Chronic Kidney Disease and Micronutrients

Citation:
 
Study Design:
Class:
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Quality Rating:
Research Purpose:

To compare the changes in the sulphur-containing amino acid concentrations with those in other amino acids in stable long-term renal transplant recipients, as well as the effects of pyridoxine, folic acid and vitamin B12 on plasma amino acid concentrations.

Inclusion Criteria:
  • Transplanted recipients with one to fourteen years of transplantation
  • Stable renal function
  • Elevated plasma cysteine-homocysteine concentrations.
Exclusion Criteria:

Not mentioned.

Description of Study Protocol:

Recruitment

Not mentioned.

Design

Time series study: Transplanted recipients and healthy control subjects were initially studied to measure plasma amino acids concentrations. Transplanted patients had received their kidneys from one to fourteen years before the time of the study. All were receiving immunosuppressive drugs together with corticosteroids. They were eating a normal diet, and none was receiving vitamin therapy at the beginning of the study. All were ambulant and measurements were made at the time of outpatient visits. After an overnight fast, plasma amino acids and serum creatinine were measured. The control subjects had routine hematological and biochemical examinations.

Vitamin Therapy

Those transplanted recipients whose cysteine-homocysteine concentration was high were arbitrarily selected for the second phase of the study. These patients were given vitamin B12 intramuscularly, and folic acid and pyridoxine orally for two weeks. Measurements were done before and after the vitamin therapy. Diet logs kept during the treatment period showed no change in protein or calorie intake. Then patients sequentially received pyridoxine (100mg per day for two weeks), then folic acid (five mg per day for two weeks) was added to it, and finally vitamin B12 (1,000mcg per day for two weeks) was added to the other two vitamins. Each vitamin period lasted for two weeks. Measurements were done in the beginning and after two-week intervals just before the addition of each cofactor. After an interval of six to nine months off all vitamin therapy, some of the patients were given folic acid for four weeks and vitamin B12 injection was added to the regimen for  two more weeks. These last series of vitamin-therapy studies were done due to the lack of measurement of erythrocyte folate in the vitamin studies and because there was some doubt about the relative contributions of folic acid and vitamin B12 to the changes in cysteine-homocysteine observed earlier. 

Intervention

Vitamin B12 (1000mcg)+pyridoxine (100mg)+folic acid (five mg). Each intervention lasted two weeks and was sequentially added to the previous intervention.

Statistical Analysis

Student's T test was used to assess the differences between the grouped data. The test was unpaired for the overall amino acid results and paired for the rest of the data.

 

 

Data Collection Summary:

Timing of Measurements

Biochemical parameters from transplanted patients and control subjects were measured after an overnight fast. Amino acid concentrations from transplanted patients with elevated cysteine-homocysteine concentrations were measured before and two weeks after the administration of the three vitamins together. For each vitamin regimen added sequentially every two weeks, blood samples to measure amino acids and creatinine were drawn before starting the regimen, and repeated at two-week intervals just before the addition of each cofactor, at the end of the six weeks of treatment and 10 weeks after cessation of  the vitamin therapy. Serum folate and vitamin B12 were measured before and after treatment. Finally, after six to nine months off the vitamin therapy, biochemical parameters were measured after two and four weeks of treatment with folic acid and after two weeks with vitamin B12.

Dependent Variables

  • Amino acids 
  • Cysteine-homocysteine
  • Serum creatinine
  • Erythrocyte folate.

Independent Variables

  • Pyridoxine
  • Vitamin B12
  • Folic acid.

 

Description of Actual Data Sample:

Initial N: Transplanted patients, 27 (13 male, 14 female)

Attrition (final N): Transplanted patients: 11 or 8. Only transplanted patients with high cysteine-homocysteine concentrations participated in the vitamin therapy.

Age: Mean age, 41.2 years±8.9 years (range, 21 to 51)

Ethnicity: Not mentioned

Other relevant demographics: The disease states leading to chronic renal failure and transplantation in the patients were: analgesic nephropathy (n=8), chronic pyelonephritis (n=4), chronic glomerulonephritis (n=12) and congenital renal malformation (n=3)

Anthropometrics: Results obtained for the neutral and acidic amino acids were compared with those measured in 25 normal subjects matched for age and sex with the renal recipients

Location: New South Wales, Australia.

 

Summary of Results:

Mean Plasma Amino Acid Concentrations in Transplant Recipients and in Normal Subjects 

Amino Acid Concentration (µmol/L)

Normal Subjects

Transplant Recipients

All patients (N=27)

Transplant Recipients

Creatinine (≤0.11 mmol/L) (n=11)

Transplant Recipients

Creatinine (>0.11 mmol/L) (n=16)

Cysteine-homocysteine

3.1±0.9

6.0±3.2***

3.5±0.7

7.6±3.2***

Alanine

232.1±61.4

367.4±89.5*** 

348.3±96.7** 

380.5±84.9***

Cystine

64.0±16.6 

91.6±29.3*** 

75.0±11.6 *

103.0±32.6***
Serine 121.4±25.3 99.9±30.8** 101.7±18.4* 98.7±37.6*
Valine 225.8±51.5 194.1±43.9* 205.1±39.0 186.5±46.7*
Leucine 124.8±34.7 106.4±26.3* 107.1±27.8 106.0±26.2
*P<0.05; **P<0.01; ***P<0.001; Normal vs. transplant recipient

 

AFTER VITAMIN ADMINISTRATION:

The plasma cysteine-homocysteine concentrations was decreased in 35% (P<0.05) after two weeks of treatment with all three vitamins administered together in patients with a high concentration of cysteine-homocysteine.

 

Effect on Mean Plasma Cysteine-Homocysteine Concentration of Treatment with Each Vitamin Added Sequentially At Intervals of Two Weeks in Transplant Recipients

  Before Treatment (n=11)

After Pyridoxine (n=11)

After Pyridoxine and Folic Acid (n=11) After Pyridoxine, Folic Acid and Vitamin B12 (n=11) 10 Weeks Post-Vitamin Treatment (n=10)

Cysteine-Homocysteine (µmol/L)

7.3±2.1 6.8±1.7 5.1±0.7* 4.3±0.8** 6.1±1.6
Serum Creatinine (mmol/L) 0.21±0.09 0.23±0.09 0.22±0.09 0.22±0.09 0.19±0.06

  *P<0.01; **P<0.001 Before treatment vs after treatment

 

Treatment with Folic Acid Followed by Vitamin B12 On Plasma Cysteine-Homocysteine Concentration In Transplant Recipients After an Interval of Six to Nine Months Off All Vitamin Therapy

  Before Treatment (n=8) After Folic Acid for Two Weeks (n=8) After Folic Acid for Four Weeks (n=8) After Folic Acid for Six Weeks, with Vitamin B12 After Two Weeks (n=8)

Cysteine-Homocysteine(µmol/L)

 9.0±3.1 6.3±1.3*  5.4±1.6*  5.7±2.0*
Serum Creatinine (mmol/L)  0.18±0.05 0.19±0.06  0.17±0.05  0.18±0.06 

*P<0.002. Before treatment vs. after treatment

Author Conclusion:

Plasma homocysteine is increased in renal transplant recipients when serum creatinine is only moderately elevated. Homocysteine concentrations are decreased by treatment with folic acid, suggesting that both reduced homocysteine excretion and relative shortages of folic acid are responsible.

Funding Source:
Government: the National Health and Medical Reseach Council of Australia
Not-for-profit
0
Foundation associated with industry:
Reviewer Comments:

The study was not well-designed. Selection of patients for the vitamin therapy is not clear, increasing biases to select those patients with better renal function. Patient exclusion/inclusion in each phase of the study is very confusing. Table three information about the time-treatment for vitamin B12 was different from the time described under results and methods. Finally, the small sample size increases risk for type 2 error and many statistical tests.

All data regarding healthy control subjects were not included in this worksheet but were reported in original article.

Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
 
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? No
2. Was the selection of study subjects/patients free from bias? No
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? No
  2.2. Were criteria applied equally to all study groups? ???
  2.3. Were health, demographics, and other characteristics of subjects described? No
  2.4. Were the subjects/patients a representative sample of the relevant population? No
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) Yes
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) No
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? No
  4.1. Were follow-up methods described and the same for all groups? No
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) No
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? No
  4.4. Were reasons for withdrawals similar across groups? ???
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? No
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? No
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? No
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? No
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? No
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? No
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? No
  6.6. Were extra or unplanned treatments described? No
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? No
  7.1. Were primary and secondary endpoints described and relevant to the question? No
  7.2. Were nutrition measures appropriate to question and outcomes of concern? No
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? No
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? ???
  7.5. Was the measurement of effect at an appropriate level of precision? No
  7.6. Were other factors accounted for (measured) that could affect outcomes? No
  7.7. Were the measurements conducted consistently across groups? No
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? No
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? No
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? No
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? No
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? No
9. Are conclusions supported by results with biases and limitations taken into consideration? ???
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? ???
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes