Measuring sucrose in blood after oral administration to detect abomasal ulcers in calves

Abstract

Abomasal ulcers are common in cattle, especially in calves, and to date, there is no reliable antemortem method for diagnosis, to our knowledge. We assessed if measuring sucrose in blood after oral administration in calves could be used to identify animals with abomasal ulcers. Terminally ill calves (n = 12; part A) and calves designated for slaughter (n = 123; part B) were given a sucrose solution per os, and blood samples were taken 15, 30, 60, 90, and 120 min (part A) or 30 and 60 min (part B) after administration. The calves were then euthanized or slaughtered, and their abomasa were examined. Serum samples were analyzed using highperformance liquid chromatography-mass spectrometry, and data were analyzed using general linear mixed models. Calves both with and without affected abomasa had increasing sucrose values over time without significant differences. Also, there was no relationship between the size of the mucosal lesion and sucrose values.

Abomasal ulcers, although frequently overlooked, are an important cause of indigestion in cattle. There is strong evidence that many cattle suffer from abomasal ulcers, and veal calves seem to be the most severely affected group; 1 in 4 deaths were caused by perforated abomasal ulcers in one study.¹ Unfortunately, ulcers are often clinically inapparent until they perforate.⁹ Innocuous stages of abomasal ulcers can typically not be diagnosed in cattle by any means, laboratory or imaging.

It has been shown that gastric damage can be detected by measuring the increase in gastric permeability, for example using the disaccharide sucrose.¹³ The sucrose molecule is large enough to be withheld from permeation by the intact gastric mucosa and is immediately hydrolyzed in the small intestine by the brush border enzyme sucrase to its components of glucose and fructose. Thus the absorption of undigested sucrose must occur prior to the small intestine and implies damage of the gastric mucosa.¹³

Sucrose can be measured using numerous methods, for example gas chromatography–flame ionization detection, as has been established in horses.⁵ However, high-performance liquid chromatography (HPLC) is the technique for routine sugars analysis recommended by AOAC International.¹⁷

Given the diagnostic gap for abomasal ulcers that could possibly undergo life-threatening progression, it would be desirable to develop a tool for early in vivo diagnosis. Therefore, we assessed if increased concentrations of sucrose could be detected in blood after oral administration in calves with abomasal ulcers, and whether this could be used in live animals to distinguish animals with abomasal lesions from animals with healthy abomasa.

Our animal handling and procedures were approved by the Austrian Federal Ministry of Education, Science and Research (protocol BMWFW-68.205/0134-WF/V/3b/2014). Our study was conducted in terminally ill calves (part A) and calves designated for slaughter (part B). For part A, 12 calves that were patients at the University Clinic for Ruminants (University of Veterinary Medicine, Vienna, Austria) were used to determine the course of the sucrose concentration in blood over time. All calves received milk for at least part of their diet (Table 1). A jugular venous catheter was placed, and a whole blood sample was taken before each calf received 1 g of sucrose (Wiener Feinkristallzucker [Viennese fine crystal sugar], AGRANA Zucker, Leopoldsdorf, Austria) per kg of body weight in a 10% solution of tap water per os (p.o.). Further blood samples were taken 15, 30, 60, 90, and 120 min later before the calf was euthanized. A postmortem examination was performed wherein the abomasum was opened along the greater curvature, thoroughly cleaned with water, and examined macroscopically. Abomasal ulcers were visually classified (types 14), and type 1 ulcers were subclassified (Table 2).^3,19 The ulcers were then photographed with a ruler to determine scale (Supplementary Figs. 1–4). In addition to healthy and ulcerated mucosa, calves with distinct discoloration of the mucosa, but without visible ulceration, were included in a third category for statistical analysis.

Table 1.

Sucrose values, area of the ulcer, and ulcer type in calves of part A of the study.

Calf	Age (d)	Breed	Sucrose (µmol/L)						Ulcer area (cm2)	Ulcer type
			0 min	15 min	30 min	60 min	90 min	120 min
1	48	S	0	0.5	2.0	13.6	34.2	47.0	1.8	1c
2	85	S	0	0.4	1.1	1.3	1.5	1.5	4.8	1a
3	68	S	0	3.5	6.3	10.9	20.9	31.0	16.6	3
4	22	S	0	14.6	20.7	31.6	39.2	46.7	0.5	1d
5	82	S	0	1.3	3.8	5.6	6.4	7.9	1.1	3
9	84	S	0	0.8	1.0	1.1	0.8	0.6	17.4	1a
10	41	S	0	2.0	3.8	8.5	14.9	20.7	NA	NA
6	24	BS	1.0	2.7	5.5	18.4	40.3	59.0	0	NA
7	16	S	0	1.3	1.9	3.9	6.5	7.9	0	NA
8	58	S	0	2.0	3.2	4.8	5.4	5.8	0	NA
11	5	HF	0	1.1	3.0	6.1	9.4	15.8	0	NA
12	118	S	0	1.3	1.6	2.3	3.8	3.0	0	NA

BS = Brown Swiss; HF = Holstein-Friesian; NA = not applicable; S = Simmental.

Table 2.

Types of abomasal ulcers, with subtypes of type 1.

Ulcer type	Description
Type 1	Non-perforating ulcer with incomplete penetration of the abomasal wall
1a	Erosion with minimal mucosal defects and loss of mucosal rugae
1b	Deeper erosions with mucosal hemorrhage, sharply demarcated with a depressed center
1c	Craters with coating of detritus or fibrin, depressed center, and bulging margins
1d	Radial wrinkles with a central point, affecting gastric folds only
Type 2	Bleeding ulcer with penetration of a major abomasal vessel and severe intraluminal hemorrhage
Type 3	Perforating ulcer with localized peritonitis as a result of adhesion to adjacent viscera
Type 4	Perforating ulcer with diffuse peritonitis as a result of the spread of ingesta through the peritoneal cavity

Modified from Smith et al.¹⁹ and Braun et al.³ (see also Supplementary Figs. 1–3).

For part B of the study, 123 veal Simmental or Simmental crossbreed calves, ~4-mo-old, intended for slaughter, were used. Compared to calves in part A, no medical or feeding history of these calves was available. At the abattoir, a whole blood sample was taken from the jugular vein using a vacutainer system (Vacutainer PrecisionGlide needles 20G and needle holders; Becton Dickinson, Vienna, Austria) before the calves also received 1 g of sucrose per kg of body weight in a 10% solution of tap water p.o. Further blood samples were taken 30 and 60 min later. The calves were then slaughtered, and their abomasa were collected and examined. Ulcers were classified (including discoloration of the mucosa as a separate category) and photographed as described above; the area of all abomasal ulcers was measured by the same person, applying a polygon method using software (Jalomed, Lübeck, Germany; Supplementary Fig. 4).

Blood samples were allowed to clot and then centrifuged. Serum was stored at −80°C until all samples were analyzed at the same time. All samples from part A and samples of 47 calves from part B (16 calves without ulcers, 15 calves with type 1b ulcers, 13 calves with discoloration of the abomasal mucosa, and 3 calves with chronic ulcers [2 type 1c and 1 healed type 3]) were analyzed using HPLC-tandem mass spectrometry (HPLC-MS/MS; Shimadzu, Kyoto, Japan). An HPLC column (apHera-NH₂-polymer; Sigma-Aldrich, St. Louis, MO) was used, and ions were detected (QTRAP 6500 LC-MS/MS system; AB Sciex, Framingham, MA). The mobile phase was 90% acetonitrile:10% water. After thawing the serum samples, 50 µL of serum were mixed with 20 µL of internal standard (¹³C₁₂ sucrose; Sigma-Aldrich) and 150 µL of acetonitrile (Merck, Darmstadt, Germany). The mix was vortexed for 15 s and centrifuged for 2 min at 3,840 × g. The supernatant was transferred into an injection vial, and 5 µL were injected into the HPLC-MS/MS device. Analysis was performed with a column temperature of 60°C with a flow of 1 mL/min and a retention time of 3.4 min. The calibration range was 0.29-73 µmol/L.

Data were analyzed with software R v.3.5.0.¹⁸ We applied linear mixed-effects models with maximum likelihood estimation (option REML=FALSE) for log₁₀ transformed values of sucrose as response with function lmer in package lme4.² As explanatory variables, we fitted “time since application of sucrose” measured in minutes as fixed continuous effect, clinic (part A) or slaughterhouse (part B), and either a 3-level fixed effect for presence or absence of ulcers as well as mucosal alterations, or area of ulcer measured on a continuous scale. Both models were taken into account because both questions were regarded as clinically relevant: can we detect an ulcer, and can we relate the measured sucrose concentration to ulcer size?

Data from parts A and B of the study were analyzed together, including only observations from shared times 0 (baseline), 30, and 60 min after administration of sucrose. To account for the covariance structure in our repeated measures data, we fitted a random intercept effect for calf. The p values for fixed effects were calculated with package lmerTest.¹⁰

Our data met the assumptions for linear mixed-effects models. Residuals and random effects of calves were normally distributed, and residuals showed variance homogeneity. We also tested random intercept and random slope models, but these were not significantly better than random slope models only. Graphs were created using the packages ggplot2 and lsmeans.^11,21 G*Power v.3.1.9.2 was used for post-hoc power calculations.⁴

Of the 12 calves in part A, 6 had abomasal ulcers, and 5 served as control animals. Calf 10 had necrosis of large parts of the abomasal mucosa as a result of ischemia and was therefore counted as part of the third category (discoloration of the abomasal mucosa without distinct ulceration). Of the 123 calves in part B, 37 (30%) had healthy abomasa, 27 (22%) had discoloration of the mucosa, and 59 (48%) calves had abomasal ulcers; of these, 39 (66%) had type 1a ulcers, 17 (29%) had type 1b ulcers, and 3 (5%) had chronic ulcers (type 1c and healed type 3 ulcer).

The calves in part A of the study had altered mucosa (ulcers or discoloration) ranging from 0.5 to 17.4 cm² in size with a median of 3.3 cm² (Table 1). In calves in part B, ulcers or discoloration ranged from 0.02 to 5.2 cm² (median 0.7 cm²).

In calves without and with ulcers or mucosal discoloration in both parts of the study, sucrose concentrations rose over time (Figs. 1, 2). The peak of the curve was not passed within the observation period of 2 h after administration (Fig. 1). Neither statistical model showed a significant relationship between the presence or area of an ulcer (Figs. 3, 4) and the sucrose values measured. The sucrose values at 90 and 120 min in part A of the study were used for a post-hoc power calculation and showed that the power (β) was 0.44.

Figures 1–4.

Sucrose concentrations over time and in relation to ulcer size. Figure 1. Calves with and without ulcers from part A of the study (NU = no ulcer, gray; U = ulcer, white). Calf 10, with necrosis of the abomasal mucosa, is included in group U. Figure 2. Calves with and without ulcers as well as mucosal discolorations from part B of the study (M = mucosal discoloration, light gray; NU = dark gray; U = white). Figure 3. Sucrose concentration in relation to the area of the ulcer in all calves (parts A and B) 60 min after sucrose administration. Figure 4. Sucrose concentration in relation to the area of the ulcer in calves from part A 120 min after sucrose administration.

Our study corroborates previous findings that many calves have abomasal ulcers.⁸ Most of the ulcers found in our pilot study in calves sent for slaughter were type 1a and therefore likely innocuous. Therefore, calves with somewhat more distinct type 1b and chronic ulcers were selected for sucrose analysis in part B. The small depth and size of the ulcers possibly allowed only limited sucrose absorption, and no statistically significant difference could be shown from calves with normal intact abomasal mucosa. The median area of ulcers in our study was much smaller than ulcers in previous studies, in which large (>2 cm diameter) and extensively coalescing ulcers were significantly associated with increased sucrose concentrations in the urine of horses.¹⁴

The more relevant reason for non-detection of differences between the groups is most likely the timing of blood sampling. Data were not available beforehand to determine optimal sampling times in calves; therefore, we based our decision on studies in other species wherein sucrose peaks occurred at 45 min in horses and at 60 min in humans and rabbits.^6,13 All of our samples were analyzed together, so information from part A could not be used to optimize the approach in part B. The peak of the sucrose curve was not passed after 120 min, but at the last 2 sampling times, the difference between the groups U (ulcer) and NU (no ulcers) became most pronounced (Fig. 1). Further studies to determine the kinetics of sucrose absorption in calves are necessary to reassess the usefulness of our experimental approach.

The question remains why sucrose could be detected in calves without abomasal ulcers. It has been suggested that the exposure of gastrointestinal epithelium to glucose derived from partially hydrolyzed sucrose could cause enhancement of cytoskeletal tension, which weakens tight junctions. This would allow paracellular absorption, possibly of molecules as large as sucrose, by solvent drag.¹² This mechanism supposedly occurs when the capacity of the transcellular uptake is saturated. Therefore, the concentration of the sucrose solution could influence the rate of its paracellular absorption. It should be determined in further studies whether administering lower concentrations of sucrose could yield more significant results regarding the difference between ulcerated and healthy abomasal mucosa.

In general, aside from these possible physiologic mechanisms, there are various factors that modify intestinal absorption, for example stress, which could have been caused by transporting the calves to the abattoir and handling them for sampling.²⁰ Another factor is age. The permeability of the gastrointestinal tract in calves decreases with age. In 6-mo-old cattle, it has been shown that the jejunum appears to have the greatest potential for paracellular permeability.¹⁵ In young calves up to 44 d of age, regions with the greatest permeability are duodenum and, interestingly, the rumen.²²

We selected calves as test subjects because of their developing rumen, and we assumed that the sucrose solution would bypass the rumen via the esophageal groove and be hydrolyzed in the small intestine. However, the sucrose solution may have failed to bypass the rumen and been partially absorbed and partially fermented there. This could have caused ruminal acidosis, and the associated decrease in pH together with the increase in osmolarity could have impaired barrier function in the rumen and further increased absorption.¹⁶

Even though sucrose as a marker for gastric damage was shown to have limited sensitivity and specificity in horses, it cannot be entirely excluded that it may prove useful in calves from the data presented herein.⁷ Further studies should be conducted with modified sampling times testing different concentrations of sucrose in calves of different ages with more severe ulcers.