Validity of the Rapid Thyroxine Absorption Test for the Differentiation Between Levothyroxine Non-compliance and Malabsorption in Thyroid-Stimulating Hormone Refractory Hypothyroidism

Introduction Thyroid-stimulating hormone refractory hypothyroidism is a common problem. This is due to either non-compliance or malabsorption with levothyroxine (LT4). The study aimed to assess the validity of the rapid LT4 absorption test in the differentiation between LT4 malabsorption and non-compliance. Methods A cross-sectional study was done from January to October 2022 at Faiha Specialized Diabetes, Endocrine, and Metabolism Center, in Basrah, Southern Iraq. Twenty-two patients with thyroid-stimulating hormone (TSH) refractory hypothyroidism were evaluated by rapid LT4 absorption test with measurements of TSH before 1000 μg LT4 intake, and free thyroxine (pmol/l) and total thyroxine before (nmol/l) (baseline TT4 and baseline FT4) and two hours after (2-HR TT4 and 2-HR FT4). The findings were compared with the following four-week-long supervised LT4 absorption test results. Results In the rapid LT4 absorption test, patients with (2-HR FT4 minus baseline FT4 ≤1.28 pmol/l (0.1 ng/dl) or 2-HR FT4 minus baseline FT4 1.28-6.43 pmol/l (0.1-0.5 ng/dl) plus 2-HR TT4 minus baseline TT4<72.08 nmol/l (5.6 µg/dl)), eight out of 10 patients were correctly diagnosed with malabsorption. And in those with (2-HR FT4 minus baseline FT4 ≥6.43 (0.5 ng/dl) or 2-HR FT4 minus baseline FT4 1.28-6.43 (0.1-0.5 ng/dl) plus 2-HR TT4 minus baseline TT4≥72.08 (5.6 µg/dl)), 11 out of 12 patients were correctly diagnosed as non-compliant. This criterion showed 88.8% sensitivity, 15.4% specificity, 80% positive predictive value, and 91.6% negative predictive value for diagnosing LT4 malabsorption. Conclusion The rapid LT4 absorption test showed good diagnostic accuracy in differentiating non-compliance from malabsorption when (2-HR FT4 minus baseline FT4) and (2-HR TT4 minus baseline TT4) were used as criteria.


Introduction
In adults, hypothyroidism is the most common thyroid disorder, and it is more common in females and with increasing age. It has a wide diverse presentation ranging from asymptomatic to severe forms presenting as myxedema coma, a life-threatening complication [1,2]. The synthetic thyroid hormone levothyroxine (LT4) is an affordable treatment for hypothyroidism with few adverse effects [3]. Persistence of hypothyroid symptoms in patients on LT4 in a dose more than 1.9 µg/kg body weight or thyroid stimulating hormone (TSH) more than 4.5 µU/ml after six weeks of dose increase is called TSH refractory hypothyroidism [4]. Noncompliance is attributed to multiple factors and limitations imposed by therapy; the patient should take the medication in a fasting state, the period between medication and meal, the daily basis of medication, and avoidance of multiple drugs familiar to the patient which interfere with LT4 absorption [5]. Weekly administration of LT4 is an alternative strategy to a daily dose of LT4 intended to improve the adherence of patients. In addition, there is resistance to conventional doses of oral LT4 in patients with inflammatory bowel disease, celiac disease, lactose intolerance, atrophic gastritis [6][7][8][9], and Helicobacter pylori infection [10]. Interference with LT4 absorption is caused by taking some foods and drinks including fibers, soy protein, coffee, and grapefruit [11,12]. Moreover, some drugs interfere with LT4 absorption when co-administered with them. Examples of these drugs are calcium elements, ferrous components, aluminum hydroxide, chromium picolinate, cholestyramine, colesevelam, gastric protectants like sucralfate, estrogen receptor inhibitors like raloxifene, and phosphate chelators like sevelamer [13].
Duodenum and jejunum are the absorption sites of 60-80% of administered LT4 doses, with maximum absorption occurring when the stomach is empty and within three hours of ingestion [14]. The gastric power of hydrogen (pH) is essential for LT4 absorption. Therefore, proton pump inhibitors, gastrectomy, and gastric bypass surgery result in LT4 malabsorption [15].
Patients who continue to suffer from biochemical and/or clinical hypothyroidism despite a high dose of LT4 may be described as non-compliance or pseudo malabsorption, a condition that is usually due to nonadherence to medications. Some of these patients may have underlying psychiatric disorders, but not a simple matter of therapy abandonment [16].
Even after careful consideration of all factors that lead to malabsorption, still there are difficulties to differentiate malabsorption from non-compliance. A large single dose and a weekly dose of LT4 are two protocols used to discriminate malabsorption from non-compliance in TSH refractory hypothyroidism [17,18].
The study aimed to assess the validity of the rapid LT4 absorption test for the differentiation between noncompliance and malabsorption in TSH refractory hypothyroidism.

Materials And Methods
A cross-sectional study was conducted at Faiha Specialized Diabetes Endocrine and Metabolism Center (FDEMC), Basrah, Southern Iraq, during the period from January 2022 to October 2022. Through the period of the study, 537 out of 1120 patients with hypothyroidism had visited the center for follow-up and had TSH above target. Clinical data were taken in the form of age, duration of hypothyroidism, cardiovascular disease, malabsorption diseases, history of thyroidectomy, and drug history. Body weight and height were measured in bare feet and light clothes for calculation of body mass index (BMI) in kilogram/squared meter (kg/m2).

Inclusion and exclusion criteria
We included patients aged 18-65 years with TSH refractory hypothyroidism who were currently taking LT4 in a dose of >1.9 µg/kg and TSH >4.5µIU/ml after six weeks of dose increase. The patients agreed to participate in the study to be involved in both the rapid and supervised long LT4 absorption protocols.
The patients were excluded if they had a history of the following conditions: cardiovascular disease, pregnancy, current use of drugs that interfere with levothyroxine absorption or oral contraceptive pills, history of malabsorption syndrome, and declared non-compliance with LT4 intake.
After evaluation, 34 patients met the inclusion criteria and agreed to participate in the study. A written informed consent was taken from each participant in accordance with the ethical standards of the FDEMC Research Committee, from which ethical approval was obtained (ref #62/38/21), and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Rapid LT4 absorption test
After an overnight fast, early morning, and with an empty stomach, each patient was given 1000 µg LT4 orally and supervised over the next hour for vomiting and any clinical deterioration. Two blood samples were taken from each patient (five ml for each sample), put in a clot activator tube, centrifuged immediately, and serum separated for analysis. The first sample (baseline) was taken before LT4 intake for analysis of TSH, FT4, and TT4. The second sample was taken two hours later (2-HR) for analysis of FT4 and TT4. The evaluations of these samples were done by the following criteria: Percentage of LT4 absorption was calculated by the following equation [19]: Volume of Distribution (VD) = 0.442 x BMI. Percent absorbed = (Peak TT4-Baseline TT4) µg/dL x 10 dl/L x VD L / Total administered LT4 dose (µg). Normal % absorbed range 50-100%, Average 80%. Peak absorption of less than 60% indicates LT4 malabsorption.
Further assessments using the increment in FT4 which is 2-HR FT4 minus baseline FT4 and the increment in TT4 which is 2-HR TT4 minus baseline TT4.

Supervised long LT4 absorption test
This test was considered the standard test for differentiating non-compliance from malabsorption in the study. It was started in each patient on the seventh day after the rapid LT4 absorption test. The total weekly dose of LT4 (1.9 µg/kg body weight multiplied by seven) was given each week for the next four weeks in the early morning on an empty stomach and the patients were supervised for the next two hours for vomiting or any clinical deterioration. At the end of the period of four weeks later, TSH measurements were done again. Based on the results, the patients were classified into two groups, non-compliance (TSH ≤4.2 µIU/ml) and malabsorption (TSH >4.2 µIU/ml).
Both tests were well tolerated by all participants without any serious side effects. However, only 22 patients completed the supervised long LT4 absorption test and were involved in the final analysis. Missing at least one-week appointment was considered an exclusion from the final analysis.

Statistical analysis
The data were analyzed by the Statistical Package for the Social Sciences (SPSS), version 26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were summarized as numbers (N) and percentages (%). Continuous variables were summarized as mean ± standard deviation (M ± SD). Comparisons of % absorption and 2-HR FT4/baseline FT4 ratio in between groups (malabsorption vs non-compliance) were done using the independent student t-test. Repeated-Measures Analysis of Variance (ANOVA) with a Greenhouse-Geisser correction was used to compare the values of FT4 and TT4 at baseline and after two hours of tests, and post hoc test with Bonferroni corrections for in between group comparisons. Receiver operating characteristic (ROC) curves were plotted for evaluation of the accuracy of (% absorption, 2-HR FT4/baseline FT4 ratio, 2-HR FT4 minus baseline FT4, and 2-HR TT4 minus baseline TT4) in the diagnosis of LT4 malabsorption. Correlations of different rapid LT4 absorption test cut-offs in between groups were done using Chi-square tests. A P-value of < 0.05 was defined as statistical significance for all above comparisons.

Results
The general characteristics of the study patients are summarized in Table 1  After a one-month follow-up with a long supervised LT4 absorption test, 13 (59%) were diagnosed as noncompliance and nine (41%) as having malabsorption.
On analysis of the results of the rapid LT4 absorption test, the means of % absorption did not differ significantly in between groups, as shown in Figure 1.

FIGURE 1: In between groups, comparisons of mean % absorption.
Abbreviations: SD, standard deviation.
A similar finding was also seen on comparison of the 2-HR FT4/baseline FT4 ratio that did not differ significantly in between groups, as shown in Figure 2.

Discussion
Poor adherence to daily administration of LT4 is the most common cause for non-responding to high doses of LT4 [21]. The LT4 absorption tests (different protocols) are used to differentiate between non-compliance and true malabsorption [17,18]. In the current study, we consider the long-supervised LT4 absorption test as a gold standard test for comparison with and validity assessment of other tests for multiple reasons. First, we gave a weekly dose of 1.9µg/kg LT4 under the supervision of a doctor to ensure compliance and safety. Second, TSH needs at least four to six weeks for normalization [22]. In the rapid LT4 absorption test, Soares et al. suggested that the ratio 2-HR FT4/baseline FT4 of more than or equal to 2.5 means LT4 noncompliance in TSH refractory hypothyroidism [20]. In the present study, neither the mean 2-HR FT4/baseline FT4 ratio nor its cut-off 2.5 could significantly differentiate between malabsorption and non-compliance. When LT4 % absorption was used as a criterion, a similar finding was seen with poor diagnostic accuracy for LT4 malabsorption. While a review of data from 16 patients from the Mayo Clinic showed that the peak % LT4 absorption was at hour four and it can provide valuable information for distinguishing malabsorption from non-compliance using a cut of less than 60% [19]. There are many reports of absolute LT4 absorption as low as 63% and as high as 195% by a similar equation [23][24][25][26]. LT4 absorption appears to be more highly absorbed in the hypothyroid patient compared to the normal individual [27]. In the present study, we tried to use the simplest possible protocol with the measurements of TT4 after two hours. Despite this factor, seven out of 17 patients with ≥60 % absorption had malabsorption on the long-supervised LT4 absorption test.
In the present study, we found that taking the 2-HR increment in FT4 and to a lesser extent TT4 had significant diagnostic accuracy to differentiate LT4 malabsorption and non-compliance. And a better result was seen when both FT4 and TT4 increments were taken together. Another study by Walker et al., 2013, included 23 patients with TSH refractory hypothyroidism showing a mean increment in FT4 after two hours about 54% ± 3% from baseline FT4 occurring in all patients in contrast to only 75% of patients showing improvement of TSH and not normalization after the fourth week of treatment with weight-related weekly doses of LT4. But these weekly doses were significantly lower than the equivalent daily dose which was already taken by patients before the study. In this study, there is no cut-off value to warrant investigating malabsorption. They conclude that using combined tests (long and rapid) significantly differentiates between malabsorption and non-compliance [17].
Moreover, another study by Ghosh et al. depends on the absolute increment in FT4 during three hours of rapid LT4 absorption test and considers an increment ≥0.4 ng/dl (5.14 pmol/l) for non-compliance with a sensitivity of 97% and specificity of 80% to exclude malabsorption [22]. In this study, they did not use three hours of FT4/baseline FT4 ratio to assess the efficiency of LT4 absorption for two reasons. First, high baseline FT4 as the patient took a dose of LT4 the day of the test made a low ratio of three hours FT4/baseline FT4 and missed non-compliance. Second, in chronic prolonged hypothyroidism whose baseline FT4 can be very low, the three hours FT4/baseline FT4 ratio will be high and falsely diagnose noncompliance. However, in the present study in all of the 22 patients, the baseline hormonal measurements were done without morning intake of LT4 and still, the 2-HR FT4/baseline FT4 ratio failed to differentiate between non-compliance and malabsorption.
The study has limitations. First, there is no well-established gold standard for the differentiation between non-compliance and malabsorption. However, in the present study, we relied on the supervised long LT4 absorption test as a comparator. Second, the study was limited to a small sample and some of the included patients did not complete the overall five weeks follow-up.

Conclusions
In conclusion, the rapid LT4 absorption test is a simple and safe test in the evaluation of patients with TSH refractory hypothyroidism. It showed a good diagnostic accuracy in the differentiation between noncompliance and malabsorption when 2-HR FT4 minus baseline FT4 and 2-HR TT4 minus baseline TT4 were used as criteria. The 2-HR FT4/baseline FT4 ratio and percentage absorption both lack diagnostic accuracy.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Faiha Specialized Diabetes, Endocrine and Metabolism Center (FDEMC) Research Ethical Committee issued approval ref #62/38/21. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.