insufficiency.

Lactose and Lactase Insufficiency

Lactose-containing dairy product consumption commonly occurs in modern western society, and lactose is often hidden within many modern foodstuffs. Modern dairy farming, particularly among the temperate latitudes, became commonplace once routine pasteurization came in to being and, as such, lactose consumption increased in many societies. However, the evolutionary nutrient ‘blueprint’ for the human species does not always appear to be well-suited for ongoing milk ingestion beyond infancy [40]. For more than half of the world’s population, adult-type hypolactasia begins after weaning and is somewhat dependent upon the regulatory factors expressed by the autosomal dominant MCM6 gene [41]. For reasons not understood, some people with adult-type hypolactasia may become symptomatic after ingestion of small amounts of dairy products, and others may tolerate 250 mL or more before admitting to significant symptoms [42–43].

Symptomatic lactose maldigestion can occur in unsuspecting people when tolerance thresholds are unknowingly exceeded or when acute comorbidities present. The symptom disparity may be due, in part, to maladaptive colonic microbiota [44, 45]. Sustained viral infections may alter the enterocyte membrane ultrastructure and downregulate lactase expression [46]. After a giardia infection and viral gastroenteritis, an affected patient may acquire lactose intolerance [47] and post-infectious chronic diarrhea may be especially problematic among the elderly and among those who are immunologically compromised [48, 49].

Infantile lactase deficiency is rare, and due to autosomal recessive mutations in the LCT gene located on chromosome 2 at position q21.3 [41]. However, malnourished infants have reduced lactase activity because of epigenetic suppression or from acquired conditions [50], such as iatrogenic mucositis, malnutrition, and short-gut syndrome. Since the mechanism for protein expression on the apical membranes is similar among all disaccharidases [51], symptoms may indicate the need for broad disaccharidase testing, and/or the reflexive institution of restriction diets or enzyme supplementation or parenteral nutritional support in severe cases. To provide symptom relief from lactase insufficiency conditions, a multi-billion dollar industry has emerged. Over-the-counter approaches include consumption of hard cheese and yogurt, use of probiotics, and per-oral lactase supplementation to serve the needs of those unable to completely avoid lactose ingestion and to help meet micronutrient needs [52, 53].

Lactase persistence is not uncommon among approximately one-third of the human population, and the reason selective lactase persistence remains poorly understood [54]. Several dominant allele genotypes have been associated with the lactase-persistence phenotype (including LCT-13910CT and LCT-13910TT) [55, 56] and epigenetic factors may play an important role in the influence of phenotypic outcomes [57]. Lactase persistence polymorphisms have been associated with increased BMI and obesity [58, 59], and, in turn, lactose tolerance may facilitate an increased risk for obesity-associated malignancies [60], but this concept remains controversial [61]. Conversely, lactase persistence may be an evolutionary flaw. The Kuopio Ischaemic Heart Disease Risk Factor Study [62] found that low-fat (<3·5 %), fermented (aka: lactose free) dairy product intake was associated with a lower risk of heart disease (hazard ratio in the highest quartile=0·74; 95 % CI 0·57, 0·97; P-trend=0·03). As such, dairy ingestion and lactase ‘insufficiency’ remain clinically important considerations beyond the realm of abdominal discomfort and as a nutritional calcium source. However, when persistent lactase activity is confirmed, alternative explanations of adverse abdominal symptoms might be sought.

Lactase Testing

When results from adequate adherence to the exclusionary-lactose diet are ambiguous, diagnostic testing might include breath hydrogen testing and perhaps in vitro enzyme assay of endoscopic mucosal biopsies. This approach may be warranted along when consideration of secondary lactase insufficiency could be due to pathogenic defects as caused by parasitic states, bacterial infections, viral infections, and iatrogenic chemo-radiotherapy. It seems unlikely that genetic testing for lactase polymorphisms could be helpful in this setting.

Breath hydrogen testing [63] may be used to establish a diagnosis of lactase insufficiency, but a diagnosis of small intestinal bacterial overgrowth (SIBO) should first be excluded. Some centers advocate the use of a preliminary glucose breath test (up to 100 gram per-oral load, which equates to the consumption of a quart of milk) to exclude SIBO prior to lactose breath testing [64]. However, by consensus, a loading meal of 75 grams of glucose may be sufficient. The theory behind the glucose breath test is that an early rise in blood glucose concentration and the absence of significant breath hydrogen changes would point against SIBO as a cause for symptoms. There are several nuances practiced in different regions, and the techniques used often vary. We follow recently published guidelines which consider that accept a 20 PPM rise in breath hydrogen expression by 90 minutes after glucose substrate ingestion points toward a diagnosis of SIBO [65]. If the glucose breath test is negative before 90 for SIBO, disaccharidase insufficiency might then be considered [66], and a per-oral lactose challenge might be performed in an attempt to reproduce symptoms.

With the combined breath testing and blood lactose tolerance testing, a 25g meal of lactose is administered and timed breath samples are obtained for hydrogen analyses. A blood glucose concentration may be measured at 1 and 2 hours after substrate ingestion [65]. An early-rise in blood glucose concentration of more than 20 mg/dL would suggest that lactose digestion occurred, but the appearance of a late-rise in breath hydrogen would indicate that some lactose was not digested proximally. The appearance of such a late breath hydrogen rise (delta >20 PPM) is also dependent upon transit time to the cecum. It is of interest that a minority of patients are colonized with a methane-producing species that rapidly convert hydrogen to methane at a 4:1 ratio [67]. As such, concurrent analysis for methane expression may identify those who are lactase deficient but do not demonstrate a positive breath hydrogen test (hydrogen non-excretors). It is unlikely that immunohistochemistry or genetic testing could offer much further information or perspective when considering lactase deficiency and the response to per-oral lactase supplementation.

Sucrose and Sucrase Testing

The majority of the habitual western diet is composed of starch and augmented with high sucrose consumption. Sucrose role in the human diet increased with human agriculture production and trade. Sucrose consumption in the United States currently approximates 11-million metric tons, which is a 1-million metric ton increase since 2009 [68]. Sucrose is hydrolyzed by sucrase to free glucose and fructose by the distal aspect of the sucrase-isomaltase gene product [69]. The release of free fructose can be problematic for some people with decreased transport capacity, a mechanistic process not well understood [70]. Sucrose ingestion is a prime source of fructose and is dependent upon small intestinal glycolysis for absorption. The major fructose co-transporter in the small intestine is thought to be GLUT5 and is subject to up- and down-regulation depending upon load conditions. Most adults can tolerate up to 25 grams of fructose, [71] but incomplete absorption and symptoms of intolerance may suggest that other factors, such as female gender, may be involved [72]. Very few cases of genetically determined fructose malabsorption have been reported to date [73]. The extreme rarity of GLUT5 mutation suggests that fructose intolerance, when present, is probably multifactorial. Over-the-counter glucose-isomerase supplementation is available to attempt symptom relief, but clinical efficacy has not yet been established.

Sucrase-isomaltase (SI) is expressed as a dimeric gene product [cytogenetic location: 3q26.1] that is anchored to the mucosal brush border with the n-terminus of proximal isomaltase component and the sucrase component vulnerable to luminal proteolytic cleavage [74, 75]. Congenital sucrase-isomaltase deficiency (CSID) syndrome, like lactase deficiency, has been linked to dietary sucrose and starch intolerance [69]. Since dietary sucrose and starches are pervasive, the clinical consequences of this type of disaccharidase insufficiency are, for some, difficult to avoid. Failure to proximally hydrolyze sucrose and starches effectively relegates them to prebiotic status and may have deleterious effects that could alter the colonic microbiota and/or cause symptoms. CSID is also a model syndrome to study the maldigestion of carbohydrates at the mucosal level [76]. The condition has significant symptom similarity with enteritis and in severe cases, can result in malnutrition [77].

With considerable diligence, sucrose can be significantly reduced from the habitual diet as a means of alleviating CSID symptoms [78, 79], but supplemental sacrosidase is available and may have some utility beyond restriction diets [80–82]. The restriction of fructose might also be considered, but there is a general lack of strong evidence supporting the restriction of individual carbohydrates [20, 83]. Fructose load breath tests may be helpful, but this is a challenging consideration to make since the test reproducibility may be poor due to rapid metabolic considerations [84]. Tracer labeled (¹³C) fructose substrates are available, but are too costly to use early in the screening algorithm.

Starch, Dextrins, Isomaltase and Glucoamylase Testing

Starch maldigestion is another aspect of the sucrase-isomaltase insufficiency condition for consideration. The incomplete digestion of starch-derived dextrins was recognized as an important factor in the CSID condition more than forty years ago [85]. Since then, more than 40 different CSID mutations have been identified, and there is recent evidence to indicate that variable phenotypes may be more common than are currently recognized [86]. Commercial genetic screening is available to confirm genetic forms, but routine use of genetic screening remains cost-prohibitive. If a restriction diet were to be adhered to and followed by a dietary challenge that yielded a clinical response, disaccharidase testing could be performed to provide patients with a concrete diagnosis. Specifically, a sucrose-load breath hydrogen test or a stable isotope tracer (13C-carbon) breath test might be useful to support the diagnosis since isomaltase is co-expressed with sucrase [87]. Assay of sucrase activity should proportionally parallel palatinase activity (the in vitro surrogate α1–6 substrate for isomaltose) since sucrase and isomaltase are initially expressed as a single gene product [88]. Immunohistochemistry might also be considered as a semi-quantitative means to assess SI expression, but that would not completely address glycolytic capacity in all cases. A visual analog scale that is similar to the Visual Analog Pain Scale and the Bristol Stool Form Scale [89] is suggested for immunohistochemistry assessment SI expression. Figure 1 proposes a scale similar to where zero represents an absence of detectable SI expression by immunohistochemistry, and +4 equates to robust SI expression from the intestinal crypts to the tips of the small intestinal villae. Further validation work is needed before this grading approach might be considered.

Figure 1.

A Semi-quantitative analog grading scale of immunohistochemistry staining is proposed for sucrase-isomaltase expression along the apical mucosal surface (brown). The relatively unavailability of activity assays makes immunohistochemistry staining and attractive alternative and worthy of further development. The images selected about are representative of correctly handled samples and were compared with the reported Dahlqvist assay outcomes (μM/min/gram-protein). Enzyme activity results were normalized using total protein content determinations which are problematic since biopsies may contain various amounts of protein depending upon the forceps depth and concurrent retrieval of adventitia. Such adjustments that could easily skew the reported disaccharidase activity outcomes; immunohistochemistry staining may offer a more practical and cost-effective alternative.

Isomaltase has considerable starchy oligosaccharide hydrolytic capacity, and the spectrum of clinical impairment is varied, much like lactose intolerance [90]. Several recent findings suggest that certain CSID variants, with reduced SI enzymatic activity, may contribute to the development of IBS symptoms [91, 92]. However, in a recent small study of patients with IBS with diarrhea (IBS-D) [93], SI mutation carriers only experienced a modest benefit from the use of the low FODMAP (Fermentable Oligosaccharide, Disaccharide, Monosaccharide, and Polyols) diet when compared with non-SI carriers. This finding should have been expected since most starch substrates are not part of the FODMAP constellation [94]. The SI C-terminus plays the role of the sustaining but limiting mucosal glucosidase activity [69. 75]. The two gene products carefully orchestrate starch digestion and determining how much fermentable substrate reaches the colon; therefore, gene product aberrancies are expected to have far reaching clinical manifestations. The importance of homozygous or heterozygous SI gene variants in the pathogenesis of IBS or other functional gastrointestinal disorders remains to be validated.

Maldigestion of starch is seldom assessed in adults, perhaps because it is widely assumed that pancreatic α-amylase secretion and its associated activity is ample, especially when fat absorption is concurrently normal. Limited testing for starch hydrolysis is available, such as secretin or breath testing [95, 96], and empiric amylase supplementation has been shown to successfully treat cases of post-prandial diarrhea [97]. After excluding pancreatic insufficiency, inflammatory conditions and celiac disease, indirect disaccharidase substrate testing for maltose, palatinose (used in lieu of isomaltose), sucrose, and dextrins (very-short-chain, soluble fragments derived from amylase action on starch) could be considered for still unexplained symptoms in adults.

Maltase-glucoamylase is a chromosome-7 gene product (band location 7q34) with significant sequence homology with sucrase-isomaltase [98]. It is capable of hydrolyzing longer maltooligosaccharides, such as maltotetrose and maltopentose [69, 99]. The C-terminal of the enzyme is the most highly glucosidase but is inhibited by starch dextrins produced by alpha amylase [69]. It appears to be somewhat of a fail-safe enzyme that provides scavenger functionality to complete starch maldigestion. This mechanism appears to explain the antidotal reports of improved symptoms with dietary titration of dietary starch to match personal capacity. Unlike sucrase [92], with more than 700 coding single-nucleotide polymorphisms and mutations reported, isolated maltase-glucoamylase (MGAM) deficiency has not been confirmed to date. However, a second pseudogene (MGAM2) has been reported with significant base-pair homology to MGAM, [100], but it is unclear if a complimentary functional gene product can be ascribed. Aberrant maltase activities are difficult to interpret because the maltase-glucoamylase gene-product and the isomaltase component of the sucrase-isomaltase have overlapping maltase (glucosidase) activities. However, decreased maltase activity, with preserved sucrase and palatinase activities, might suggest MGAM mutations if the quality of the sample is good.

The pan-disaccharidase-deficiency condition has been rarely reported without clear etiologies [101, 102]. Mucosal injury from infection, antineoplastic agents, and acarbose (an antidiabetic alpha glucosidase inhibitor) would be expected to result in a transient condition that affects all ingested food substrates [103]. Dietary management approaches, including elemental diets, have been used until the condition improved. N-butyl-deoxynojirimycin (miglustat), an imino sugar and synthetic analog of D-glucose, is a medication used for treatment of lysosomal storage diseases. Miglustat has been shown to interfere with N-glycosylation of the proteins in the ER that delays intracellular trafficking of disaccharidase molecules to the apical surface [104]; and this epiphenomenon suggests a host of investigative models for maldigestion worthy of future investigation of disaccharidase deficiency should specific replacement enzyme therapy become available for clinical testing.

One might argue that starch and oligosaccharide maldigestion is the greater issue than sucrose in CSID, but this assessment is too often overlooked since starch has such an important role in the habitual diet and there is no recognized approach to addressing glucose polymer oligosaccharide maldigestion in humans. Stable 13C-isotope labeled substrates composed of sucrose or alpha-limit dextrins (soluble oligosaccharide substrates no longer vulnerable to alpha-amylase), may be prepared and used as a more specific breath test substrate [76]. Assay of small intestinal biopsies are difficult to interpret due to substrate-activity overlap. A pediatric study demonstrated that assay outcomes did not correlated with disaccharidase tolerance [105]. Furthermore, the samples taken by forceps are likely to vary in depth and breadth of mucosa such that reported activity ratio outcomes may not be truly representative due to variable total protein content (divisor). Breath testing with stable isotope-tracer-tagged alpha limit dextrin provides oxidative values that represent combined digestive capacity of isomaltase (palatinase) and maltase-glucoamylase. Crude starch substrate would be dependent upon alpha-amylase action as well as disaccharidases. Sucrose substrate breath tests results also reflect in vivo activity that test could be most useful to assess the complimentary portion of the SI dimer. Sacrosidase supplementation therapy is available to be used in concert with strict dietary fructose restriction for best results [106]. Should other candidate therapies advance to replace mucosal isomaltase and glucoamylase activities in vivo [107, 108], per-oral therapy activity could be easily assessed and efficacy might be determined.

Summary/conclusion

This communiqué reviewed the rationale for disaccharidase testing in clinical practice when there is a need to assess unexplained chronic and recurrent abdominal discomfort and associated symptoms. Disaccharidase insufficiency hides in plain-sight and lies just beyond the reach of most routine endoscopist’s forceps, but sample integrity and representation remain obstacles to routine use enzyme activity assays. Disaccharidase insufficiency is a real and underappreciated problem for which technology has advanced the evaluative tools to investigate with greater specificity. Immunohistochemistry staining is an attractive and practical alternative and worthy of further development. Commercial microscopes and intensity algorithms are available to quantify enzyme expression and implementation would not significantly extend procedure time or increase clinical risks. However, it is yet unclear how these tools should be accommodated into mainstream practice since therapies beyond dietary restrictions are few. Furthermore, the microbiome response to disaccharidase deficiencies is an involving field and should provide insights into maladies beyond functional bowel syndrome and mucositis.

If one believes that lactase insufficiency frequently exists, and the associated gastrointestinal symptoms of lactose intolerance impact the activities of daily living for millions of patients, the same credence should be paid to the other disaccharidases such as sucrase-isomaltase that relate to more important foodstuffs. One should consider that all disaccharidases share the same basic mechanism of protein expression when unexplained signs and symptoms appear that resemble lactase insufficiency. Therefore, testing for SI and maltase-glucoamylase expression appears to have an important role in the evaluation of unexplained abdominal discomfort and related symptoms and enzyme testing should have a great role in the evaluation of patients. Unfortunately, the biopsy enzyme activity assays cannot universally be relied upon for definitive assessments and substrate-specific breath testing is not widely available, but would be welcome. The dilemma is ‘what would one do with the disaccharidase activity data?’ since, at present, there is relatively little that can be done beyond dietary modifications to address any specific maldigestive issues. Recommendations for dietary modification have a role in patient management and disaccharidase testing outcomes would justify such testing.

Summary Bullet Points.

• Lactase persistence beyond early childhood with several dominant alleles is frequent, but recent studies point to such persistence being a marker for increased risk for obesity and obesity-associated malignancies.

• More than 40 sucrase-isomaltase-gene variants coding for reduced enzymatic activity have been reported and indicates deficiency conditions are more common than previously thought may contribute to the development of IBS-like symptoms.

• Disaccharidase testing is challenging to perform routinely, and outcomes currently rely on non-standardized mucosal samples snap-frozen on liquid nitrogen or indirect estimates of activity that are based upon by-products of maldigestion.

• Disaccharidase testing, especially for sucrase-isomaltase, that might be better served by the use of immunohistochemistry with results applied to a semi-quantitative comparative scale, but further validation work is needed.