NSA: Serum Proteins (2009)


Van Binsbergen CJ, Odink J, Van den Berg H, Koppeschaar H, Coelingh Bennink HJ. Eur J Clin Nutr. Nutritional status in anorexia nervosa: Clinical chemistry, vitamins, iron and zinc. 1988 Nov; 42(11): 929-937.

PubMed ID: 3074921
Study Design:
Cross-Sectional Study
D - Click here for explanation of classification scheme.
Quality Rating:
Neutral NEUTRAL: See Quality Criteria Checklist below.
Research Purpose:

To investigate the status of vitamins, iron and zinc in a group of patients with anorexia nervosa (AN), and compare the status to that of lean women and a group of women of normal weight. A routine clinical chemical profile was determined.

Inclusion Criteria:

Women with AN:

  • Ages between 18 and 35 years old
  • Underweight 25% or more below ideal body weight (IBW) for age and height, according to the Metropolitan Life Insurance Company statistical bulletin (1959)
  • Amenorrhea for at least six months duration
  • Acrocyanosis, lanugo
  • Distorted attitude towards eating, food and weight
  • Fear of becoming obese in spite of extreme thinness
  • Met diagnostic criteria by Feighner et al, 1972 and criteria described in the diagnostic statistical manual of mental disorders (DSM III) of the American Psychiatric Association (1080).

A group of 20 healthy, age-matched women as controls:

  • Weighing 80% to 90% of IBW (10 of the women; lean control group)
  • Weighing 90% of 120% of IBW (10 of the women; normal weight control group)
  • Regular ovulatory cycles (indicated by a biphasic basal temperature curve) ranging between 26 and 35 days.
Exclusion Criteria:

Women with AN:

  • Oral contraception or other medication use
  • Medical or psychiatric illness that would account for the anorexia and weight loss.

A group of 20 healthy, age-matched women as controls:

  • Acrocyanosis, lanugo
  • Distorted attitudes towards eating, food or weight
  • Extreme fear of becoming obese.
Description of Study Protocol:


Patients with AN attending the Departments of Psychiatry and of Obstetrics and Gynecology of the State University Hospital of Utrecht between 1983 and 1986 were approached. The healthy matches were recruited through advertisement.


Cross-sectional analysis. 


Subjects fasted overnight and reported to clinic for data collection measures.

Statistical Analysis

Data were investigated with one-way analysis of variance (ANOVA) with factor group.  Significance was set at P<0.05.

Data Collection Summary:

Timing of Measurements

After an overnight fast, subjects presented at clinic for a blood sample. Prior to the study, no dietary prescriptions were given. It is not reported when the subject met with the dietitian for diet history collection.

Dependent Variables

  • Energy intake: Dietitian used the cross-check dietary history method of Burke (1947); subjects were asked about their customary diet (in-household measures), and afterwards, the dietary intake during the last month was cross-checked
  • Regularity of meals: Dietitian collected
  • Occurrence of self-induced vomiting and laxative use: Dietitian collected
  • Leucocyte, lympocyte, platelets in blood
  • Hemoglobin (Hb), hematocrit (Ht), total protein
  • Albumin, prealbumin, triglycerides, L-y-glutamyl transferase (y-GT), alkaline phosphotase (ALP), glutamate pyruvate  transaminase (GPT), creatinine, urea
  • Sodium, potassium, calcium, iron, zinc, total iron binding capacity (TIBC) and iron saturation
  • Cholesterol and HDL-cholesterol in serum: Determined with kits
  • Retinol binding protein (RBP)
  • Serum all-trans retinol, carotene (total carotenoids), and alpha-tocopherol
  • 25-hydroxy-vitamin D extracted from serum
  • Thiamin, flavin adenine dinucleotide (FAD), pyridoxal-5'-phosphate (PLP) and vitamin C (L-ascorbic acid) in whole blood
  • Vitamin B12 (true cyanocobalamin) and folate (5-methyl-tetrahydrofolic acid) in serum
  • Basal transketolase (ETK), glutathione reductase (EGR), and glutamate oxaloacetate transaminase (EGOT) activities in erythrocytes.

Independent Variables

 AN, lean or normal weight grouping.



Description of Actual Data Sample:
  • Initial N: (20 AN, 10 lean, 10 healthy)
  • Age: AN 24.7, lean 26.1, normal 25.1 years (means).


BMI: O<0.001

  • AN: 14.4
  • Lean: 18.1
  • Normal: 20.8.


The Netherlands.

Summary of Results:

 Key Findings

  • Patients with AN had higher activities of y-GT and SGPT and a higher concentration of prealbumin in serum and lower leukocyte and lymphocyte counts in blood. No other routine clinical chemical parameters were significantly different between groups.
  • AN patients had higher serum vitamin B12 and retinol levels. No significant differences were found for thiamin, vitamin B6, vitamin C, folate, vitamin E and vitamin D. AN patients had a lower level of FAD in blood and a lower stimulation ratio of the alpha-EGR.
  • Patients with AN had higher serum ferritin concentration and lower TIBC; Hb, Hct and iron saturation were not significantly different. No significant difference was found in the concentration of zinc in plasma.







Statistical Significance of Group Difference






Energy Intake (kcal)





Basal Temperature (degrees Celsius) at day nine of cycle





Leucocytes (109 per L) 4.30 5.36 5.68 P<0.05
Lymphocytes (109 per L) 1.48 1.83 2.01 P<0.05
Platelets (109 per L) 173 171 168 NS
y-GT (U per L) 17.5 7.3 8.5 P<0.001
GPT (U per L) 20.0 7.7 8.6 P<0.01
ALP (U per L) 40.3 42.0 39.4 NS
Creatinine (micromol per L) 72.1 66.7 72.9 NS
Urea (mmol per L) 5.19 4.10 4.31 NS
Sodium (mmol per L) 141 141 141 NS
Potassium (mmol per L) 3.71 4.06 4.11 NS
Calcium (mmol per L) 2.19 2.15 2.23 NS
Total protein (g per L) 65.2 66.0 65.9 NS
Albumin (g per L) 40.7 41.9 41.2 NS
Prealbumin (mg per L) 301 252 244 P<0.05
RBP (mg per L) 53.5 51.7 51.4 NS
Triglycerides (mmol per L) 0.65 0.63 0.74 NS
Cholesterol (mmol per L) 4.93 4.26 4.34 NS
HDL cholesterol (mmol per L) 1.49 1.47 1.47 NS
Thiamin (nmol per L) 122 107 126 NS
ETK (U per mmol Hb) 12.0 11.3 10.2 NS
alpha-ETK 1.11 1.10 1.10 NS
FAD (nmol per L) 245 279 328 P<0.001
EGR (U per L mmol Hb) 104.4 96.5 97.2 NS
alpha-EGR 1.03 1.18 1.18 P<0.001
PLP (nmol per L) 80.3 81.2 89.2 NS
EGOT (U per mmol Hb) 67.7 64.2 63.9 NS
Vitamin B12 (pmol per L) 475 274 301 P<0.01
Vitamin C (micromol per L) 46.1 52.9 55.5 NS
Folate (nmol per L) 8.74 8.32 7.64 NS
Retinol (micromol per L) 1.59 1.32 1.18 P<0.05
Carotene (micromol per L) 2.30 2.24 2.14 NS
25-OH-vitamin D (nmol per L) 40.8 51.5 56.8 NS
alpha-tocopherol (micromol per L) 25.3 23.4 22.1 NS
Hb (nmol per L) 7.6 7.9 8.0 NS
Hct 0.37 0.39 0.39 NS
Iron (micromol per L) 14.9 15.6 16.4 NS
TIBC (micromol per L) 49.7 53.8 62.6 P<0.001
Iron saturation 0.308 0.297 0.274 NS
Ferritin (microg per L) 68 39 12 P<0.01
Zinc (micromol per L) 12.3 12.4 13.7 NS


Author Conclusion:

Patients with AN had poor intake of nutrients and energy, but the results obtained did not indicate an inadequate status of vitamins, iron and zinc.

Funding Source:
University/Hospital: Dept of OB/GYN, University Hospital, Utrecht; Dept of Clinical Biochemistry, TNO-GIVO Toxicology and Nutrition Institute, Zeist, Dept of Endocrinology and Internal Medicine, University Hospital, Utrecht
Reviewer Comments:


  • Small sample size
  • AN patients denied high doses of vitamin use prior to the study, but it cannot excluded because AN patients may be less than truthful about eating behaviors
  • Vitamin concentrations may not reflect vitamin stores
  • Cross-sectional; cannot assume cause and effect.
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) N/A
  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) N/A
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? Yes
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? Yes
  2.2. Were criteria applied equally to all study groups? Yes
  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? No
  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? No
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  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? No
  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.) No
  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? N/A
  4.1. Were follow-up methods described and the same for all groups? N/A
  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%.) N/A
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? N/A
  4.4. Were reasons for withdrawals similar across groups? N/A
  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? Yes
  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? Yes
  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? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? N/A
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? Yes
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? N/A
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? N/A
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? N/A
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? N/A
  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? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? Yes
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? Yes
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? No
  7.7. Were the measurements conducted consistently across groups? Yes
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? Yes
  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)? N/A
  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? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? No
10. Is bias due to study's funding or sponsorship unlikely? No
  10.1. Were sources of funding and investigators' affiliations described? No
  10.2. Was the study free from apparent conflict of interest? No